Confocal bioimaging the living cornea with autofluorescence and specific fluorescent probes

NASA Astrophysics Data System (ADS)

Masters, Barry R.; Paddock, Stephen W.

1990-08-01

Confocal bioimaging of the fine structure of the living rabbit cornea with both reflected light and fluorescent light has been demonstrated with a laser scanning confocal imaging system. Kalman averaging was used to reduce the noise in the images. Superficial epithelial, basal epithelial cells, stromal keratocytes, and endothelial cells were imaged. These cells and their subcellular structures were imaged in the two modes for comparison. The superficial epithelial cells were imaged by their autofluorescence (488/520 nm). This fluorescence signal may be due to the mitochondrial flavoproteins and can be used as a noninvasive indicator of cellular oxidative function. Thiazole orange was used to stain cell nuclei for fluorescence imaging. DiOC6 was used to stain the endoplasmic reticulum for fluorescence imaging. Fluorescein- conjugated phalloidin was used to stain actin for fluorescence imaging.

Laser line scanning for fluorescence reflectance imaging: a phantom study and in vivo validation of the enhancement of contrast and resolution.

PubMed

Fantoni, Frédéric; Hervé, Lionel; Poher, Vincent; Gioux, Sylvain; Mars, Jérôme I; Dinten, Jean-Marc

2014-01-01

Intraoperative fluorescence imaging in reflectance geometry is an attractive imaging modality to noninvasively monitor fluorescence-targeted tumors. In some situations, this kind of imaging suffers from poor resolution due to the diffusive nature of photons in tissue. The objective of the proposed technique is to tackle this limitation. It relies on the scanning of the medium with a laser line illumination and the acquisition of images at each position of excitation. The detection scheme proposed takes advantage of the stack of images acquired to enhance the resolution and the contrast of the final image. The experimental protocol is described to fully understand why we overpass the classical limits and validate the scheme on tissue-like phantoms and in vivo with a preliminary testing. The results are compared with those obtained with a classical wide-field illumination.

Dual-detector X-ray fluorescence imaging of ancient artifacts with surface relief

PubMed Central

Smilgies, Detlef-M.; Powers, Judson A.; Bilderback, Donald H.; Thorne, Robert E.

2012-01-01

Interpretation of X-ray fluorescence images of archeological artifacts is complicated by the presence of surface relief and roughness. Using two symmetrically arranged fluorescence detectors in a back-reflection geometry, the proper X-ray fluorescence yield can be distinguished from intensity variations caused by surface topography. This technique has been applied to the study of Roman inscriptions on marble. PMID:22713888

Label-free NIR reflectance imaging as a complimentary tool for two-photon fluorescence microscopy: multimodal investigation of stroke (Conference Presentation)

NASA Astrophysics Data System (ADS)

Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S.

2016-03-01

Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.

Performance of different reflectance and diffuse optical imaging tomographic approaches in fluorescence molecular imaging of small animals

NASA Astrophysics Data System (ADS)

Dinten, Jean-Marc; Petié, Philippe; da Silva, Anabela; Boutet, Jérôme; Koenig, Anne; Hervé, Lionel; Berger, Michel; Laidevant, Aurélie; Rizo, Philippe

2006-03-01

Optical imaging of fluorescent probes is an essential tool for investigation of molecular events in small animals for drug developments. In order to get localization and quantification information of fluorescent labels, CEA-LETI has developed efficient approaches in classical reflectance imaging as well as in diffuse optical tomographic imaging with continuous and temporal signals. This paper presents an overview of the different approaches investigated and their performances. High quality fluorescence reflectance imaging is obtained thanks to the development of an original "multiple wavelengths" system. The uniformity of the excitation light surface area is better than 15%. Combined with the use of adapted fluorescent probes, this system enables an accurate detection of pathological tissues, such as nodules, beneath the animal's observed area. Performances for the detection of ovarian nodules on a nude mouse are shown. In order to investigate deeper inside animals and get 3D localization, diffuse optical tomography systems are being developed for both slab and cylindrical geometries. For these two geometries, our reconstruction algorithms are based on analytical expression of light diffusion. Thanks to an accurate introduction of light/matter interaction process in the algorithms, high quality reconstructions of tumors in mice have been obtained. Reconstruction of lung tumors on mice are presented. By the use of temporal diffuse optical imaging, localization and quantification performances can be improved at the price of a more sophisticated acquisition system and more elaborate information processing methods. Such a system based on a pulsed laser diode and a time correlated single photon counting system has been set up. Performances of this system for localization and quantification of fluorescent probes are presented.

Fluorescence Imaging Topography Scanning System for intraoperative multimodal imaging

PubMed Central

Quang, Tri T.; Kim, Hye-Yeong; Bao, Forrest Sheng; Papay, Francis A.; Edwards, W. Barry; Liu, Yang

2017-01-01

Fluorescence imaging is a powerful technique with diverse applications in intraoperative settings. Visualization of three dimensional (3D) structures and depth assessment of lesions, however, are oftentimes limited in planar fluorescence imaging systems. In this study, a novel Fluorescence Imaging Topography Scanning (FITS) system has been developed, which offers color reflectance imaging, fluorescence imaging and surface topography scanning capabilities. The system is compact and portable, and thus suitable for deployment in the operating room without disturbing the surgical flow. For system performance, parameters including near infrared fluorescence detection limit, contrast transfer functions and topography depth resolution were characterized. The developed system was tested in chicken tissues ex vivo with simulated tumors for intraoperative imaging. We subsequently conducted in vivo multimodal imaging of sentinel lymph nodes in mice using FITS and PET/CT. The PET/CT/optical multimodal images were co-registered and conveniently presented to users to guide surgeries. Our results show that the developed system can facilitate multimodal intraoperative imaging. PMID:28437441

Nanoscale cellular imaging with scanning angle interference microscopy.

PubMed

DuFort, Christopher; Paszek, Matthew

2014-01-01

Fluorescence microscopy is among the most widely utilized tools in cell and molecular biology due to its ability to noninvasively obtain time-resolved images of live cells with molecule-specific contrast. In this chapter, we describe a simple high-resolution technique, scanning angle interference microscopy (SAIM), for the imaging and localization of fluorescent molecules with nanometer precision along the optical axis. In SAIM, samples above a reflective surface are sequentially scanned with an excitation laser at varying angles of incidence. Interference patterns generated between the incident and reflected lights result in an emission intensity that depends on the height of a fluorophore above the silicon surface and the angle of the incident radiation. The measured fluorescence intensities are then fit to an optical model to localize the labeled molecules along the z-axis with 5-10 nm precision and diffraction-limited lateral resolution. SAIM is easily implemented on widely available commercial total internal reflection fluorescence microscopes, offering potential for widespread use in cell biology. Here, we describe the setup of SAIM and its application for imaging cellular structures near (<1 μm) the sample substrate. © 2014 Elsevier Inc. All rights reserved.

Probing Membrane Order and Topography in Supported Lipid Bilayers by Combined Polarized Total Internal Reflection Fluorescence-Atomic Force Microscopy

PubMed Central

Oreopoulos, John; Yip, Christopher M.

2009-01-01

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C20 or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions. PMID:19254557

Development of a widefield reflectance and fluorescence imaging device for the detection of skin and oral cancer

NASA Astrophysics Data System (ADS)

Pratavieira, S.; Santos, P. L. A.; Bagnato, V. S.; Kurachi, C.

2009-06-01

Oral and skin cancers constitute a major global health problem that cause great impact in patients. The most common screening method for oral cancer is visual inspection and palpation of the mouth. Visual examination relies heavily on the experience and skills of the physician to identify and delineate early premalignant and cancer changes, which is not simple due to the similar characteristics of early stage cancers and benign lesions. Optical imaging has the potential to address these clinical challenges. Contrast between normal and neoplastic areas may be increased, distinct to the conventional white light, when using illumination and detection conditions. Reflectance imaging can detect local changes in tissue scattering and absorption and fluorescence imaging can probe changes in the biochemical composition. These changes have shown to be indicatives of malignant progression. Widefield optical imaging systems are interesting because they may enhance the screening ability in large regions allowing the discrimination and the delineation of neoplastic and potentially of occult lesions. Digital image processing allows the combination of autofluorescence and reflectance images in order to objectively identify and delineate the peripheral extent of neoplastic lesions in the skin and oral cavity. Combining information from different imaging modalities has the potential of increasing diagnostic performance, due to distinct provided information. A simple widefiled imaging device based on fluorescence and reflectance modes together with a digital image processing was assembled and its performance tested in an animal study.

Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina.

PubMed

Zawadzki, Robert J; Zhang, Pengfei; Zam, Azhar; Miller, Eric B; Goswami, Mayank; Wang, Xinlei; Jonnal, Ravi S; Lee, Sang-Hyuck; Kim, Dae Yu; Flannery, John G; Werner, John S; Burns, Marie E; Pugh, Edward N

2015-06-01

Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.

Improving confocal microscopy with solid-state semiconductor excitation sources

NASA Astrophysics Data System (ADS)

Sivers, Nelson L.

To efficiently excite the fluorescent dyes used in imaging biological samples with a confocal microscope, the wavelengths of the exciting laser must be near the fluorochrome absorption peak. However, this causes imaging problems when the fluorochrome absorption and emission spectra overlap significantly, i.e. have small Stokes shifts, which is the case for most fluorochromes that emit in the red to infrared. As a result, the reflected laser excitation cannot be distinguished from the information-containing fluorescence signal. However, cryogenically cooling the exciting laser diode enabled the laser emission wavelengths to be tuned to shorter wavelengths, decreasing the interference between the laser and the fluorochrome's fluorescence. This reduced the amount of reflected laser light in the confocal image. However, the cooled laser diode's shorter wavelength signal resulted in slightly less efficient fluorochrome excitation. Spectrophotometric analysis showed that as the laser diodes were cooled, their output power increased, which more than compensated for the lower fluorochrome excitation and resulted in significantly more intense fluorescence. Thus, by tuning the laser diode emission wavelengths away from the fluorescence signal, less reflected laser light and more fluorescence information reached the detector, creating images with better signal to noise ratios. Additionally, new, high, luminous flux, light-emitting diodes (LEDs) are now powerful enough to create confocal fluorescence signals comparable to those produced by the traditional laser excitation sources in fluorescence confocal microscopes. The broader LED spectral response effectively excited the fluorochrome, yet was spectrally limited enough for standard filter sets to separate the LED excitation from the fluorochrome fluorescence signal. Spectrophotometric analysis of the excitation and fluorescence spectra of several fluorochromes showed that high-powered, LED-induced fluorescence contained the same spectral information and could be more intense than that produced by lasers. An alternative, LED-based, confocal microscope is proposed in this thesis that would be capable of exciting multiple fluorochromes in a single specimen, producing images of several distinct cellular components simultaneously. The inexpensive, LED-based, confocal microscope would require lower peak excitation intensities to produce fluorescence signals equal to those produced by laser excitation, reducing cellular damage and slowing fluorochrome photobleaching.

Multimodal quantitative phase and fluorescence imaging of cell apoptosis

NASA Astrophysics Data System (ADS)

Fu, Xinye; Zuo, Chao; Yan, Hao

2017-06-01

Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

Fluorescence tomography characterization for sub-surface imaging with protoporphyrin IX

PubMed Central

Kepshire, Dax; Davis, Scott C.; Dehghani, Hamid; Paulsen, Keith D.; Pogue, Brian W.

2009-01-01

Optical imaging of fluorescent objects embedded in a tissue simulating medium was characterized using non-contact based approaches to fluorescence remittance imaging (FRI) and sub-surface fluorescence diffuse optical tomography (FDOT). Using Protoporphyrin IX as a fluorescent agent, experiments were performed on tissue phantoms comprised of typical in-vivo tumor to normal tissue contrast ratios, ranging from 3.5:1 up to 10:1. It was found that tomographic imaging was able to recover interior inclusions with high contrast relative to the background; however, simple planar fluorescence imaging provided a superior contrast to noise ratio. Overall, FRI performed optimally when the object was located on or close to the surface and, perhaps most importantly, FDOT was able to recover specific depth information about the location of embedded regions. The results indicate that an optimal system for localizing embedded fluorescent regions should combine fluorescence reflectance imaging for high sensitivity and sub-surface tomography for depth detection, thereby allowing more accurate localization in all three directions within the tissue. PMID:18545571

Enhancing in vivo tumor boundary delineation with structured illumination fluorescence molecular imaging and spatial gradient mapping

NASA Astrophysics Data System (ADS)

Sun, Jessica; Miller, Jessica P.; Hathi, Deep; Zhou, Haiying; Achilefu, Samuel; Shokeen, Monica; Akers, Walter J.

2016-08-01

Fluorescence imaging, in combination with tumor-avid near-infrared (NIR) fluorescent molecular probes, provides high specificity and sensitivity for cancer detection in preclinical animal models, and more recently, assistance during oncologic surgery. However, conventional camera-based fluorescence imaging techniques are heavily surface-weighted such that surface reflection from skin or other nontumor tissue and nonspecific fluorescence signals dominate, obscuring true cancer-specific signals and blurring tumor boundaries. To address this challenge, we applied structured illumination fluorescence molecular imaging (SIFMI) in live animals for automated subtraction of nonspecific surface signals to better delineate accumulation of an NIR fluorescent probe targeting α4β1 integrin in mice bearing subcutaneous plasma cell xenografts. SIFMI demonstrated a fivefold improvement in tumor-to-background contrast when compared with other full-field fluorescence imaging methods and required significantly reduced scanning time compared with diffuse optical spectroscopy imaging. Furthermore, the spatial gradient mapping enhanced highlighting of tumor boundaries. Through the relatively simple hardware and software modifications described, SIFMI can be integrated with clinical fluorescence imaging systems, enhancing intraoperative tumor boundary delineation from the uninvolved tissue.

Differential high-speed digital micromirror device based fluorescence speckle confocal microscopy.

PubMed

Jiang, Shihong; Walker, John

2010-01-20

We report a differential fluorescence speckle confocal microscope that acquires an image in a fraction of a second by exploiting the very high frame rate of modern digital micromirror devices (DMDs). The DMD projects a sequence of predefined binary speckle patterns to the sample and modulates the intensity of the returning fluorescent light simultaneously. The fluorescent light reflecting from the DMD's "on" and "off" pixels is modulated by correlated speckle and anticorrelated speckle, respectively, to form two images on two CCD cameras in parallel. The sum of the two images recovers a widefield image, but their difference gives a near-confocal image in real time. Experimental results for both low and high numerical apertures are shown.

k-Space Image Correlation Spectroscopy: A Method for Accurate Transport Measurements Independent of Fluorophore Photophysics

PubMed Central

Kolin, David L.; Ronis, David; Wiseman, Paul W.

2006-01-01

We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or “blinking”. Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of α5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection. PMID:16861272

Teaching about photosynthesis with simple equipment: analysis of light-induced changes in fluorescence and reflectance of plant leaves.

PubMed

Björn, Lars Olof; Li, Shaoshan

2013-10-01

Solar energy absorbed by plants results in either reflection or absorption. The latter results in photosynthesis, fluorescence, or heat. Measurements of fluorescence changes have been used for monitoring processes associated with photosynthesis. A simple method to follow changes in leaf fluorescence and leaf reflectance associated with nonphotochemical quenching and light acclimation of leaves is described. The main equipment needed consists of a green-light emitting laser pointer, a digital camera, and a personal computer equipped with the camera acquisition software and the programs ImageJ and Excel. Otherwise, only commonly available cheap materials are required.

A novel multiwavelength fluorescence image-guided surgery imaging system

NASA Astrophysics Data System (ADS)

Volpi, D.; Tullis, I. D. C.; Laios, A.; Pathiraja, P. N. J.; Haldar, K.; Ahmed, A. A.; Vojnovic, B.

2014-02-01

We describe the development and performance analysis of two clinical near-infrared fluorescence image-guided surgery (FIGS) devices that aim to overcome some of the limitations of current FIGS systems. The devices operate in a widefield-imaging mode and can work (1) in conjunction with a laparoscope, during minimally invasive surgery, and (2) as a hand-held, open surgery imaging system. In both cases, narrow-band excitation light, delivered at multiple wavelengths, is efficiently combined with white reflectance light. Light is delivered to ~100 cm2 surgical field at 1-2 mW/cm2 for white light and 3-7 mW/cm2 (depending on wavelength) of red - near infrared excitation, at a typical working distance of 350 mm for the hand-held device and 100 mm for the laparoscope. A single, sensitive, miniaturized color camera collects both fluorescence and white reflectance light. The use of a single imager eliminates image alignment and software overlay complexity. A novel filtering and illumination arrangement allows simultaneous detection of white reflectance and fluorescence emission from multiple dyes in real-time. We will present both fluorescence detection sensitivity modeling and practical performance data. We have demonstrated the efficiency and the advantages of the devices both pre-clinically and during live surgery on humans. Both the hand-held and the laparoscopic systems have proved to be reliable and beneficial in an ongoing clinical trial involving sentinel lymph node detection in gynecological cancers. We will show preliminary results using two clinically approved dyes, Methylene blue and indocyanine green. We anticipate that this technology can be integrated and routinely used in a larger variety of surgical procedures.

Confocal Microscopy and Molecular-Specific Optical Contrast Agents for the Detection of Oral Neoplasia

PubMed Central

Carlson, Alicia L.; Gillenwater, Ann M.; Williams, Michelle D.; El-Naggar, Adel K.; Richards-Kortum, R. R.

2009-01-01

Using current clinical diagnostic techniques, it is difficult to visualize tumor morphology and architecture at the cellular level, which is necessary for diagnostic localization of pathologic lesions. Optical imaging techniques have the potential to address this clinical need by providing real-time, sub-cellular resolution images. This paper describes the use of dual mode confocal microscopy and optical molecular-specific contrast agents to image tissue architecture, cellular morphology, and sub-cellular molecular features of normal and neoplastic oral tissues. Fresh tissue slices were prepared from 33 biopsies of clinically normal and abnormal oral mucosa obtained from 14 patients. Reflectance confocal images were acquired after the application of 6% acetic acid, and fluorescence confocal images were acquired after the application of a fluorescence contrast agent targeting the epidermal growth factor receptor (EGFR). The dual imaging modes provided images similar to light microscopy of hematoxylin and eosin and immunohistochemistry staining, but from thick fresh tissue slices. Reflectance images provided information on the architecture of the tissue and the cellular morphology. The nuclear-to-cytoplasmic (N/C) ratio from the reflectance images was at least 7.5 times greater for the carcinoma than the corresponding normal samples, except for one case of highly keratinized carcinoma. Separation of carcinoma from normal and mild dysplasia was achieved using this ratio (p<0.01). Fluorescence images of EGFR expression yielded a mean fluorescence labeling intensity (FLI) that was at least 2.7 times higher for severe dysplasia and carcinoma samples than for the corresponding normal sample, and could be used to distinguish carcinoma from normal and mild dysplasia (p<0.01). Analyzed together, the N/C ratio and the mean FLI may improve the ability to distinguish carcinoma from normal squamous epithelium. PMID:17877424

Online Multitasking Line-Scan Imaging Techniques for Simultaneous Safety and Quality Evaluation of Apples

NASA Astrophysics Data System (ADS)

Kim, Moon Sung; Lee, Kangjin; Chao, Kaunglin; Lefcourt, Alan; Cho, Byung-Kwan; Jun, Won

We developed a push-broom, line-scan imaging system capable of simultaneous measurements of reflectance and fluorescence. The system allows multitasking inspections for quality and safety attributes of apples due to its dynamic capabilities in simultaneously capturing fluorescence and reflectance, and selectivity in multispectral bands. A multitasking image-based inspection system for online applications has been suggested in that a single imaging device that could perform a multitude of both safety and quality inspection needs. The presented multitask inspection approach in online applications may provide an economically viable means for a number of food processing industries being able to adapt to operate and meet the dynamic and specific inspection and sorting needs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirvonen, Liisa M.; Le Marois, Alix; Suhling, Klaus, E-mail: klaus.suhling@kcl.ac.uk

We perform wide-field time-correlated single photon counting-based fluorescence lifetime imaging (FLIM) with a crossed delay line anode image intensifier, where the pulse propagation time yields the photon position. This microchannel plate-based detector was read out with conventional fast timing electronics and mounted on a fluorescence microscope with total internal reflection (TIR) illumination. The picosecond time resolution of this detection system combines low illumination intensity of microwatts with wide-field data collection. This is ideal for fluorescence lifetime imaging of cell membranes using TIR. We show that fluorescence lifetime images of living HeLa cells stained with membrane dye di-4-ANEPPDHQ exhibit a reducedmore » lifetime near the coverslip in TIR compared to epifluorescence FLIM.« less

Dye-Enhanced Multimodal Confocal Imaging of Brain Cancers

NASA Astrophysics Data System (ADS)

Wirth, Dennis; Snuderl, Matija; Sheth, Sameer; Curry, William; Yaroslavsky, Anna

2011-04-01

Background and Significance: Accurate high resolution intraoperative detection of brain tumors may result in improved patient survival and better quality of life. The goal of this study was to evaluate dye enhanced multimodal confocal imaging for discriminating normal and cancerous brain tissue. Materials and Methods: Fresh thick brain specimens were obtained from the surgeries. Normal and cancer tissues were investigated. Samples were stained in methylene blue and imaged. Reflectance and fluorescence signals were excited at 658nm. Fluorescence emission and polarization were registered from 670 nm to 710 nm. The system provided lateral resolution of 0.6 μm and axial resolution of 7 μm. Normal and cancer specimens exhibited distinctively different characteristics. H&E histopathology was processed from each imaged sample. Results and Conclusions: The analysis of normal and cancerous tissues indicated clear differences in appearance in both the reflectance and fluorescence responses. These results confirm the feasibility of multimodal confocal imaging for intraoperative detection of small cancer nests and cells.

Fast widefield techniques for fluorescence and phase endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.

Endomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast wide-field imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated.

Application of a reflective microscope objective for multiphoton microscopy.

PubMed

Kabir, Mohammad M; Choubal, Aakash M; Toussaint, Kimani C

2018-04-20

Reflective objectives (ROs) mitigate chromatic aberration across a broad wavelength range. Yet, a systematic performance characterisation of ROs has not been done. In this paper, we compare the performance of a 0.5 numerical-aperture (NA) reflective objective (RO) with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments spanning ∼1 octave in the visible and NIR wavelengths, the SO leads to defocusing errors of 25-40% for TPF images of subdiffraction fluorescent beads and 10-12% for SHG images of collagen fibres. The corresponding error for the RO is ∼4% for both imaging modalities. This work emphasises the potential utility of ROs for multimodal multiphoton microscopy applications. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Sprayable enzyme-activatable fluorescent probes: kinetic mapping using dynamic fluorescence imaging can help detecting tiny cancer foci (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kobayashi, Hisataka

2017-02-01

Optical fluorescence-guided imaging is increasingly used to guide surgery and endoscopic procedures. Sprayable enzyme-activatable probes are particularly useful because of high target-to-background ratios that increase sensitivity for tiny cancer foci. However, green fluorescent activatable probes suffers from interference from autofluorescence found in biological tissue. Dynamic imaging followed by the kinetic analysis could be detected local enzyme activity and used to differentiate specific fluorescence arising from an activated probe in a tumor from autofluorescence in background tissues especially when low concentrations of the dye are applied to detect tiny cancer foci. Serial fluorescence imaging was performed using various concentrations of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) which was sprayed on the peritoneal surface with tiny implants of SHIN3-dsRed ovarian cancer tumors. Temporal differences in signal between specific green fluorescence in cancer foci and non-specific autofluorescence in background tissue was measured and processed into three kinetic maps reflecting maximum fluorescence signal (MF), wash-in rate (WIR), and area under the curve (AUC), respectively. Especially at lower concentrations, kinetic maps derived from dynamic fluorescence imaging were clearly superior to unprocessed images for detection small cancer foci.

Near-Infrared Confocal Laser Reflectance Cytoarchitectural Imaging of the Substantia Nigra and Cerebellum in the Fresh Human Cadaver.

PubMed

Cheyuo, Cletus; Grand, Walter; Balos, Lucia L

2017-01-01

Cytoarchitectural neuroimaging remains critical for diagnosis of many brain diseases. Fluorescent dye-enhanced, near-infrared confocal in situ cellular imaging of the brain has been reported. However, impermeability of the blood-brain barrier to most fluorescent dyes limits clinical utility of this modality. The differential degree of reflectance from brain tissue with unenhanced near-infrared imaging may represent an alternative technique for in situ cytoarchitectural neuroimaging. We assessed the utility of unenhanced near-infrared confocal laser reflectance imaging of the cytoarchitecture of the cerebellum and substantia nigra in 2 fresh human cadaver brains using a confocal near-infrared laser probe. Cellular images based on near-infrared differential reflectance were captured at depths of 20-180 μm from the brain surface. Parts of the cerebellum and substantia nigra imaged using the probe were subsequently excised and stained with hematoxylin and eosin for histologic correlation. Near-infrared reflectance imaging revealed the 3-layered cytoarchitecture of the cerebellum, with Purkinje cells appearing hyperreflectant. In the substantia nigra, neurons appeared hyporeflectant with hyperreflectant neuromelanin cytoplasmic inclusions. Cytoarchitecture of the cerebellum and substantia nigra revealed on near-infrared imaging closely correlated with the histology on hematoxylin-eosin staining. We showed that unenhanced near-infrared reflectance imaging of fresh human cadaver brain can reliably identify and distinguish neurons and detailed cytoarchitecture of the cerebellum and substantia nigra. Copyright © 2016 Elsevier Inc. All rights reserved.

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

PubMed

Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

2014-01-01

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

Snapshot imaging Fraunhofer line discriminator for detection of plant fluorescence

NASA Astrophysics Data System (ADS)

Gupta Roy, S.; Kudenov, M. W.

2015-05-01

Non-invasive quantification of plant health is traditionally accomplished using reflectance based metrics, such as the normalized difference vegetative index (NDVI). However, measuring plant fluorescence (both active and passive) to determine photochemistry of plants has gained importance. Due to better cost efficiency, lower power requirements, and simpler scanning synchronization, detecting passive fluorescence is preferred over active fluorescence. In this paper, we propose a high speed imaging approach for measuring passive plant fluorescence, within the hydrogen alpha Fraunhofer line at ~656 nm, using a Snapshot Imaging Fraunhofer Line Discriminator (SIFOLD). For the first time, the advantage of snapshot imaging for high throughput Fraunhofer Line Discrimination (FLD) is cultivated by our system, which is based on a multiple-image Fourier transform spectrometer and a spatial heterodyne interferometer (SHI). The SHI is a Sagnac interferometer, which is dispersion compensated using blazed diffraction gratings. We present data and techniques for calibrating the SIFOLD to any particular wavelength. This technique can be applied to quantify plant fluorescence at low cost and reduced complexity of data collection.

Fluorescence-guided tumor visualization using a custom designed NIR attachment to a surgical microscope for high sensitivity imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kittle, David S.; Patil, Chirag G.; Mamelak, Adam; Hansen, Stacey; Perry, Jeff; Ishak, Laura; Black, Keith L.; Butte, Pramod V.

2016-03-01

Current surgical microscopes are limited in sensitivity for NIR fluorescence. Recent developments in tumor markers attached with NIR dyes require newer, more sensitive imaging systems with high resolution to guide surgical resection. We report on a small, single camera solution enabling advanced image processing opportunities previously unavailable for ultra-high sensitivity imaging of these agents. The system captures both visible reflectance and NIR fluorescence at 300 fps while displaying full HD resolution video at 60 fps. The camera head has been designed to easily mount onto the Zeiss Pentero microscope head for seamless integration into surgical procedures.

Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

PubMed

Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D

2015-01-27

Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy.

Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer

NASA Astrophysics Data System (ADS)

Jabbour, Joey M.; Cheng, Shuna; Malik, Bilal H.; Cuenca, Rodrigo; Jo, Javier A.; Wright, John; Cheng, Yi-Shing Lisa; Maitland, Kristen C.

2013-04-01

Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm2 tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, <1 μm lateral and 3.5 μm axial resolution. FLIM-RCM imaging was performed on a tissue phantom, normal porcine buccal mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.

Experimental investigation of a HOPG crystal fan for x-ray fluorescence molecular imaging

NASA Astrophysics Data System (ADS)

Rosentreter, Tanja; Müller, Bernhard; Schlattl, Helmut; Hoeschen, Christoph

2017-03-01

Imaging x-ray fluorescence generally generates a conflict between the best image quality or highest sensitivity and lowest possible radiation dose. Consequently many experimental studies investigating the feasibility of this molecular imaging method, deal with either monochromatic x-ray sources that are not practical in clinical environment or accept high x-ray doses in order to maintain the advantage of high sensitivity and producing high quality images. In this work we present a x-ray fluorescence imaging setup using a HOPG crystal fan construction consisting of a Bragg reflecting analyzer array together with a scatter reducing radial collimator. This method allows for the use of polychromatic x-ray tubes that are in general easily accessible in contrast to monochromatic x-ray sources such as synchrotron facilities. Moreover this energy-selecting device minimizes the amount of Compton scattered photons while simultaneously increasing the fluorescence signal yield, thus significantly reducing the signal to noise ratio. The aim is to show the feasibility of this approach by measuring the Bragg reflected Kα fluorescence signal of an object containing an iodine solution using a large area detector with moderate energy resolution. Contemplating the anisotropic energy distribution of background scattered x-rays we compare the detection sensitivity, applying two different detector angular configurations. Our results show that even for large area detectors with limited energy resolution, iodine concentrations of 0.12 % can be detected. However, the potentially large scan times and therefore high radiation dose need to be decreased in further investigations.

Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane

PubMed Central

Devauges, Viviane; Matthews, Daniel R.; Aluko, Justin; Nedbal, Jakub; Levitt, James A.; Poland, Simon P.; Coban, Oana; Weitsman, Gregory; Monypenny, James; Ng, Tony; Ameer-Beg, Simon M.

2014-01-01

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor. PMID:25360776

Dye-enhanced reflectance and fluorescence confocal microscopy as an optical pathology tool

NASA Astrophysics Data System (ADS)

Yaroslavsky, Anna N.; Salomatina, Elena; Novak, John; Amat-Roldan, Ivan; Castano, Ana; Hamblin, Michael

2006-02-01

Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the ex vivo trial with fresh thick non-melanoma skin cancer excisions and in vivo trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding en face frozen H&E sections. The results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both in vivo and in vitro. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The developed technique may provide an efficient real-time optical tool for detecting skin pathology.

Tumor cell differentiation by label-free microscopy

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert; Weber, Petra; Wagner, Michael

2013-05-01

Autofluorescence and Raman measurements of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of the tumor (control) cells and the less malignant cells are similar, differences can be deduced from fluorescence spectra and nanosecond decay times. In particular, upon excitation around 375nm, the fluorescence ratio of the protein bound and the free coenzyme NADH depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. Slight differences are also observed in the Raman spectra of these cell lines, mainly originating from small granules (lysosomes) surrounding the cell nucleus. While larger numbers of fluorescence and Raman spectra are evaluated by multivariate statistical methods, additional information is obtained from spectral images and fluorescence lifetime images (FLIM).

Compact 3D printed module for fluorescence and label-free imaging using evanescent excitation

NASA Astrophysics Data System (ADS)

Pandey, Vikas; Gupta, Shalini; Elangovan, Ravikrishnan

2018-01-01

Total internal reflection fluorescence (TIRF) microscopy is widely used for selective excitation and high-resolution imaging of fluorophores, and more recently label-free nanosized objects, with high vertical confinement near a liquid-solid interface. Traditionally, high numerical aperture objectives (>1.4) are used to simultaneously generate evanescent waves and collect fluorescence emission signals which limits their use to small area imaging (<0.1 mm2). Objective-based TIRFs are also expensive as they require dichroic mirrors and efficient notch filters to prevent specular reflection within the objective lenses. We have developed a compact 3D module called cTIRF that can generate evanescent waves in microscope glass slides via a planar waveguide illumination. The module can be attached as a fixture to any existing optical microscope, converting it into a TIRF and enabling high signal-to-noise ratio (SNR) fluorescence imaging using any magnification objective. As the incidence optics is perpendicular to the detector, label-free evanescent scattering-based imaging of submicron objects can also be performed without using emission filters. SNR is significantly enhanced in this case as compared to cTIRF alone, as seen through our model experiments performed on latex beads and mammalian cells. Extreme flexibility and the low cost of our approach makes it scalable for limited resource settings.

Two-Dimensional Standing Wave Total Internal Reflection Fluorescence Microscopy: Superresolution Imaging of Single Molecular and Biological Specimens

PubMed Central

Chung, Euiheon; Kim, Daekeun; Cui, Yan; Kim, Yang-Hyo; So, Peter T. C.

2007-01-01

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or ∼100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy. PMID:17483188

Preliminary study of diagnostic spectroscopic imaging for nasopharyngeal carcinoma

NASA Astrophysics Data System (ADS)

Li, Buhong; Xie, Shusen; Zhang, Xiaodong; Li, Depin

2003-12-01

The optical biopsy system for nasopharyngeal carcinoma based on the technique of laser-induced exogenous fluorescence has been successful developed. Ar+ laser was selected as the excitation light source based on the measurement of the Emission-Excitation Matrix of Hematoporphyrin Monomethyl Ether. Tissue-simulating optical phantoms diluted with different concentration of HMME were used to simulated nasopharyngeal carcinoma lesions in the performance test for the drug-fluorescence optical biopsy system, especially for the comparison of fluorescence image contrast between the excitation wavelength of 488nm and 514.5nm, respectively. Experimental results show that the fluorescence image contrast of simulated nasopharyngeal carcinoma lesions excited by the light at the wavelength of 488nm is about three fold higher than that at 514.5nm, and the sensitivity and resolution of the fluorescence and reflection twilight image can satisfy the needs for clinical diagnosis and localization.

Microplate-compatible total internal reflection fluorescence microscopy for receptor pharmacology

NASA Astrophysics Data System (ADS)

Chen, Minghan; Zaytseva, Natalya V.; Wu, Qi; Li, Min; Fang, Ye

2013-05-01

We report the use of total internal reflection fluorescence (TIRF) microscopy for analyzing receptor pharmacology and the development of a microplate-compatible TIRF imaging system. Using stably expressed green fluorescence protein tagged β2-adrenergic receptor as the reporter, we found that the activation of different receptors results in distinct kinetic signatures of the TIRF intensity of cells. These TIRF signatures closely resemble the characteristics of their respective label-free dynamic mass redistribution signals in the same cells. This suggests that TIRF in microplate can be used for profiling and screening drugs.

Topography of Cells Revealed by Variable-Angle Total Internal Reflection Fluorescence Microscopy.

PubMed

Cardoso Dos Santos, Marcelina; Déturche, Régis; Vézy, Cyrille; Jaffiol, Rodolphe

2016-09-20

We propose an improved version of variable-angle total internal reflection fluorescence microscopy (vaTIRFM) adapted to modern TIRF setup. This technique involves the recording of a stack of TIRF images, by gradually increasing the incident angle of the light beam on the sample. A comprehensive theory was developed to extract the membrane/substrate separation distance from fluorescently labeled cell membranes. A straightforward image processing was then established to compute the topography of cells with a nanometric axial resolution, typically 10-20 nm. To highlight the new opportunities offered by vaTIRFM to quantify adhesion process of motile cells, adhesion of MDA-MB-231 cancer cells on glass substrate coated with fibronectin was examined. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Stereoscopic Integrated Imaging Goggles for Multimodal Intraoperative Image Guidance

PubMed Central

Mela, Christopher A.; Patterson, Carrie; Thompson, William K.; Papay, Francis; Liu, Yang

2015-01-01

We have developed novel stereoscopic wearable multimodal intraoperative imaging and display systems entitled Integrated Imaging Goggles for guiding surgeries. The prototype systems offer real time stereoscopic fluorescence imaging and color reflectance imaging capacity, along with in vivo handheld microscopy and ultrasound imaging. With the Integrated Imaging Goggle, both wide-field fluorescence imaging and in vivo microscopy are provided. The real time ultrasound images can also be presented in the goggle display. Furthermore, real time goggle-to-goggle stereoscopic video sharing is demonstrated, which can greatly facilitate telemedicine. In this paper, the prototype systems are described, characterized and tested in surgeries in biological tissues ex vivo. We have found that the system can detect fluorescent targets with as low as 60 nM indocyanine green and can resolve structures down to 0.25 mm with large FOV stereoscopic imaging. The system has successfully guided simulated cancer surgeries in chicken. The Integrated Imaging Goggle is novel in 4 aspects: it is (a) the first wearable stereoscopic wide-field intraoperative fluorescence imaging and display system, (b) the first wearable system offering both large FOV and microscopic imaging simultaneously, (c) the first wearable system that offers both ultrasound imaging and fluorescence imaging capacities, and (d) the first demonstration of goggle-to-goggle communication to share stereoscopic views for medical guidance. PMID:26529249

Comparative study of protoporphyrin IX fluorescence image enhancement methods to improve an optical imaging system for oral cancer detection

NASA Astrophysics Data System (ADS)

Jiang, Ching-Fen; Wang, Chih-Yu; Chiang, Chun-Ping

2011-07-01

Optoelectronics techniques to induce protoporphyrin IX fluorescence with topically applied 5-aminolevulinic acid on the oral mucosa have been developed to noninvasively detect oral cancer. Fluorescence imaging enables wide-area screening for oral premalignancy, but the lack of an adequate fluorescence enhancement method restricts the clinical imaging application of these techniques. This study aimed to develop a reliable fluorescence enhancement method to improve PpIX fluorescence imaging systems for oral cancer detection. Three contrast features, red-green-blue reflectance difference, R/B ratio, and R/G ratio, were developed first based on the optical properties of the fluorescence images. A comparative study was then carried out with one negative control and four biopsy confirmed clinical cases to validate the optimal image processing method for the detection of the distribution of malignancy. The results showed the superiority of the R/G ratio in terms of yielding a better contrast between normal and neoplastic tissue, and this method was less prone to errors in detection. Quantitative comparison with the clinical diagnoses in the four neoplastic cases showed that the regions of premalignancy obtained using the proposed method accorded with the expert's determination, suggesting the potential clinical application of this method for the detection of oral cancer.

Use of a white light supercontinuum laser for confocal interference-reflection microscopy

PubMed Central

Chiu, L-D; Su, L; Reichelt, S; Amos, WB

2012-01-01

Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460–700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser. PMID:22432542

Characterization of a reflective objective with multiphoton microscopy

NASA Astrophysics Data System (ADS)

Kabir, Mohammad M.; Choubal, Aakash M.; Sivaguru, Mayandi; Toussaint, Kimani C.

2018-02-01

Reflective objectives (ROs) can reduce chromatic aberration across a wide wavelength range in multiphoton microscopy (MPM). However, a systematic characterization of the performance of ROs has not been carried out. In this paper, we analyze the performance of a 0.5 numerical-aperture (NA) RO and compare it with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments extending 1 octave in visible and NIR wavelengths, the SO introduces defocusing errors of 25% for TPF images of sub-diffraction fluorescent beads and 10% for SHG images of collagen fibers. For both imaging systems, the RO provides a corresponding error of 4%. This work highlights the potential usefulness of ROs for multimodal MPM applications.

Multimodal imaging system for dental caries detection

NASA Astrophysics Data System (ADS)

Liang, Rongguang; Wong, Victor; Marcus, Michael; Burns, Peter; McLaughlin, Paul

2007-02-01

Dental caries is a disease in which minerals of the tooth are dissolved by surrounding bacterial plaques. A caries process present for some time may result in a caries lesion. However, if it is detected early enough, the dentist and dental professionals can implement measures to reverse and control caries. Several optical, nonionized methods have been investigated and used to detect dental caries in early stages. However, there is not a method that can singly detect the caries process with both high sensitivity and high specificity. In this paper, we present a multimodal imaging system that combines visible reflectance, fluorescence, and Optical Coherence Tomography (OCT) imaging. This imaging system is designed to obtain one or more two-dimensional images of the tooth (reflectance and fluorescence images) and a three-dimensional OCT image providing depth and size information of the caries. The combination of two- and three-dimensional images of the tooth has the potential for highly sensitive and specific detection of dental caries.

Fluorescence excitation and imaging of single molecules near dielectric-coated and bare surfaces: a theoretical study.

PubMed

Axelrod, Daniel

2012-08-01

Microscopic fluorescent samples of interest to cell and molecular biology are commonly embedded in an aqueous medium near a solid surface that is coated with a thin film such as a lipid multilayer, collagen, acrylamide, or a cell wall. Both excitation and emission of fluorescent single molecules near film-coated surfaces are strongly affected by the proximity of the coated surface, the film thickness, its refractive index and the fluorophore's orientation. For total internal reflection excitation, multiple reflections in the film can lead to resonance peaks in the evanescent intensity versus incidence angle curve. For emission, multiple reflections arising from the fluorophore's near field emission can create a distinct intensity pattern in both the back focal plane and the image plane of a high aperture objective. This theoretical analysis discusses how these features can be used to report film thickness and refractive index, and fluorophore axial position and orientation. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

Real-time visualization of melanin granules in normal human skin using combined multiphoton and reflectance confocal microscopy.

PubMed

Majdzadeh, Ali; Lee, Anthony M D; Wang, Hequn; Lui, Harvey; McLean, David I; Crawford, Richard I; Zloty, David; Zeng, Haishan

2015-05-01

Recent advances in biomedical optics have enabled dermal and epidermal components to be visualized at subcellular resolution and assessed noninvasively. Multiphoton microscopy (MPM) and reflectance confocal microscopy (RCM) are noninvasive imaging modalities that have demonstrated promising results in imaging skin micromorphology, and which provide complementary information regarding skin components. This study assesses whether combined MPM/RCM can visualize intracellular and extracellular melanin granules in the epidermis and dermis of normal human skin. We perform MPM and RCM imaging of in vivo and ex vivo skin in the infrared domain. The inherent three-dimensional optical sectioning capability of MPM/RCM is used to image high-contrast granular features across skin depths ranging from 50 to 90 μm. The optical images thus obtained were correlated with conventional histologic examination including melanin-specific staining of ex vivo specimens. MPM revealed highly fluorescent granular structures below the dermal-epidermal junction (DEJ) region. Histochemical staining also demonstrated melanin-containing granules that correlate well in size and location with the granular fluorescent structures observed in MPM. Furthermore, the MPM fluorescence excitation wavelength and RCM reflectance of cell culture-derived melanin were equivalent to those of the granules. This study suggests that MPM can noninvasively visualize and quantify subepidermal melanin in situ. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multifunction Imaging and Spectroscopic Instrument

NASA Technical Reports Server (NTRS)

Mouroulis, Pantazis

2004-01-01

A proposed optoelectronic instrument would perform several different spectroscopic and imaging functions that, heretofore, have been performed by separate instruments. The functions would be reflectance, fluorescence, and Raman spectroscopies; variable-color confocal imaging at two different resolutions; and wide-field color imaging. The instrument was conceived for use in examination of minerals on remote planets. It could also be used on Earth to characterize material specimens. The conceptual design of the instrument emphasizes compactness and economy, to be achieved largely through sharing of components among subsystems that perform different imaging and spectrometric functions. The input optics for the various functions would be mounted in a single optical head. With the exception of a targeting lens, the input optics would all be aimed at the same spot on a specimen, thereby both (1) eliminating the need to reposition the specimen to perform different imaging and/or spectroscopic observations and (2) ensuring that data from such observations can be correlated with respect to known positions on the specimen. The figure schematically depicts the principal components and subsystems of the instrument. The targeting lens would collect light into a multimode optical fiber, which would guide the light through a fiber-selection switch to a reflection/ fluorescence spectrometer. The switch would have four positions, enabling selection of spectrometer input from the targeting lens, from either of one or two multimode optical fibers coming from a reflectance/fluorescence- microspectrometer optical head, or from a dark calibration position (no fiber). The switch would be the only moving part within the instrument.

Fluorescence microscopy.

PubMed

Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

2014-10-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

Fluorescence Microscopy

PubMed Central

Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

2016-01-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

Multispectral near-IR reflectance imaging of simulated early occlusal lesions: Variation of lesion contrast with lesion depth and severity

PubMed Central

Simon, Jacob C.; Chan, Kenneth H.; Darling, Cynthia L.; Fried, Daniel

2014-01-01

Background and Objectives Early demineralization appears with high contrast at near-IR wavelengths due to a ten to twenty fold difference in the magnitude of light scattering between sound and demineralized enamel. Water absorption in the near-IR has a significant effect on the lesion contrast and the highest contrast has been measured in spectral regions with higher water absorption. The purpose of this study was to determine how the lesion contrast changes with lesion severity and depth for different spectral regions in the near-IR and compare that range of contrast with visible reflectance and fluorescence. Materials and Methods Forty-four human molars were used in this in vitro study. Teeth were painted with an acid-resistant varnish, leaving a 4×4 mm window on the occlusal surface of each tooth exposed for demineralization. Artificial lesions were produced in the unprotected windows after 12–48 hr exposure to a demineralizing solution at pH-4.5. Near-IR reflectance images were acquired over several near-IR spectral distributions, visible light reflectance, and fluorescence with 405-nm excitation and detection at wavelengths greater than 500-nm. Crossed polarizers were used for reflectance measurements to reduce interference from specular reflectance. Cross polarization optical coherence tomography (CP-OCT) was used to non-destructively assess the depth and severity of demineralization in each sample window. Matching two dimensional CP-OCT images of the lesion depth and integrated reflectivity were compared with the reflectance and fluorescence images to determine how accurately the variation in the lesion contrast represents the variation in the lesion severity. Results Artificial lesions appear more uniform on tooth surfaces exposed to an acid challenge at visible wavelengths than they do in the near-IR. Measurements of the lesion depth and severity using CP-OCT show that the lesion severity varies markedly across the sample windows and that the lesion contrast in the visible does not accurately reflect the large variation in the lesion severity. Reflectance measurements at certain near-IR wavelengths more accurately reflect variation in the depth and severity of the lesions. Conclusion The results of the study suggest that near-IR reflectance measurements at longer wavelengths coincident with higher water absorption are better suited for imaging early caries lesions. PMID:24375543

Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro

To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic {beta} cells, the regulated biphasic exocytosis from two typesmore » of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.« less

Study on high power ultraviolet laser oil detection system

NASA Astrophysics Data System (ADS)

Jin, Qi; Cui, Zihao; Bi, Zongjie; Zhang, Yanchao; Tian, Zhaoshuo; Fu, Shiyou

2018-03-01

Laser Induce Fluorescence (LIF) is a widely used new telemetry technology. It obtains information about oil spill and oil film thickness by analyzing the characteristics of stimulated fluorescence and has an important application in the field of rapid analysis of water composition. A set of LIF detection system for marine oil pollution is designed in this paper, which uses 355nm high-energy pulsed laser as the excitation light source. A high-sensitivity image intensifier is used in the detector. The upper machine sends a digital signal through a serial port to achieve nanoseconds range-gated width control for image intensifier. The target fluorescence spectrum image is displayed on the image intensifier by adjusting the delay time and the width of the pulse signal. The spectral image is coupled to CCD by lens imaging to achieve spectral display and data analysis function by computer. The system is used to detect the surface of the floating oil film in the distance of 25m to obtain the fluorescence spectra of different oil products respectively. The fluorescence spectra of oil products are obvious. The experimental results show that the system can realize high-precision long-range fluorescence detection and reflect the fluorescence characteristics of the target accurately, with broad application prospects in marine oil pollution identification and oil film thickness detection.

Neurosurgical confocal endomicroscopy: A review of contrast agents, confocal systems, and future imaging modalities

PubMed Central

Zehri, Aqib H.; Ramey, Wyatt; Georges, Joseph F.; Mooney, Michael A.; Martirosyan, Nikolay L.; Preul, Mark C.; Nakaji, Peter

2014-01-01

Background: The clinical application of fluorescent contrast agents (fluorescein, indocyanine green, and aminolevulinic acid) with intraoperative microscopy has led to advances in intraoperative brain tumor imaging. Their properties, mechanism of action, history of use, and safety are analyzed in this report along with a review of current laser scanning confocal endomicroscopy systems. Additional imaging modalities with potential neurosurgical utility are also analyzed. Methods: A comprehensive literature search was performed utilizing PubMed and key words: In vivo confocal microscopy, confocal endomicroscopy, fluorescence imaging, in vivo diagnostics/neoplasm, in vivo molecular imaging, and optical imaging. Articles were reviewed that discussed clinically available fluorophores in neurosurgery, confocal endomicroscopy instrumentation, confocal microscopy systems, and intraoperative cancer diagnostics. Results: Current clinically available fluorescent contrast agents have specific properties that provide microscopic delineation of tumors when imaged with laser scanning confocal endomicroscopes. Other imaging modalities such as coherent anti-Stokes Raman scattering (CARS) microscopy, confocal reflectance microscopy, fluorescent lifetime imaging (FLIM), two-photon microscopy, and second harmonic generation may also have potential in neurosurgical applications. Conclusion: In addition to guiding tumor resection, intraoperative fluorescence and microscopy have the potential to facilitate tumor identification and complement frozen section analysis during surgery by providing real-time histological assessment. Further research, including clinical trials, is necessary to test the efficacy of fluorescent contrast agents and optical imaging instrumentation in order to establish their role in neurosurgery. PMID:24872922

Hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic imaging

NASA Astrophysics Data System (ADS)

Chen, Zhenyue; Deán-Ben, Xosé Luís.; Gottschalk, Sven; Razansky, Daniel

2018-02-01

Fluorescence imaging is widely employed in all fields of cell and molecular biology due to its high sensitivity, high contrast and ease of implementation. However, the low spatial resolution and lack of depth information, especially in strongly-scattering samples, restrict its applicability for deep-tissue imaging applications. On the other hand, optoacoustic imaging is known to deliver a unique set of capabilities such as high spatial and temporal resolution in three dimensions, deep penetration and spectrally-enriched imaging contrast. Since fluorescent substances can generate contrast in both modalities, simultaneous fluorescence and optoacoustic readings can provide new capabilities for functional and molecular imaging of living organisms. Optoacoustic images can further serve as valuable anatomical references based on endogenous hemoglobin contrast. Herein, we propose a hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic tomography, both operating in reflection mode, which synergistically combines the advantages of stand-alone systems. Validation of the spatial resolution and sensitivity of the system were first carried out in tissue mimicking phantoms while in vivo imaging was further demonstrated by tracking perfusion of an optical contrast agent in a mouse brain in the hybrid imaging mode. Experimental results show that the proposed system effectively exploits the contrast mechanisms of both imaging modalities, making it especially useful for accurate monitoring of fluorescence-based signal dynamics in highly scattering samples.

Compact fluorescence and white-light imaging system for intraoperative visualization of nerves

NASA Astrophysics Data System (ADS)

Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; tan Hehir, Cristina

2012-02-01

Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

A compact fluorescence and white light imaging system for intraoperative visualization of nerves

NASA Astrophysics Data System (ADS)

Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; Tan Hehir, Cristina

2012-03-01

Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

Design and validation of a diffuse reflectance and spectroscopic microendoscope with poly(dimethylsioxane)-based phantoms

PubMed Central

Greening, Gage J.; Powless, Amy J.; Hutcheson, Joshua A.; Prieto, Sandra P.; Majid, Aneeka A.; Muldoon, Timothy J.

2015-01-01

Many cases of epithelial cancer originate in basal layers of tissue and are initially undetected by conventional microendoscopy techniques. We present a bench-top, fiber-bundle microendoscope capable of providing high resolution images of surface cell morphology. Additionally, the microendoscope has the capability to interrogate deeper into material by using diffuse reflectance and broadband diffuse reflectance spectroscopy. The purpose of this multimodal technique was to overcome the limitation of microendoscopy techniques that are limited to only visualizing morphology at the tissue or cellular level. Using a custom fiber optic probe, high resolution surface images were acquired using topical proflavine to fluorescently stain non-keratinized epithelia. A 635 nm laser coupled to a 200 μm multimode fiber delivers light to the sample and the diffuse reflectance signal was captured by a 1 mm image guide fiber. Finally, a tungsten-halogen lamp coupled to a 200 μm multimode fiber delivers broadband light to the sample to acquire spectra at source-detector separations of 374, 729, and 1051 μm. To test the instrumentation, a high resolution proflavine-induced fluorescent image of resected healthy mouse colon was acquired. Additionally, five monolayer poly(dimethylsiloxane)-based optical phantoms with varying absorption and scattering properties were created to acquire diffuse reflectance profiles and broadband spectra. PMID:25983372

Design and validation of a diffuse reflectance and spectroscopic microendoscope with poly(dimethylsiloxane)-based phantoms

NASA Astrophysics Data System (ADS)

Greening, Gage J.; Powless, Amy J.; Hutcheson, Joshua A.; Prieto, Sandra P.; Majid, Aneeka A.; Muldoon, Timothy J.

2015-03-01

Many cases of epithelial cancer originate in basal layers of tissue and are initially undetected by conventional microendoscopy techniques. We present a bench-top, fiber-bundle microendoscope capable of providing high resolution images of surface cell morphology. Additionally, the microendoscope has the capability to interrogate deeper into material by using diffuse reflectance and broadband diffuse reflectance spectroscopy. The purpose of this multimodal technique was to overcome the limitation of microendoscopy techniques that are limited to only visualizing morphology at the tissue or cellular level. Using a custom fiber optic probe, high resolution surface images were acquired using topical proflavine to fluorescently stain non-keratinized epithelia. A 635 nm laser coupled to a 200 μm multimode fiber delivers light to the sample and the diffuse reflectance signal was captured by a 1 mm image guide fiber. Finally, a tungsten-halogen lamp coupled to a 200 μm multimode fiber delivers broadband light to the sample to acquire spectra at source-detector separations of 374, 729, and 1051 μm. To test the instrumentation, a high resolution proflavine-induced fluorescent image of resected healthy mouse colon was acquired. Additionally, five monolayer poly(dimethylsiloxane)-based optical phantoms with varying absorption and scattering properties were created to acquire diffuse reflectance profiles and broadband spectra.

Lensfree fluorescent on-chip imaging of transgenic Caenorhabditis elegans over an ultra-wide field-of-view.

PubMed

Coskun, Ahmet F; Sencan, Ikbal; Su, Ting-Wei; Ozcan, Aydogan

2011-01-06

We demonstrate lensfree on-chip fluorescent imaging of transgenic Caenorhabditis elegans (C. elegans) over an ultra-wide field-of-view (FOV) of e.g., >2-8 cm(2) with a spatial resolution of ∼10 µm. This is the first time that a lensfree on-chip platform has successfully imaged fluorescent C. elegans samples. In our wide-field lensfree imaging platform, the transgenic samples are excited using a prism interface from the side, where the pump light is rejected through total internal reflection occurring at the bottom facet of the substrate. The emitted fluorescent signal from C. elegans samples is then recorded on a large area opto-electronic sensor-array over an FOV of e.g., >2-8 cm(2), without the use of any lenses, thin-film interference filters or mechanical scanners. Because fluorescent emission rapidly diverges, such lensfree fluorescent images recorded on a chip look blurred due to broad point-spread-function of our platform. To combat this resolution challenge, we use a compressive sampling algorithm to uniquely decode the recorded lensfree fluorescent patterns into higher resolution images, demonstrating ∼10 µm resolution. We tested the efficacy of this compressive decoding approach with different types of opto-electronic sensors to achieve a similar resolution level, independent of the imaging chip. We further demonstrate that this wide FOV lensfree fluorescent imaging platform can also perform sequential bright-field imaging of the same samples using partially-coherent lensfree digital in-line holography that is coupled from the top facet of the same prism used in fluorescent excitation. This unique combination permits ultra-wide field dual-mode imaging of C. elegans on a chip which could especially provide a useful tool for high-throughput screening applications in biomedical research.

Lensfree Fluorescent On-Chip Imaging of Transgenic Caenorhabditis elegans Over an Ultra-Wide Field-of-View

PubMed Central

Ozcan, Aydogan

2011-01-01

We demonstrate lensfree on-chip fluorescent imaging of transgenic Caenorhabditis elegans (C. elegans) over an ultra-wide field-of-view (FOV) of e.g., >2–8 cm2 with a spatial resolution of ∼10µm. This is the first time that a lensfree on-chip platform has successfully imaged fluorescent C. elegans samples. In our wide-field lensfree imaging platform, the transgenic samples are excited using a prism interface from the side, where the pump light is rejected through total internal reflection occurring at the bottom facet of the substrate. The emitted fluorescent signal from C. elegans samples is then recorded on a large area opto-electronic sensor-array over an FOV of e.g., >2–8 cm2, without the use of any lenses, thin-film interference filters or mechanical scanners. Because fluorescent emission rapidly diverges, such lensfree fluorescent images recorded on a chip look blurred due to broad point-spread-function of our platform. To combat this resolution challenge, we use a compressive sampling algorithm to uniquely decode the recorded lensfree fluorescent patterns into higher resolution images, demonstrating ∼10 µm resolution. We tested the efficacy of this compressive decoding approach with different types of opto-electronic sensors to achieve a similar resolution level, independent of the imaging chip. We further demonstrate that this wide FOV lensfree fluorescent imaging platform can also perform sequential bright-field imaging of the same samples using partially-coherent lensfree digital in-line holography that is coupled from the top facet of the same prism used in fluorescent excitation. This unique combination permits ultra-wide field dual-mode imaging of C. elegans on a chip which could especially provide a useful tool for high-throughput screening applications in biomedical research. PMID:21253611

Fluorescence imaging in the last two decades

PubMed Central

Miyawaki, Atsushi

2013-01-01

In commemoration of the 20th anniversary of the molecular cloning of the gene for the green fluorescent protein from the jellyfish Aequorea victoria, I would like to reflect on the development of new fluorescence imaging technology in the last two decades. As this technology has become increasingly diversified, it has become more and more of a challenge to come up with a comprehensive and exhaustive review of it. Here I will focus on optogenetics and large-scale, three-dimensional reconstruction. Those two technological innovations have been achieved in the neuroscience community owing to the combined efforts of molecular biologists and light microscopists. In addition, modern fluorescence imaging has indeed improved our understanding of the spatiotemporal regulation of fundamental biological functions at cellular level. As an example, I will introduce some findings we made regarding the movement of biomolecules across the nuclear membrane. The above-mentioned imaging approaches are possible today but were impossible two decades ago. PMID:23393311

In vivo tomographic imaging of lung colonization of tumour in mouse with simultaneous fluorescence and X-ray CT.

PubMed

Zhang, Bin; Gao, Fuping; Wang, Mengjiao; Cao, Xu; Liu, Fei; Wang, Xin; Luo, Jianwen; Wang, Guangzhi; Bai, Jing

2014-01-01

Non-invasive in vivo imaging of diffuse and wide-spread colonization within the lungs, rather than distinct solid primary tumors, is still a challenging work. In this work, a lung colonization mouse model bearing A549 human lung tumor was simultaneously scanned by a dual-modality fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) system in vivo. A two steps method which incorporates CT structural information into the FMT reconstruction procedure is employed to provide concurrent anatomical and functional information. By using the target-specific fluorescence agent, the fluorescence tomographic results show elevated fluorescence intensity deep within the lungs which is colonized with diffuse and wide-spread tumors. The results were confirmed with ex vivo fluorescence reflectance imaging and histological examination of the lung tissues. With FMT reconstruction combined with the CT information, the dual-modality FMT/micro-CT system is expected to offer sensitive and noninvasive imaging of diffuse tumor colonization within the lungs in vivo. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Near-Membrane Refractometry Using Supercritical Angle Fluorescence.

PubMed

Brunstein, Maia; Roy, Lopamudra; Oheim, Martin

2017-05-09

Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet truly quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the "footprint" region of the cell during image acquisition. Our RI measurement is based on the determination on a back-focal plane image of the critical angle separating evanescent and far-field fluorescence emission components. We validate our method by imaging mouse embryonic fibroblasts and BON cells. By targeting various dyes and fluorescent-protein chimeras to vesicles, the plasma membrane, as well as mitochondria and the endoplasmic reticulum, we demonstrate local RI measurements with subcellular resolution on a standard TIRF microscope, with a removable Bertrand lens as the only modification. Our technique has important applications for imaging axial vesicle dynamics and the mitochondrial energy state or detecting metabolically more active cancer cells. Copyright © 2017. Published by Elsevier Inc.

Clinical application of a multi-modal endoscope with fluorescent peptide in detection of colorectal neoplasia (Conference Presentation)

NASA Astrophysics Data System (ADS)

Dai, Zhenzhen; Joshi, Bishnu P.; Gao, Zhenghong; Lee, Jeonghoon; Ghimire, Navin; Prabhu, Anoop; Wamsteker, Erik J.; Kwon, Richard S.; Elta, Grace H.; Appelman, Henry D.; Owens, Scott R.; Kuick, Rork; Turgeon, Kim K.; Wang, Thomas D.

2017-02-01

Early detection of precursor lesions for colorectal cancer can greatly improve survival. Pre-neoplasia can appear flat with conventional white light endoscopy. Sessile serrated adenomas (SSA) are precursor lesions found primarily in the proximal colon and frequently appear flat and indistinct. We performed a clinical study of n=37 patients using a multimodal endoscopy with a FITC-labeled peptide specific for SSA. Lesions were imaged with white light, reflectance and fluorescence. White light images were acquired before the peptide was applied and were used to help localize regions of abnormal tissues rightly. Co-registered fluorescence and reflectance images were combined to get ratio images thus the distance was corrected. We calculated the target/background ratio (T/B ratio) to quantify the images and found 2.3-fold greater fluorescence intensity for SSA compared with normal tissues. We found the T/B ratio for SSA to be significantly greater than that for normal colonic mucosa with 89.47% sensitivity and 91.67% specificity at the threshold of 1.22. An ROC curve for SSA and normal mucosa was also plotted with area under curve (AUC) of 0.93. The result also shows that SSA and adenoma are statistically significant and can be identified with 78.95% sensitivity and 90.48% specificity at the threshold of 1.66. An ROC curve was plotted with AUC of 0.88. Therefore, our result shows that the application of a multimodal endoscope with fluorescently labeled peptide can quantify images and works especially good for the detection of SSA which is a premalignant flat lesion conferring a high risk of subsequently leading to a colon cancer.

Dynamic Measurement of Tumor Vascular Permeability and Perfusion using a Hybrid System for Simultaneous Magnetic Resonance and Fluorescence Imaging.

PubMed

Ren, Wuwei; Elmer, Andreas; Buehlmann, David; Augath, Mark-Aurel; Vats, Divya; Ripoll, Jorge; Rudin, Markus

2016-04-01

Assessing tumor vascular features including permeability and perfusion is essential for diagnostic and therapeutic purposes. The aim of this study was to compare fluorescence and magnetic resonance imaging (MRI)-based vascular readouts in subcutaneously implanted tumors in mice by simultaneous dynamic measurement of tracer uptake using a hybrid fluorescence molecular tomography (FMT)/MRI system. Vascular permeability was measured using a mixture of extravascular imaging agents, GdDOTA and the dye Cy5.5, and perfusion using a mixture of intravascular agents, Endorem and a fluorescent probe (Angiosense). Dynamic fluorescence reflectance imaging (dFRI) was integrated into the hybrid system for high temporal resolution. Excellent correspondence between uptake curves of Cy5.5/GdDOTA and Endorem/Angiosense has been found with correlation coefficients R > 0.98. The two modalities revealed good agreement regarding permeability coefficients and centers-of-gravity of the imaging agent distribution. The FMT/dFRI protocol presented is able to accurately map physiological processes and poses an attractive alternative to MRI for characterizing tumor neoangiogenesis.

Ratiometric spectral imaging for fast tumor detection and chemotherapy monitoring in vivo

PubMed Central

Hwang, Jae Youn; Gross, Zeev; Gray, Harry B.; Medina-Kauwe, Lali K.; Farkas, Daniel L.

2011-01-01

We report a novel in vivo spectral imaging approach to cancer detection and chemotherapy assessment. We describe and characterize a ratiometric spectral imaging and analysis method and evaluate its performance for tumor detection and delineation by quantitatively monitoring the specific accumulation of targeted gallium corrole (HerGa) into HER2-positive (HER2 +) breast tumors. HerGa temporal accumulation in nude mice bearing HER2 + breast tumors was monitored comparatively by a. this new ratiometric imaging and analysis method; b. established (reflectance and fluorescence) spectral imaging; c. more commonly used fluorescence intensity imaging. We also tested the feasibility of HerGa imaging in vivo using the ratiometric spectral imaging method for tumor detection and delineation. Our results show that the new method not only provides better quantitative information than typical spectral imaging, but also better specificity than standard fluorescence intensity imaging, thus allowing enhanced in vivo outlining of tumors and dynamic, quantitative monitoring of targeted chemotherapy agent accumulation into them. PMID:21721808

Evanescent field microscopy techniques for studying dynamics at the surface of living cells

NASA Astrophysics Data System (ADS)

Sund, Susan E.

This thesis presents two distinct optical microscopy techniques for applications in cell biophysics: (a)the extension to living cells of an established technique, total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) for the first time in imaging mode; and (b)the novel development of polarized total internal reflection fluorescence (p- TIRF) to study membrane orientation in living cells. Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about the relevant chemical kinetic rates in vivo. TIR/FRAP, an established technique which can measure reversible biomolecular kinetic rates at surfaces, is extended here to measure kinetic parameters of microinjected rhodamine actin at the cytofacial surface of the plasma membrane of living cultured smooth muscle cells. For the first time, spatial imaging (with a CCD camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging allows production of spatially resolved images of kinetic data, and calculation of correlation distances, cell-wide gradients, and kinetic parameter dependence on initial fluorescence intensity. In living cells, membrane curvature occurs both in easily imaged large scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method, p-TIRF, is introduced here to visualize such regions. The method is based on fluorescence of the oriented membrane probe diI- C18-(3) (diI) excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane. A theoretical background of the technique and experimental verifications are presented in samples of protein solutions, model lipid bilayers, and living cells. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time- course maps of membrane orientations on diI labeled macrophages from which low visibility membrane structures can be identified and quantified. The TIR images are sharpened and contrast-enhanced by deconvoluting them with an experimentally-measured point spread function.

Demeclocycline as a contrast agent for detecting brain neoplasms using confocal microscopy

NASA Astrophysics Data System (ADS)

Wirth, Dennis; Smith, Thomas W.; Moser, Richard; Yaroslavsky, Anna N.

2015-04-01

Complete resection of brain tumors improves life expectancy and quality. Thus, there is a strong need for high-resolution detection and microscopically controlled removal of brain neoplasms. The goal of this study was to test demeclocycline as a contrast enhancer for the intraoperative detection of brain tumors. We have imaged benign and cancerous brain tumors using multimodal confocal microscopy. The tumors investigated included pituitary adenoma, meningiomas, glioblastomas, and metastatic brain cancers. Freshly excised brain tissues were stained in 0.75 mg ml-1 aqueous solution of demeclocyline. Reflectance images were acquired at 402 nm. Fluorescence signals were excited at 402 nm and registered between 500 and 540 nm. After imaging, histological sections were processed from the imaged specimens and compared to the optical images. Fluorescence images highlighted normal and cancerous brain cells, while reflectance images emphasized the morphology of connective tissue. The optical and histological images were in accordance with each other for all types of tumors investigated. Demeclocyline shows promise as a contrast agent for intraoperative detection of brain tumors.

Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array

PubMed Central

Luthman, Anna Siri; Dumitru, Sebastian; Quiros‐Gonzalez, Isabel; Joseph, James

2017-01-01

Abstract The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter‐based imaging systems. Hyperspectral imaging (HSI) facilitates the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for HSI is typically complex, bulky and expensive. We sought to overcome these limitations using a novel robust and low cost HSI camera based on a spectrally resolved detector array (SRDA). We integrated this HSI camera into a wide‐field reflectance‐based imaging system operating in the near‐infrared range to assess the suitability for in vivo imaging of exogenous fluorescent contrast agents. Using this fluorescence HSI (fHSI) system, we were able to accurately resolve the presence and concentration of at least 7 fluorescent dyes in solution. We also demonstrate high spectral unmixing precision, signal linearity with dye concentration and at depth in tissue mimicking phantoms, and delineate 4 fluorescent dyes in vivo. Our approach, including statistical background removal, could be directly generalised to broader spectral ranges, for example, to resolve tissue reflectance or autofluorescence and in future be tailored to video rate applications requiring snapshot HSI data acquisition. PMID:28485130

Analysis of hyperspectral fluorescence images for poultry skin tumor inspection

NASA Astrophysics Data System (ADS)

Kong, Seong G.; Chen, Yud-Ren; Kim, Intaek; Kim, Moon S.

2004-02-01

We present a hyperspectral fluorescence imaging system with a fuzzy inference scheme for detecting skin tumors on poultry carcasses. Hyperspectral images reveal spatial and spectral information useful for finding pathological lesions or contaminants on agricultural products. Skin tumors are not obvious because the visual signature appears as a shape distortion rather than a discoloration. Fluorescence imaging allows the visualization of poultry skin tumors more easily than reflectance. The hyperspectral image samples obtained for this poultry tumor inspection contain 65 spectral bands of fluorescence in the visible region of the spectrum at wavelengths ranging from 425 to 711 nm. The large amount of hyperspectral image data is compressed by use of a discrete wavelet transform in the spatial domain. Principal-component analysis provides an effective compressed representation of the spectral signal of each pixel in the spectral domain. A small number of significant features are extracted from two major spectral peaks of relative fluorescence intensity that have been identified as meaningful spectral bands for detecting tumors. A fuzzy inference scheme that uses a small number of fuzzy rules and Gaussian membership functions successfully detects skin tumors on poultry carcasses. Spatial-filtering techniques are used to significantly reduce false positives.

A comparison of hyperspectral reflectance and fluorescence imaging techniques for detection of contaminants on leafy greens

USDA-ARS?s Scientific Manuscript database

Ensuring the supply of safe, contaminant free fresh fruit and vegetables is of importance to consumers, suppliers and governments worldwide. In this study, three hyperspectral imaging (HSI) configurations coupled with two multivariate image analysis techniques are compared for detection of fecal con...

Workflow for high-content, individual cell quantification of fluorescent markers from universal microscope data, supported by open source software.

PubMed

Stockwell, Simon R; Mittnacht, Sibylle

2014-12-16

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators. Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software(1) to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.

PubMed

Yamamura, Hisao; Suzuki, Yoshiaki; Imaizumi, Yuji

2015-05-01

Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca(2+) events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca(2+) signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Hyperspectral imaging technique for detection of poultry fecal residues on food processing equipments

NASA Astrophysics Data System (ADS)

Cho, Byoung-Kwan; Kim, Moon S.; Chen, Yud-Ren

2005-11-01

Emerging concerns about safety and security in current mass production of food products necessitate rapid and reliable inspection for contaminant-free products. Diluted fecal residues on poultry processing plant equipment surface, not easily discernable from water by human eye, are contamination sources for poultry carcasses. Development of sensitive detection methods for fecal residues is essential to ensure safe production of poultry carcasses. Hyperspectral imaging techniques have shown good potential for detecting of the presence of fecal and other biological substances on food and processing equipment surfaces. In this study, use of high spatial resolution hyperspectral reflectance and fluorescence imaging (with UV-A excitation) is presented as a tool for selecting a few multispectral bands to detect diluted fecal and ingesta residues on materials used for manufacturing processing equipment. Reflectance and fluorescence imaging methods were compared for potential detection of a range of diluted fecal residues on the surfaces of processing plant equipment. Results showed that low concentrations of poultry feces and ingesta, diluted up to 1:100 by weight with double distilled water, could be detected using hyperspectral fluorescence images with an accuracy of 97.2%. Spectral bands determined in this study could be used for developing a real-time multispectral inspection device for detection of harmful organic residues on processing plant equipment.

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

2016-04-01

Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

Very High Spectral Resolution Imaging Spectroscopy: the Fluorescence Explorer (FLEX) Mission

NASA Technical Reports Server (NTRS)

Moreno, Jose F.; Goulas, Yves; Huth, Andreas; Middleton, Elizabeth; Miglietta, Franco; Mohammed, Gina; Nedbal, Ladislav; Rascher, Uwe; Verhoef, Wouter; Drusch, Matthias

2016-01-01

The Fluorescence Explorer (FLEX) mission has been recently selected as the 8th Earth Explorer by the European Space Agency (ESA). It will be the first mission specifically designed to measure from space vegetation fluorescence emission, by making use of very high spectral resolution imaging spectroscopy techniques. Vegetation fluorescence is the best proxy to actual vegetation photosynthesis which can be measurable from space, allowing an improved quantification of vegetation carbon assimilation and vegetation stress conditions, thus having key relevance for global mapping of ecosystems dynamics and aspects related with agricultural production and food security. The FLEX mission carries the FLORIS spectrometer, with a spectral resolution in the range of 0.3 nm, and is designed to fly in tandem with Copernicus Sentinel-3, in order to provide all the necessary spectral / angular information to disentangle emitted fluorescence from reflected radiance, and to allow proper interpretation of the observed fluorescence spatial and temporal dynamics.

Autofluorescence imaging can identify preinvasive or clinically occult lesions in fallopian tube epithelium: a promising step towards screening and early detection.

PubMed

McAlpine, J N; El Hallani, S; Lam, S F; Kalloger, S E; Luk, M; Huntsman, D G; MacAulay, C; Gilks, C B; Miller, D M; Lane, P M

2011-03-01

Optical imaging systems are robust, portable, relatively inexpensive, and have proven utility in detecting precancerous lesions in the lung, esophagus, colon, oral cavity and cervix. We describe the use of light-induced endogenous fluorescence (autofluorescence) in identifying preinvasive and occult carcinomas in ex vivo samples of human fallopian tube (FT) epithelium. Women undergoing surgery for an i) ovarian mass, ii) a history suggestive of hereditary breast-ovarian cancer, or iii) known serous ovarian cancer following neoadjuvant chemotherapy (NAC) were approached for informed consent. Immediately following surgery, FT's were photographed in reflectance and fluorescence at high resolution. Images included: (1) white-light reflectance of luminal/epithelial surface; (2) narrow-band green reflectance (570 nm) (3) green autofluorescence (405/436 nm excitation); and (4) blue autofluorescence (405 nm excitation). Areas revealing a loss of natural tissue fluorescence or marked increase in tissue microvasculature were recorded and compared to final histopathologic diagnosis (SEE-FIM protocol). Fifty-six cases involving one or both fallopian tubes underwent reflectance and fluorescence visualization. Nine cases were excluded, either secondary to non-ovarian primary pathology (7) or excessive trauma (2) rendering tissue interpretation impossible. Of the 47 cases remaining, there were 11 high grade serous (HGS) and 9 non-serous ovarian carcinomas undergoing primary debulking surgery, 5 serous carcinomas having received NAC, 8 benign ovarian tumors, and 14 women undergoing risk-reducing bilateral salpingo-oophorectomy (RRBSO). Methodology was feasible, efficient, and reproducible. TIC or carcinoma was identified in 7/11 HGS, 3/5 NAC, and 1/14 RRBSO. Optical images were reviewed to determine test positive or negative based on standardized criteria. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for the entire cohort (73%; 83%; 57%; 91%) and in a subgroup that excluded non-serous histology (87.5%; 92%; 78%; 96%). Abnormal FT lesions can be identified using ex vivo optical imaging technologies. With this platform, we will move towards genomic interrogation of identified lesions, and developing in vivo screening modalities via falloposcopy. Copyright © 2010 Elsevier Inc. All rights reserved.

Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching

PubMed Central

Fu, Yan; Winter, Peter W.; Rojas, Raul; Wang, Victor; McAuliffe, Matthew; Patterson, George H.

2016-01-01

We report superresolution optical sectioning using a multiangle total internal reflection fluorescence (TIRF) microscope. TIRF images were constructed from several layers within a normal TIRF excitation zone by sequentially imaging and photobleaching the fluorescent molecules. The depth of the evanescent wave at different layers was altered by tuning the excitation light incident angle. The angle was tuned from the highest (the smallest TIRF depth) toward the critical angle (the largest TIRF depth) to preferentially photobleach fluorescence from the lower layers and allow straightforward observation of deeper structures without masking by the brighter signals closer to the coverglass. Reconstruction of the TIRF images enabled 3D imaging of biological samples with 20-nm axial resolution. Two-color imaging of epidermal growth factor (EGF) ligand and clathrin revealed the dynamics of EGF-activated clathrin-mediated endocytosis during internalization. Furthermore, Bayesian analysis of images collected during the photobleaching step of each plane enabled lateral superresolution (<100 nm) within each of the sections. PMID:27044072

Wide-field lensless fluorescent microscopy using a tapered fiber-optic faceplate on a chip.

PubMed

Coskun, Ahmet F; Sencan, Ikbal; Su, Ting-Wei; Ozcan, Aydogan

2011-09-07

We demonstrate lensless fluorescent microscopy over a large field-of-view of ~60 mm(2) with a spatial resolution of <4 µm. In this on-chip fluorescent imaging modality, the samples are placed on a fiber-optic faceplate that is tapered such that the density of the fiber-optic waveguides on the top facet is >5 fold larger than the bottom one. Placed on this tapered faceplate, the fluorescent samples are pumped from the side through a glass hemisphere interface. After excitation of the samples, the pump light is rejected through total internal reflection that occurs at the bottom facet of the sample substrate. The fluorescent emission from the sample is then collected by the smaller end of the tapered faceplate and is delivered to an opto-electronic sensor-array to be digitally sampled. Using a compressive sampling algorithm, we decode these raw lensfree images to validate the resolution (<4 µm) of this on-chip fluorescent imaging platform using microparticles as well as labeled Giardia muris cysts. This wide-field lensfree fluorescent microscopy platform, being compact and high-throughput, might provide a valuable tool especially for cytometry, rare cell analysis (involving large area microfluidic systems) as well as for microarray imaging applications.

Laser fluorescence bronchoscope for localization of occult lung tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Profio, A.E.; Doiron, D.R.; King, E.G.

1979-11-01

A system for imaging occult bronchogenic carcinoma by the fluorescence of previously-injected, tumor-specific compound hematoporphyrin-derivative has been assembled and successfully used to locate a tumor l mm thick. The violet excitation source is a krypton ion laser coupled to fused quartz fiber light conductor. An electrostatic image intensifier attached to a standard flexible fiberoptic bronchoscope provides a bright image even at relatively low irradiance. A red secondary filter rejects most reflected background and autofluorescence. Sensitivity and contrast capability of the system should permit detection of a tumor less than 0.1 mm thick.

In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice.

PubMed

de la Rosa, Xavier; Santalucía, Tomàs; Fortin, Pierre-Yves; Purroy, Jesús; Calvo, Maria; Salas-Perdomo, Angélica; Justicia, Carles; Couillaud, Franck; Planas, Anna M

2013-02-01

Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core.

Versatile microfluidic total internal reflection (TIR)-based devices: application to microbeads velocity measurement and single molecule detection with upright and inverted microscope.

PubMed

Le, Nam Cao Hoai; Yokokawa, Ryuji; Dao, Dzung Viet; Nguyen, Thien Duy; Wells, John C; Sugiyama, Susumu

2009-01-21

A poly(dimethylsiloxane) (PDMS) chip for Total Internal Reflection (TIR)-based imaging and detection has been developed using Si bulk micromachining and PDMS casting. In this paper, we report the applications of the chip on both inverted and upright fluorescent microscopes and confirm that two types of sample delivery platforms, PDMS microchannel and glass microchannel, can be easily integrated depending on the magnification of an objective lens needed to visualize a sample. Although any device configuration can be achievable, here we performed two experiments to demonstrate the versatility of the microfluidic TIR-based devices. The first experiment was velocity measurement of Nile red microbeads with nominal diameter of 500 nm in a pressure-driven flow. The time-sequenced fluorescent images of microbeads, illuminated by an evanescent field, were cross-correlated by a Particle Image Velocimetry (PIV) program to obtain near-wall velocity field of the microbeads at various flow rates from 500 nl/min to 3000 nl/min. We then evaluated the capabilities of the device for Single Molecule Detection (SMD) of fluorescently labeled DNA molecules from 30 bp to 48.5 kbp and confirm that DNA molecules as short as 1105 bp were detectable. Our versatile, integrated device could provide low-cost and fast accessibility to Total Internal Reflection Fluorescent Microscopy (TIRFM) on both conventional upright and inverted microscopes. It could also be a useful component in a Micro-Total Analysis System (micro-TAS) to analyze nanoparticles or biomolecules near-wall transport or motion.

Optical Reflectance and Fluorescence for Detecting Nitrogen Needs in Zea mays L.

NASA Technical Reports Server (NTRS)

McMurtrey, J. E.; Middleton, E. M.; Corp. L. A.; Campbell, P. K. Entcheva; Butcher, L. M.; Daughtry, C. S. T.

2003-01-01

Nitrogen (N) status in field grown corn (Zea mays L.) was assessed using spectral techniques. Passive reflectance remote sensing and, both passive and active fluorescence sensing methods were investigated. Reflectance and fluorescence methods are reported to detect changes in the primary plant pigments (chlorophylls a and b; carotenoids) in higher plant species. As a general rule, foliar chlorophyll a (Chl a) and chlorophyll b (Chl b) usually exist in approx.3:l ratio. In plants under stress, Chl b content is affected before Chl a reductions occur. For reflectance, a version of the chlorophyll absorption in reflectance index (CARI) method was tested with narrow bands from the Airborne Imaging Spectroradiometer for Applications (ASIA). CARI minimizes the effects of soil background on the signal from green canopies. A modified CARI (MCARI) was used to track total Chl a levels in the red dip of the spectrum from the corn canopy. A second MCARI was used to track the auxiliary plant pigments (Chl b and the carotenoids) in the yellow/orange/red edge part of the reflectance spectrum. The difference between these two MCARI indices detected variations in N levels across the field plot canopies using ASIA data. At the leaf level, ratios of fluorescence emissions in the blue, green, red and far-red wavelengths sensed responses that were associated with the plant pigments, and were indicative of energy transfer in the photosynthetic process. N stressed corn stands could be distinguish from those with optimally applied N with fluorescence emission spectra obtained from individual corn leaves. Both reflectance and fluorescence methods are sensitive in detecting corn N needs and may be especially powerful in monitoring crop conditions if both types of information can be combined.

A programmable light engine for quantitative single molecule TIRF and HILO imaging.

PubMed

van 't Hoff, Marcel; de Sars, Vincent; Oheim, Martin

2008-10-27

We report on a simple yet powerful implementation of objective-type total internal reflection fluorescence (TIRF) and highly inclined and laminated optical sheet (HILO, a type of dark-field) illumination. Instead of focusing the illuminating laser beam to a single spot close to the edge of the microscope objective, we are scanning during the acquisition of a fluorescence image the focused spot in a circular orbit, thereby illuminating the sample from various directions. We measure parameters relevant for quantitative image analysis during fluorescence image acquisition by capturing an image of the excitation light distribution in an equivalent objective backfocal plane (BFP). Operating at scan rates above 1 MHz, our programmable light engine allows directional averaging by circular spinning the spot even for sub-millisecond exposure times. We show that restoring the symmetry of TIRF/HILO illumination reduces scattering and produces an evenly lit field-of-view that affords on-line analysis of evanescnt-field excited fluorescence without pre-processing. Utilizing crossed acousto-optical deflectors, our device generates arbitrary intensity profiles in BFP, permitting variable-angle, multi-color illumination, or objective lenses to be rapidly exchanged.

Imaging intracellular protein dynamics by spinning disk confocal microscopy

PubMed Central

Stehbens, Samantha; Pemble, Hayley; Murrow, Lindsay; Wittmann, Torsten

2012-01-01

The palette of fluorescent proteins has grown exponentially over the last decade, and as a result live imaging of cells expressing fluorescently tagged proteins is becoming more and more main stream. Spinning disk confocal microscopy (SDC) is a high speed optical sectioning technique, and a method of choice to observe and analyze intracellular fluorescent protein dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low noise scientific grade cooled charged-coupled device (CCD) cameras, and can achieve frame rates of up 1000 frames per second. In this chapter we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy, and provide a rationale for specific design choices. We also give guidelines how other imaging techniques such as total internal reflection (TIRF) microscopy or spatially controlled photoactivation can be coupled with SDC imaging, and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction. PMID:22264541

Liposomal encapsulation of a near-infrared fluorophore enhances fluorescence quenching and reliable whole body optical imaging upon activation in vivo.

PubMed

Tansi, Felista L; Rüger, Ronny; Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Kaiser, Werner A; Hilger, Ingrid

2013-11-11

In the past decade, there has been significant progress in the development of water soluble near-infrared fluorochromes for use in a wide range of imaging applications. Fluorochromes with high photo and thermal stability, sensitivity, adequate pharmacological properties and absorption/emission maxima within the near infrared window (650-900 nm) are highly desired for in vivo imaging, since biological tissues show very low absorption and auto-fluorescence at this spectrum window. Taking these properties into consideration, a myriad of promising near infrared fluorescent probes has been developed recently. However, a hallmark of most of these probes is a rapid clearance in vivo, which hampers their application. It is hypothesized that encapsulation of the near infrared fluorescent dye DY-676-COOH, which undergoes fluorescence quenching at high concentrations, in the aqueous interior of liposomes will result in protection and fluorescence quenching, which upon degradation by phagocytes in vivo will lead to fluorescence activation and enable imaging of inflammation. Liposomes prepared with high concentrations of DY-676-COOH reveal strong fluorescence quenching. It is demonstrated that the non-targeted PEGylated fluorescence-activatable liposomes are taken up predominantly by phagocytosis and degraded in lysosomes. Furthermore, in zymosan-induced edema models in mice, the liposomes are taken up by monocytes and macrophages which migrate to the sites of inflammation. Opposed to free DY-676-COOH, prolonged stability and retention of liposomal-DY-676-COOH is reflected in a significant increase in fluorescence intensity of edema. Thus, protected delivery and fluorescence quenching make the DY-676-COOH-loaded liposomes a highly promising contrast agent for in vivo optical imaging of inflammatory diseases. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Successful Translation of Fluorescence Navigation During Oncologic Surgery: A Consensus Report.

PubMed

Rosenthal, Eben L; Warram, Jason M; de Boer, Esther; Basilion, James P; Biel, Merrill A; Bogyo, Matthew; Bouvet, Michael; Brigman, Brian E; Colson, Yolonda L; DeMeester, Steven R; Gurtner, Geoffrey C; Ishizawa, Takeaki; Jacobs, Paula M; Keereweer, Stijn; Liao, Joseph C; Nguyen, Quyen T; Olson, James M; Paulsen, Keith D; Rieves, Dwaine; Sumer, Baran D; Tweedle, Michael F; Vahrmeijer, Alexander L; Weichert, Jamey P; Wilson, Brian C; Zenn, Michael R; Zinn, Kurt R; van Dam, Gooitzen M

2016-01-01

Navigation with fluorescence guidance has emerged in the last decade as a promising strategy to improve the efficacy of oncologic surgery. To achieve routine clinical use, the onus is on the surgical community to objectively assess the value of this technique. This assessment may facilitate both Food and Drug Administration approval of new optical imaging agents and reimbursement for the imaging procedures. It is critical to characterize fluorescence-guided procedural benefits over existing practices and to elucidate both the costs and the safety risks. This report is the result of a meeting of the International Society of Image Guided Surgery (www.isigs.org) on February 6, 2015, in Miami, Florida, and reflects a consensus of the participants' opinions. Our objective was to critically evaluate the imaging platform technology and optical imaging agents and to make recommendations for successful clinical trial development of this highly promising approach in oncologic surgery. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Open-source do-it-yourself multi-color fluorescence smartphone microscopy

PubMed Central

Sung, Yulung; Campa, Fernando; Shih, Wei-Chuan

2017-01-01

Fluorescence microscopy is an important technique for cellular and microbiological investigations. Translating this technique onto a smartphone can enable particularly powerful applications such as on-site analysis, on-demand monitoring, and point-of-care diagnostics. Current fluorescence smartphone microscope setups require precise illumination and imaging alignment which altogether limit its broad adoption. We report a multi-color fluorescence smartphone microscope with a single contact lens-like add-on lens and slide-launched total-internal-reflection guided illumination for three common tasks in investigative fluorescence microscopy: autofluorescence, fluorescent stains, and immunofluorescence. The open-source, simple and cost-effective design has the potential for do-it-yourself fluorescence smartphone microscopy. PMID:29188104

High contrast two-photon imaging of fingermarks

NASA Astrophysics Data System (ADS)

Stoltzfus, Caleb R.; Rebane, Aleksander

2016-04-01

Optically-acquired fingermarks are widely used as evidence across law enforcement agencies as well as in the courts of law. A common technique for visualizing latent fingermarks on nonporous surfaces consists of cyanoacrylate fuming of the fingerprint material, followed by impregnation with a fluorescent dye, which under ultra violet (UV) illumination makes the fingermarks visible and thus accessible for digital recording. However, there exist critical circumstances, when the image quality is compromised due to high background scattering, high auto-fluorescence of the substrate material, or other detrimental photo-physical and photo-chemical effects such as light-induced damage to the sample. Here we present a novel near-infrared (NIR), two-photon induced fluorescence imaging modality, which significantly enhances the quality of the fingermark images, especially when obtained from highly reflective and/or scattering surfaces, while at the same time reducing photo-damage to sensitive forensic samples.

Fluorescence confocal microscopy for pathologists.

PubMed

Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

2014-03-01

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on surgical specimens other than the skin and to evaluate the diagnostic capability of this technology from pathologists' viewpoint.

A new phenomenon?

NASA Astrophysics Data System (ADS)

Ganci, S.

2017-03-01

This letter describes an interesting optical phenomenon engaging the reflected images of a new tube LED lamp (an array of high luminosity LEDs substituting the old fluorescent lamps) on any convex glossy black surface.

Characterisation of a resolution enhancing image inversion interferometer.

PubMed

Wicker, Kai; Sindbert, Simon; Heintzmann, Rainer

2009-08-31

Image inversion interferometers have the potential to significantly enhance the lateral resolution and light efficiency of scanning fluorescence microscopes. Self-interference of a point source's coherent point spread function with its inverted copy leads to a reduction in the integrated signal for off-axis sources compared to sources on the inversion axis. This can be used to enhance the resolution in a confocal laser scanning microscope. We present a simple image inversion interferometer relying solely on reflections off planar surfaces. Measurements of the detection point spread function for several types of light sources confirm the predicted performance and suggest its usability for scanning confocal fluorescence microscopy.

Reflecting microscope system with a 0.99 numerical aperture designed for three-dimensional fluorescence imaging of individual molecules at cryogenic temperatures

PubMed Central

Inagawa, H.; Toratani, Y.; Motohashi, K.; Nakamura, I.; Matsushita, M.; Fujiyoshi, S.

2015-01-01

We have developed a cryogenic fluorescence microscope system, the core of which is a reflecting objective that consists of spherical and aspherical mirrors. The use of an aspherical mirror allows the reflecting objective to have a numerical aperture (NA) of up to 0.99, which is close to the maximum possible NA of 1.03 in superfluid helium. The performance of the system at a temperature of 1.7 K was tested by recording a three-dimensional fluorescence image of individual quantum dots using excitation wavelengths (λex) of 532 nm and 635 nm. At 1.7 K, the microscope worked with achromatic and nearly diffraction-limited performance. The 1/e2 radius (Γ) of the point spread function of the reflecting objective in the lateral (xy) direction was 0.212 ± 0.008 μm at λex = 532 nm and was less than 1.2 times the simulated value for a perfectly polished objective. The radius Γ in the axial (z) direction was 0.91 ± 0.04 μm at λex = 532 nm and was less than 1.4 times the simulated value of Γ. The chromatic aberrations between the two wavelengths were one order of magnitude smaller than Γ in each direction. PMID:26239746

Time-lapse total internal reflection fluorescence video of acetylcholine receptor cluster formation on myotubes.

PubMed

Wang, M D; Axelrod, D

1994-09-01

To study when and where acetylcholine receptor (AChR) clusters appear on developing rat myotubes in primary culture, we have made time-lapse movies of total internal reflection fluorescence (TIRF) overlaid with schlieren transmitted light images. The receptors, including the ones newly incorporated into the membrane, were labeled with rhodamine alpha-bungarotoxin (R-BT) continuously present in the medium. Since TIRF illuminates only cell-substrate contact regions where almost all of the AChR clusters are located, background fluorescence from fluorophores either in the bulk solution or inside the cells can be suppressed. Also, because TIRF minimizes the exposure of the cell interior to light, the healthy survival of the culture during imaging procedures is much enhanced relative to standard epi- (or trans-) illumination. During the experiment, cells were kept alive on the microscope stage at 37 degrees C in an atmosphere of 10% CO2. Two digital images were recorded by a CCD camera every 20 min: the schlieren image of the cells and the TIRF image of the clusters. After background subtraction, the cluster image was displayed in pseudocolors, overlaid onto the cell images, and recorded as 3 frames on a videotape. The final movies are thus able to summarize a week-long experiment in less than a minute. These movies and images show that clusters form often shortly after the myoblast fusion but sometimes much later, and the formation takes place very rapidly (a few hours). The clusters have an average lifetime of around a day, much shorter than the lifetime of a typical myotube. The brightest and largest clusters tend to be the longest-lived. The cluster formation seems to be associated with the contacts of myotubes at the glass substrate, but not with cell-cell contacts or myoblast fusion into myotubes. New AChR continuously appear in preexisting clusters: after photobleaching, the fluorescence of some clusters recovers within an hour.

Adaptive Spot Detection With Optimal Scale Selection in Fluorescence Microscopy Images.

PubMed

Basset, Antoine; Boulanger, Jérôme; Salamero, Jean; Bouthemy, Patrick; Kervrann, Charles

2015-11-01

Accurately detecting subcellular particles in fluorescence microscopy is of primary interest for further quantitative analysis such as counting, tracking, or classification. Our primary goal is to segment vesicles likely to share nearly the same size in fluorescence microscopy images. Our method termed adaptive thresholding of Laplacian of Gaussian (LoG) images with autoselected scale (ATLAS) automatically selects the optimal scale corresponding to the most frequent spot size in the image. Four criteria are proposed and compared to determine the optimal scale in a scale-space framework. Then, the segmentation stage amounts to thresholding the LoG of the intensity image. In contrast to other methods, the threshold is locally adapted given a probability of false alarm (PFA) specified by the user for the whole set of images to be processed. The local threshold is automatically derived from the PFA value and local image statistics estimated in a window whose size is not a critical parameter. We also propose a new data set for benchmarking, consisting of six collections of one hundred images each, which exploits backgrounds extracted from real microscopy images. We have carried out an extensive comparative evaluation on several data sets with ground-truth, which demonstrates that ATLAS outperforms existing methods. ATLAS does not need any fine parameter tuning and requires very low computation time. Convincing results are also reported on real total internal reflection fluorescence microscopy images.

Towards automated early cancer detection: Non-invasive, fluorescence-based approaches for quantitative assessment of cells and tissue to identify pre-cancers

NASA Astrophysics Data System (ADS)

Levitt, Jonathan Michael

Cancer is the second leading cause of death globally, second only to heart disease. As in many diseases, patient survival is directly related to how early lesions are detected. Using conventional screening methods, the early changes associated with cancer, which occur on the microscopic scale, can easily go overlooked. Due to the inherent drawbacks of conventional techniques we present non-invasive, optically based methods to acquire high resolution images from live samples and assess cellular function associated with the onset of disease. Specifically, we acquired fluorescence images from NADH and FAD to quantify morphology and metabolic activity. We first conducted studies to monitor monolayers of keratinocytes in response to apoptosis which has been shown to be disrupted during cancer progression. We found that as keratinocytes undergo apoptosis there are populations of mitochondria that exhibit a higher metabolic activity that become progressively confined to a gradually smaller perinuclear region. To further assess the changes associated with early cancer growth we developed automated methods to rapidly quantify fluorescence images and extract morphological and metabolic information from life tissue. In this study, we simultaneously quantified mitochondrial organization, metabolic activity, nuclear size distribution, and the localization of the structural protein keratin, to differentiate between normal and pre-cancerous engineered tissues. We found the degree mitochondrial organization, as determined from the fractal derived Hurst parameter, was well correlated to level of cellular differentiation. We also found that the metabolic activity in the pre-cancerous cells was greater and more consistent throughout tissue depths in comparison to normal tissue. Keratin localization, also quantified from the fluorescence images, we found it to be confined to the uppermost layers of normal tissue while it was more evenly distributed in the precancerous tissues. To allow for evaluation of the early cancerous changes in vivo, we developed video-rate confocal reflectance/multi-photon fluorescence microscope as a clinical prototype. This device was specifically designed to rapidly acquire and assess non-invasively acquire fluorescence images using the automated methods we have developed. We have demonstrated the ability of this microscope to simultaneously acquire fluorescence, confocal reflectance, and second-harmonic generation images as well as assess blood flow in vivo.

Design, manufacturing and alignment of a fluorescence imaging spectrometer based on refractive optics and a transmission grating

NASA Astrophysics Data System (ADS)

Lousberg, G. P.; Lemagne, F.; Gloesener, P.; Flebus, C.; Rougelot, S.; Coatantiec, C.; Harnisch, B.

2017-11-01

In the framework of the Fluorescence Explorer (FLEX) phase A/B1 study, an elegant breadboard (EBB) of an imaging spectrometer is designed, manufactured and aligned by AMOS, with Airbus Defence&Space as the prime Contractor of the study. The FLEX mission is one of the two candidates of the 8th Earth Explorer mission. The main constituting instrument of the FLEX mission is an imaging spectrometer observing vegetation fluorescence and reflectance with a high- and a low-resolution channels in the 500 nm -780 nm band. As part of the system feasibility study of the mission, a breadboard of the high-resolution channel of the instrument is designed and manufactured with a high representativeness of a future flight concept. The high-resolution channel is referred to as FIMAS (Fluorescence IMAging Spectrometer). The main purpose of the EBB is to demonstrate (1) the manufacturability of the instrument and (2) the compliance of the optical performances with respect to the science requirements (including spatial and spectral resolution and stray-light).

Deep UV Native Fluorescence Imaging of Antarctic Cryptoendolithic Communities

NASA Technical Reports Server (NTRS)

Storrie-Lombardi, M. C.; Douglas, S.; Sun, H.; McDonald, G. D.; Bhartia, R.; Nealson, K. H.; Hug, W. F.

2001-01-01

An interdisciplinary team at the Jet Propulsion Laboratory Center for Life Detection has embarked on a project to provide in situ chemical and morphological characterization of Antarctic cryptoendolithic microbial communities. We present here in situ deep ultraviolet (UV) native fluorescence and environmental scanning electron microscopy images transiting 8.5 mm into a sandstone sample from the Antarctic Dry Valleys. The deep ultraviolet imaging system employs 224.3, 248.6, and 325 nm lasers to elicit differential fluorescence and resonance Raman responses from biomolecules and minerals. The 224.3 and 248.6 nm lasers elicit a fluorescence response from the aromatic amino and nucleic acids. Excitation at 325 nm may elicit activity from a variety of biomolecules, but is more likely to elicit mineral fluorescence. The resultant fluorescence images provide in situ chemical and morphological maps of microorganisms and the associated organic matrix. Visible broadband reflectance images provide orientation against the mineral background. Environmental scanning electron micrographs provided detailed morphological information. The technique has made possible the construction of detailed fluorescent maps extending from the surface of an Antarctic sandstone sample to a depth of 8.5 mm. The images detect no evidence of microbial life in the superficial 0.2 mm crustal layer. The black lichen component between 0.3 and 0.5 mm deep absorbs all wavelengths of both laser and broadband illumination. Filamentous deep ultraviolet native fluorescent activity dominates in the white layer between 0.6 mm and 5.0 mm from the surface. These filamentous forms are fungi that continue into the red (iron-rich) region of the sample extending from 5.0 to 8.5 mm. Using differential image subtraction techniques it is possible to identify fungal nuclei. The ultraviolet response is markedly attenuated in this region, apparently from the absorption of ultraviolet light by iron-rich particles coating the filaments. Below 8.5 mm the filamentous morphology of the upper layers gives way to punctate 1-2 micron particles evidencing fluorescent activity following excitation at both deep ultraviolet wavelengths.

Fiber-optic confocal reflectance microscope with miniature objective for in vivo imaging of human tissues.

PubMed

Sung, Kung-Bin; Liang, Chen; Descour, Michael; Collier, Tom; Follen, Michele; Richards-Kortum, Rebecca

2002-10-01

We have built a fiber-optic confocal reflectance microscope capable of imaging human tissues in near real time. Miniaturization of the objective lens and the mechanical components for positioning and axially scanning the objective enables the device to be used in inner organs of the human body. The lateral resolution is 2 micrometers and axial resolution is 10 micrometers. Confocal images of fixed tissue biopsies and the human lip in vivo have been obtained at 15 frames/s without any fluorescent stains. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope.

Sentinel lymph node detection in gynecologic malignancies by a handheld fluorescence camera

NASA Astrophysics Data System (ADS)

Hirsch, Ole; Szyc, Lukasz; Muallem, Mustafa Zelal; Ignat, Iulia; Chekerov, Radoslav; Macdonald, Rainer; Sehouli, Jalid; Braicu, Ioana; Grosenick, Dirk

2017-02-01

Near-infrared fluorescence imaging using indocyanine green (ICG) as a tracer is a promising technique for mapping the lymphatic system and for detecting sentinel lymph nodes (SLN) during cancer surgery. In our feasibility study we have investigated the application of a custom-made handheld fluorescence camera system for the detection of lymph nodes in gynecological malignancies. It comprises a low cost CCD camera with enhanced NIR sensitivity and two groups of LEDs emitting at wavelengths of 735 nm and 830 nm for interlaced recording of fluorescence and reflectance images of the tissue, respectively. With the help of our system, surgeons can observe fluorescent tissue structures overlaid onto the anatomical image on a monitor in real-time. We applied the camera system for intraoperative lymphatic mapping in 5 patients with vulvar cancer, 5 patients with ovarian cancer, 3 patients with cervical cancer, and 3 patients with endometrial cancer. ICG was injected at four loci around the primary malignant tumor during surgery. After a residence time of typically 15 min fluorescence images were taken in order to visualize the lymph nodes closest to the carcinomas. In cases with vulvar cancer about half of the lymph nodes detected by routinely performed radioactive SLN mapping have shown fluorescence in vivo as well. In the other types of carcinomas several lymph nodes could be detected by fluorescence during laparotomy. We conclude that our low cost camera system has sufficient sensitivity for lymphatic mapping during surgery.

Non-invasive, Contrast-enhanced Spectral Imaging of Breast Cancer Signatures in Preclinical Animal Models In vivo

PubMed Central

Ramanujan, V Krishnan; Ren, Songyang; Park, Sangyong; Farkas, Daniel L

2011-01-01

We report here a non-invasive multispectral imaging platform for monitoring spectral reflectance and fluorescence images from primary breast carcinoma and metastatic lymph nodes in preclinical rat model in vivo. The system is built around a monochromator light source and an acousto-optic tunable filter (AOTF) for spectral selection. Quantitative analysis of the measured reflectance profiles in the presence of a widely-used lymphazurin dye clearly demonstrates the capability of the proposed imaging platform to detect tumor-associated spectral signatures in the primary tumors as well as metastatic lymphatics. Tumor-associated changes in vascular oxygenation and interstitial fluid pressure are reasoned to be the physiological sources of the measured reflectance profiles. We also discuss the translational potential of our imaging platform in intra-operative clinical setting. PMID:21572915

A Patch-Based Method for Repetitive and Transient Event Detection in Fluorescence Imaging

PubMed Central

Boulanger, Jérôme; Gidon, Alexandre; Kervran, Charles; Salamero, Jean

2010-01-01

Automatic detection and characterization of molecular behavior in large data sets obtained by fast imaging in advanced light microscopy become key issues to decipher the dynamic architectures and their coordination in the living cell. Automatic quantification of the number of sudden and transient events observed in fluorescence microscopy is discussed in this paper. We propose a calibrated method based on the comparison of image patches expected to distinguish sudden appearing/vanishing fluorescent spots from other motion behaviors such as lateral movements. We analyze the performances of two statistical control procedures and compare the proposed approach to a frame difference approach using the same controls on a benchmark of synthetic image sequences. We have then selected a molecular model related to membrane trafficking and considered real image sequences obtained in cells stably expressing an endocytic-recycling trans-membrane protein, the Langerin-YFP, for validation. With this model, we targeted the efficient detection of fast and transient local fluorescence concentration arising in image sequences from a data base provided by two different microscopy modalities, wide field (WF) video microscopy using maximum intensity projection along the axial direction and total internal reflection fluorescence microscopy. Finally, the proposed detection method is briefly used to statistically explore the effect of several perturbations on the rate of transient events detected on the pilot biological model. PMID:20976222

A Method of High Throughput Monitoring Crop Physiology Using Chlorophyll Fluorescence and Multispectral Imaging.

PubMed

Wang, Heng; Qian, Xiangjie; Zhang, Lan; Xu, Sailong; Li, Haifeng; Xia, Xiaojian; Dai, Liankui; Xu, Liang; Yu, Jingquan; Liu, Xu

2018-01-01

We present a high throughput crop physiology condition monitoring system and corresponding monitoring method. The monitoring system can perform large-area chlorophyll fluorescence imaging and multispectral imaging. The monitoring method can determine the crop current condition continuously and non-destructively. We choose chlorophyll fluorescence parameters and relative reflectance of multispectral as the indicators of crop physiological status. Using tomato as experiment subject, the typical crop physiological stress, such as drought, nutrition deficiency and plant disease can be distinguished by the monitoring method. Furthermore, we have studied the correlation between the physiological indicators and the degree of stress. Besides realizing the continuous monitoring of crop physiology, the monitoring system and method provide the possibility of machine automatic diagnosis of the plant physiology. Highlights: A newly designed high throughput crop physiology monitoring system and the corresponding monitoring method are described in this study. Different types of stress can induce distinct fluorescence and spectral characteristics, which can be used to evaluate the physiological status of plants.

Evaluation of automated threshold selection methods for accurately sizing microscopic fluorescent cells by image analysis.

PubMed Central

Sieracki, M E; Reichenbach, S E; Webb, K L

1989-01-01

The accurate measurement of bacterial and protistan cell biomass is necessary for understanding their population and trophic dynamics in nature. Direct measurement of fluorescently stained cells is often the method of choice. The tedium of making such measurements visually on the large numbers of cells required has prompted the use of automatic image analysis for this purpose. Accurate measurements by image analysis require an accurate, reliable method of segmenting the image, that is, distinguishing the brightly fluorescing cells from a dark background. This is commonly done by visually choosing a threshold intensity value which most closely coincides with the outline of the cells as perceived by the operator. Ideally, an automated method based on the cell image characteristics should be used. Since the optical nature of edges in images of light-emitting, microscopic fluorescent objects is different from that of images generated by transmitted or reflected light, it seemed that automatic segmentation of such images may require special considerations. We tested nine automated threshold selection methods using standard fluorescent microspheres ranging in size and fluorescence intensity and fluorochrome-stained samples of cells from cultures of cyanobacteria, flagellates, and ciliates. The methods included several variations based on the maximum intensity gradient of the sphere profile (first derivative), the minimum in the second derivative of the sphere profile, the minimum of the image histogram, and the midpoint intensity. Our results indicated that thresholds determined visually and by first-derivative methods tended to overestimate the threshold, causing an underestimation of microsphere size. The method based on the minimum of the second derivative of the profile yielded the most accurate area estimates for spheres of different sizes and brightnesses and for four of the five cell types tested. A simple model of the optical properties of fluorescing objects and the video acquisition system is described which explains how the second derivative best approximates the position of the edge. Images PMID:2516431

Nanoscale characterization of vesicle adhesion by normalized total internal reflection fluorescence microscopy.

PubMed

Cardoso Dos Santos, Marcelina; Vézy, Cyrille; Jaffiol, Rodolphe

2016-06-01

We recently proposed a straightforward fluorescence microscopy technique to study adhesion of Giant Unilamellar Vesicles. This technique is based on dual observations which combine epi-fluorescence microscopy and total internal reflection fluorescence (TIRF) microscopy: TIRF images are normalized by epi-fluorescence ones. By this way, it is possible to map the membrane/substrate separation distance with a nanometric resolution, typically ~20 nm, with a maximal working range of 300-400 nm. The purpose of this paper is to demonstrate that this technique is useful to quantify vesicle adhesion from ultra-weak to strong membrane-surface interactions. Thus, we have examined unspecific and specific adhesion conditions. Concerning unspecific adhesion, we have controlled the strength of electrostatic forces between negatively charged vesicles and various functionalized surfaces which exhibit a positive or a negative effective charge. Specific adhesion was highlighted with lock-and-key forces mediated by the well defined biotin/streptavidin recognition. Copyright © 2016 Elsevier B.V. All rights reserved.

In vivo fluorescence imaging of hepatocellular carcinoma xenograft using near-infrared labeled epidermal growth factor receptor (EGFR) peptide

PubMed Central

Li, Z.; Zhou, Q.; Zhou, J.; Duan, X.; Zhu, J.; Wang, T. D.

2016-01-01

Minimally-invasive surgery of hepatocellular carcinoma (HCC) can be limited by poor tumor visualization with white light. We demonstrate systemic administration of a Cy5.5-labeled peptide specific for epidermal growth factor receptor (EGFR) to target HCC in vivo in a mouse xenograft model. We attached a compact imaging module to the proximal end of a medical laparoscope to collect near-infrared fluorescence and reflectance images concurrently at 15 frames/sec. We measured a mean target-to-background ratio of 2.99 ± 0.22 from 13 surgically exposed subcutaneous human HCC tumors in vivo in 5 mice. This integrated imaging methodology is promising to guide laparoscopic resection of HCC. PMID:27699089

Single-Molecule Light-Sheet Imaging of Suspended T Cells.

PubMed

Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

2018-05-08

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

Foliar Reflectance and Fluorescence Responses for Plants Under Nitrogen Stress Determined with Active and Passive Systems

NASA Technical Reports Server (NTRS)

Middleton, E. M.; McMurtrey, J. E.; Campbell, P. K. Entcheva; Corp, L. A.; Butcher, L. M.; Chappelle, E. W.

2003-01-01

Vegetation productivity is driven by nitrogen (N) availability in soils. Both excessive and low soil N induce physiological changes in plant foliage. In 2001, we examined the use of spectral fluorescence and reflectance measurements to discriminate among plants provided different N fertilizer application rates: 20%, 50%, 100% and 150% of optimal N levels. A suite of optical, fluorescence, and biophysical measurements were collected on leaves from field grown corn (Zea mays L.) and soybean plants (Glycine max L.) grown in pots (greenhouse + ambient sunlight daily). Three types of steady state laser-induced fluorescence measurements were made on adaxial and abaxial surfaces: 1) fluorescence images in four 10 nm bands (blue, green, red, far-red) resulting from broad irradiance excitation; 2) emission spectra (5 nm resolution) produced by excitation at single wavelengths (280,380 or 360, and 532 nm); and 3) excitation spectra (2 nm resolution), with emission wavelengths fixed at wavelengths centered on selected solar Fraunhofer lines (532,607,677 and 745 nm). Two complementary sets of high resolution (less than 2 nm) optical spectra were acquired for both adaxial and abaxial leaf surfaces: 1) optical properties (350-2500 nm) for reflectance, transmittance, and absorptance; and 2) reflectance spectra (500-1000 nm) acquired with and without a short pass filter at 665 nm to determine the fluorescence contribution to apparent reflectance in the 650-750 spectrum, especially at the 685 and 740 nm chlorophyll fluorescence (ChIF) peaks. The strongest relationships between foliar chemistry and optical properties were demonstrated for C/N content and two optical parameters associated with the red edge inflection point. Select optical properties and ChIF parameters were highly correlated for both species. A significant contribution of ChIF to apparent reflectance was observed, averaging 10-25% at 685 nm and 2 - 6% at 740 nm over all N treatments. Discrimination of N treatment groups was possible with specific fluorescence band ratios (e.g., F740/F525 obtained with 380EX). From all measurements assessing fluorescence, higher ChIF and blue/green emissions were measured from the abaxial leaf surfaces; Abaxial surfaces also produced higher reflectances in the 400-800 nm spectrum. Fluorescence information collected in Fraunhofer regions located on the shoulders of ChIF features compared favorably with peak emissions. This supports the potential capability of a future space-born interferometer sensor to capture plant canopy fluorescence.

Sun-induced fluorescence - a new probe of photosynthesis: First maps from the imaging spectrometer HyPlant.

PubMed

Rascher, U; Alonso, L; Burkart, A; Cilia, C; Cogliati, S; Colombo, R; Damm, A; Drusch, M; Guanter, L; Hanus, J; Hyvärinen, T; Julitta, T; Jussila, J; Kataja, K; Kokkalis, P; Kraft, S; Kraska, T; Matveeva, M; Moreno, J; Muller, O; Panigada, C; Pikl, M; Pinto, F; Prey, L; Pude, R; Rossini, M; Schickling, A; Schurr, U; Schüttemeyer, D; Verrelst, J; Zemek, F

2015-12-01

Variations in photosynthesis still cause substantial uncertainties in predicting photosynthetic CO2 uptake rates and monitoring plant stress. Changes in actual photosynthesis that are not related to greenness of vegetation are difficult to measure by reflectance based optical remote sensing techniques. Several activities are underway to evaluate the sun-induced fluorescence signal on the ground and on a coarse spatial scale using space-borne imaging spectrometers. Intermediate-scale observations using airborne-based imaging spectroscopy, which are critical to bridge the existing gap between small-scale field studies and global observations, are still insufficient. Here we present the first validated maps of sun-induced fluorescence in that critical, intermediate spatial resolution, employing the novel airborne imaging spectrometer HyPlant. HyPlant has an unprecedented spectral resolution, which allows for the first time quantifying sun-induced fluorescence fluxes in physical units according to the Fraunhofer Line Depth Principle that exploits solar and atmospheric absorption bands. Maps of sun-induced fluorescence show a large spatial variability between different vegetation types, which complement classical remote sensing approaches. Different crop types largely differ in emitting fluorescence that additionally changes within the seasonal cycle and thus may be related to the seasonal activation and deactivation of the photosynthetic machinery. We argue that sun-induced fluorescence emission is related to two processes: (i) the total absorbed radiation by photosynthetically active chlorophyll; and (ii) the functional status of actual photosynthesis and vegetation stress. © 2015 John Wiley & Sons Ltd.

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

PubMed

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-07-02

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Novel image processing method study for a label-free optical biosensor

NASA Astrophysics Data System (ADS)

Yang, Chenhao; Wei, Li'an; Yang, Rusong; Feng, Ying

2015-10-01

Optical biosensor is generally divided into labeled type and label-free type, the former mainly contains fluorescence labeled method and radioactive-labeled method, while fluorescence-labeled method is more mature in the application. The mainly image processing methods of fluorescent-labeled biosensor includes smooth filtering, artificial gridding and constant thresholding. Since some fluorescent molecules may influence the biological reaction, label-free methods have been the main developing direction of optical biosensors nowadays. The using of wider field of view and larger angle of incidence light path which could effectively improve the sensitivity of the label-free biosensor also brought more difficulties in image processing, comparing with the fluorescent-labeled biosensor. Otsu's method is widely applied in machine vision, etc, which choose the threshold to minimize the intraclass variance of the thresholded black and white pixels. It's capacity-constrained with the asymmetrical distribution of images as a global threshold segmentation. In order to solve the irregularity of light intensity on the transducer, we improved the algorithm. In this paper, we present a new image processing algorithm based on a reflectance modulation biosensor platform, which mainly comprises the design of sliding normalization algorithm for image rectification and utilizing the improved otsu's method for image segmentation, in order to implement automatic recognition of target areas. Finally we used adaptive gridding method extracting the target parameters for analysis. Those methods could improve the efficiency of image processing, reduce human intervention, enhance the reliability of experiments and laid the foundation for the realization of high throughput of label-free optical biosensors.

Fluorescent diamond nanoparticle as a probe of intracellular traffic in primary neurons in culture

NASA Astrophysics Data System (ADS)

Le, Xuan Loc; Lepagnol-Bestel, Aude-Marie; Adam, Marie-Pierre; Thomas, Alice; Dantelle, Géraldine; Chang, Cheng-Chun; Mohan, Nitin; Chang, Huan-Cheng; Treussart, François; Simonneau, Michel

2012-03-01

Neurons display dendritic spines plasticity and morphology anomalies in numerous psychiatric and neurodegenerative diseases. These changes are associated to abnormal dendritic traffic that can be evidenced by fluorescence microscopy. As a fluorescent probe we propose to use fluorescent diamond nanoparticles with size of < 50 nm. Color centers embedded inside the diamond nanoparticles are perfectly photostable emitters allowing for long-term tracking. Nanodiamond carbon surface is also well suited for biomolecule functionalization to target specific cellular compartments. We show that fluorescent nanodiamonds can be spontaneously internalized in neurons in culture and imaged by confocal and Total Internal Reflection (TIRF) microscopy with a high signal over background ratio.

On the demands on imaging spectrometry for the monitoring of global vegetation fluorescence from space

NASA Astrophysics Data System (ADS)

Kraft, S.; Del Bello, U.; Drusch, M.; Gabriele, A.; Harnisch, B.; Moreno, J.

2013-09-01

Vegetation fluorescence when measured from space contributes only a tiny fraction of the signal coming on top of the reflected radiance by the Earth surface and the atmosphere. As a consequence, imaging spectrometers have to provide sufficient throughput and radiometric accuracy to enable accurate global monitoring of the daily to seasonal variations of the Earth's vegetation breath, which is particularly challenging if ground resolutions of a few hundred meters are targeted. Since fluorescence retrieval algorithms have to make corrections for atmospheric effects, it is necessary to provide sufficient spectral resolution, so that signal alterations due to the main parameters such as surface pressure, atmospheric temperature profile, vertical distribution of aerosols concentration, and water vapour content can be accurately modelled. ESA's Earth Explorer 8 candidate mission FLEX carries a Fluorescence Imaging Spectrometer (FLORIS), which has been designed and optimised to enable such measurement. The spectrometer will measure in a spectral range between 500 and 780 nm and provide high spectral resolution of 0.3 nm in particular at the Oxygen-A and -B bands. It will also cover the photochemical reflection features between 500 and 600 nm, the Chlorophyll absorption region between 600 and 677 nm, and the red-edge in the region of 697 to 755 nm. FLEX will fly in formation with Sentinel-3 in order to further enhance the spectral coverage from measurements made by the Sentinel-3 instruments OLCI and SLSTR, particularly for cloud screening and proper characterization of the atmospheric status.

LED-based interference-reflection microscopy combined with optical tweezers for quantitative three-dimensional microtubule imaging.

PubMed

Simmert, Steve; Abdosamadi, Mohammad Kazem; Hermsdorf, Gero; Schäffer, Erik

2018-05-28

Optical tweezers combined with various microscopy techniques are a versatile tool for single-molecule force spectroscopy. However, some combinations may compromise measurements. Here, we combined optical tweezers with total-internal-reflection-fluorescence (TIRF) and interference-reflection microscopy (IRM). Using a light-emitting diode (LED) for IRM illumination, we show that single microtubules can be imaged with high contrast. Furthermore, we converted the IRM interference pattern of an upward bent microtubule to its three-dimensional (3D) profile calibrated against the optical tweezers and evanescent TIRF field. In general, LED-based IRM is a powerful method for high-contrast 3D microscopy.

Evanescent excitation and emission in fluorescence microscopy.

PubMed

Axelrod, Daniel

2013-04-02

Evanescent light-light that does not propagate but instead decays in intensity over a subwavelength distance-appears in both excitation (as in total internal reflection) and emission (as in near-field imaging) forms in fluorescence microscopy. This review describes the physical connection between these two forms as a consequence of geometrical squeezing of wavefronts, and describes newly established or speculative applications and combinations of the two. In particular, each can be used in analogous ways to produce surface-selective images, to examine the thickness and refractive index of films (such as lipid multilayers or protein layers) on solid supports, and to measure the absolute distance of a fluorophore to a surface. In combination, the two forms can further increase selectivity and reduce background scattering in surface images. The polarization properties of each lead to more sensitive and accurate measures of fluorophore orientation and membrane micromorphology. The phase properties of the evanescent excitation lead to a method of creating a submicroscopic area of total internal reflection illumination or enhanced-resolution structured illumination. Analogously, the phase properties of evanescent emission lead to a method of producing a smaller point spread function, in a technique called virtual supercritical angle fluorescence. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Planar laser-induced fluorescence measurements of high-enthalpy free jet flow with nitric oxide

NASA Technical Reports Server (NTRS)

Palmer, Jennifer L.; Mcmillin, Brian K.; Hanson, Ronald K.

1992-01-01

Planar laser-induced fluorescence (PLIF) measurements of property fields in a high-enthalpy, supersonic, underexpanded free jet generated in a reflection-type shock tunnel are reported. PLIF images showing velocity and temperature sensitivity are presented. The inferred radial velocity and relative rotational temperature fields are found to be in agreement with those predicted by a numerical simulation of the flowfield using the method of characteristics.

In Vivo and Ex Vivo Confocal Microscopy for Dermatologic and Mohs Surgeons.

PubMed

Longo, Caterina; Ragazzi, Moira; Rajadhyaksha, Milind; Nehal, Kishwer; Bennassar, Antoni; Pellacani, Giovanni; Malvehy Guilera, Josep

2016-10-01

Confocal microscopy is a modern imaging device that has been extensively applied in skin oncology. More specifically, for tumor margin assessment, it has been used in two modalities: reflectance mode (in vivo on skin patient) and fluorescence mode (on freshly excised specimen). Although in vivo reflectance confocal microscopy is an add-on tool for lentigo maligna mapping, fluorescence confocal microscopy is far superior for basal cell carcinoma and squamous cell carcinoma margin assessment in the Mohs setting. This article provides a comprehensive overview of the use of confocal microscopy for skin cancer margin evaluation. Copyright © 2016 Elsevier Inc. All rights reserved.

Near-infrared fluorescence imaging of experimentally collagen-induced arthritis in rats using the nonspecific dye tetrasulfocyanine in comparison with gadolinium-based contrast-enhanced magnetic resonance imaging, histology, and clinical score

NASA Astrophysics Data System (ADS)

Gemeinhardt, Ines; Puls, Dorothee; Gemeinhardt, Ole; Taupitz, Matthias; Wagner, Susanne; Schnorr, Beatrix; Licha, Kai; Schirner, Michael; Ebert, Bernd; Petzelt, Diethard; Macdonald, Rainer; Schnorr, Jörg

2012-10-01

Using 15 rats with collagen-induced arthritis (30 joints) and 7 control rats (14 joints), we correlated the intensity of near-infrared fluorescence (NIRF) of the nonspecific dye tetrasulfocyanine (TSC) with magnetic resonance imaging (MRI), histopathology, and clinical score. Fluorescence images were obtained in reflection geometry using a NIRF camera system. Normalized fluorescence intensity (INF) was determined after intravenous dye administration on different time points up to 120 min. Contrast-enhanced MRI using gadodiamide was performed after NIRF imaging. Analyses were performed in a blinded fashion. Histopathological and clinical scores were determined for each ankle joint. INF of moderate and high-grade arthritic joints were significantly higher (p<0.005) than the values of control and low-grade arthritic joints between 5 and 30 min after TSC-injection. This result correlated well with post-contrast MRI signal intensities at about 5 min after gadodiamide administration. Furthermore, INF and signal increase on contrast-enhanced MRI showed high correlation with clinical and histopathological scores. Sensitivities and specificities for detection of moderate and high-grade arthritic joints were slightly lower for NIRF imaging (89%/81%) than for MRI (100%/91%). NIRF imaging using TSC, which is characterized by slower plasma clearance compared to indocyanine green (ICG), has the potential to improve monitoring of inflamed joints.

High contrast near-infrared polarized reflectance images of demineralization on tooth buccal and occlusal surfaces at lambda = 1310-nm.

PubMed

Wu, J; Fried, D

2009-03-01

Sound enamel manifests peak transparency in the near-IR (NIR) at 1310-nm, therefore the near-IR is ideally suited for high contrast imaging of dental caries. The purpose of this study was to acquire images of early demineralized enamel on the buccal and occlusal surfaces of extracted human teeth using NIR reflectance imaging and compare the contrast of those images with the contrast of images taken using other methods. Fifteen human molars were used in this in vitro study. Teeth were painted with a clear acid-resistant varnish, leaving two 2 mm x 2 mm windows on the buccal and occlusal surfaces of each tooth for demineralization. Artificial lesions were produced in the exposed windows after a 2-day exposure to a demineralizing solution at pH 4.5. Lesions were imaged using NIR transillumination, NIR and visible light reflectance, and fluorescence imaging methods. Crossed polarizers were used where appropriate to improve contrast. Polarization sensitive optical coherence tomography (PS-OCT) was also used to non-destructively assess the depth and severity of demineralization in each sample window. NIR reflectance imaging had the highest image contrast for both the buccal and occlusal groups and it was significantly higher contrast than visible light reflectance (P < 0.05). The results of the study suggest that NIR reflectance imaging is a promising new method for acquiring high contrast images of early demineralization on tooth surfaces. Copyright 2009 Wiley-Liss, Inc.

Optimization and performance evaluation of a conical mirror based fluorescence molecular tomography imaging system

NASA Astrophysics Data System (ADS)

Zhao, Yue; Zhang, Wei; Zhu, Dianwen; Li, Changqing

2016-03-01

We performed numerical simulations and phantom experiments with a conical mirror based fluorescence molecular tomography (FMT) imaging system to optimize its performance. With phantom experiments, we have compared three measurement modes in FMT: the whole surface measurement mode, the transmission mode, and the reflection mode. Our results indicated that the whole surface measurement mode performed the best. Then, we applied two different neutral density (ND) filters to improve the measurement's dynamic range. The benefits from ND filters are not as much as predicted. Finally, with numerical simulations, we have compared two laser excitation patterns: line and point. With the same excitation position number, we found that the line laser excitation had slightly better FMT reconstruction results than the point laser excitation. In the future, we will implement Monte Carlo ray tracing simulations to calculate multiple reflection photons, and create a look-up table accordingly for calibration.

Coherent total internal reflection dark-field microscopy: label-free imaging beyond the diffraction limit.

PubMed

von Olshausen, Philipp; Rohrbach, Alexander

2013-10-15

Coherent imaging is barely applicable in life-science microscopy due to multiple interference artifacts. Here, we show how these interferences can be used to improve image resolution and contrast. We present a dark-field microscopy technique with evanescent illumination via total internal reflection that delivers high-contrast images of coherently scattering samples. By incoherent averaging of multiple coherent images illuminated from different directions we can resolve image structures that remain unresolved by conventional (incoherent) fluorescence microscopy. We provide images of 190 nm beads revealing resolution beyond the diffraction limit and slightly increased object distances. An analytical model is introduced that accounts for the observed effects and which is confirmed by numerical simulations. Our approach may be a route to fast, label-free, super-resolution imaging in live-cell microscopy.

Hyperspectral imaging for detection of black tip damage in wheat kernels

NASA Astrophysics Data System (ADS)

Delwiche, Stephen R.; Yang, I.-Chang; Kim, Moon S.

2009-05-01

A feasibility study was conducted on the use of hyperspectral imaging to differentiate sound wheat kernels from those with the fungal condition called black point or black tip. Individual kernels of hard red spring wheat were loaded in indented slots on a blackened machined aluminum plate. Damage conditions, determined by official (USDA) inspection, were either sound (no damage) or damaged by the black tip condition alone. Hyperspectral imaging was separately performed under modes of reflectance from white light illumination and fluorescence from UV light (~380 nm) illumination. By cursory inspection of wavelength images, one fluorescence wavelength (531 nm) was selected for image processing and classification analysis. Results indicated that with this one wavelength alone, classification accuracy can be as high as 95% when kernels are oriented with their dorsal side toward the camera. It is suggested that improvement in classification can be made through the inclusion of multiple wavelength images.

Laser injury and in vivo multimodal imaging using a mouse model

NASA Astrophysics Data System (ADS)

Pocock, Ginger M.; Boretsky, Adam; Gupta, Praveena; Oliver, Jeff W.; Motamedi, Massoud

2011-03-01

Balb/c wild type mice were used to perform in vivo experiments of laser-induced thermal damage to the retina. A Heidelberg Spectralis HRA confocal scanning laser ophthalmoscope with a spectral domain optical coherence tomographer was used to obtain fundus and cross-sectional images of laser induced injury in the retina. Sub-threshold, threshold, and supra-threshold lesions were observed using optical coherence tomography (OCT), infrared reflectance, red-free reflectance, fluorescence angiography, and autofluorescence imaging modalities at different time points post-exposure. Lesions observed using all imaging modalities, except autofluorescence, were not visible immediately after exposure but did resolve within an hour and grew in size over a 24 hour period. There was a decrease in fundus autofluorescence at exposure sites immediately following exposure that developed into hyper-fluorescence 24-48 hours later. OCT images revealed threshold damage that was localized to the RPE but extended into the neural retina over a 24 hour period. Volumetric representations of the mouse retina were created to visualize the extent of damage within the retina over a 24 hour period. Multimodal imaging provides complementary information regarding damage mechanisms that may be used to quantify the extent of the damage as well as the effectiveness of treatments without need for histology.

Fully integrated reflection-mode photoacoustic, two-photon, and second harmonic generation microscopy in vivo

NASA Astrophysics Data System (ADS)

Song, Wei; Xu, Qiang; Zhang, Yang; Zhan, Yang; Zheng, Wei; Song, Liang

2016-08-01

The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies. Here, we developed a fully integrated multimodal microscopy that can provide photoacoustic (optical absorption), two-photon (fluorescence), and second harmonic generation (SHG) information from tissue in vivo, with intrinsically co-registered images. Moreover, using a delicately designed optical-acoustic coupling configuration, a high-frequency miniature ultrasonic transducer was integrated into a water-immersion optical objective, thus allowing all three imaging modalities to provide a high lateral resolution of ~290 nm with reflection-mode imaging capability, which is essential for studying intricate anatomy, such as that of the brain. Taking advantage of the complementary and comprehensive contrasts of the system, we demonstrated high-resolution imaging of various tissues in living mice, including microvasculature (by photoacoustics), epidermis cells, cortical neurons (by two-photon fluorescence), and extracellular collagen fibers (by SHG). The intrinsic image co-registration of the three modalities conveniently provided improved visualization and understanding of the tissue microarchitecture. The reported results suggest that, by revealing complementary tissue microstructures in vivo, this multimodal microscopy can potentially facilitate a broad range of biomedical studies, such as imaging of the tumor microenvironment and neurovascular coupling.

Intravital Confocal and Two-photon Imaging of Dual-color Cells and Extracellular Matrix Mimics

PubMed Central

Bal, Ufuk; Andresen, Volker; Baggett, Brenda; Utzinger, Urs

2013-01-01

To optimize imaging of cells in three dimensional culture we studied confocal backscattering, Second Harmonic Generation (SHG) and autofluorescence as source of contrast in extracellular matrix (ECM) mimics and evaluated the attenuation as well as bleaching of endogenous cellular fluorescence signals. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence while still providing good reflectance to detect voids in the embedding medium. We labeled breast cancer cells’ outline with DsRed2 and nucleus with eGFP. DsRed2 can be excited with confocal imaging at 568nm, and with two photon excitation (TPE) in the red and longer NIR. eGFP was excited at 488nm for confocal and in the NIR for TPE. While there is small difference in the bleaching rate for eGFP between confocal and TPE we observed significant difference for DsRed2 where bleaching is strongest during TPE in the red wavelengths and smallest during confocal imaging. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence becomes twice as strong compared to confocal imaging. PMID:23380006

Multispectral Scanner for Monitoring Plants

NASA Technical Reports Server (NTRS)

Gat, Nahum

2004-01-01

A multispectral scanner has been adapted to capture spectral images of living plants under various types of illumination for purposes of monitoring the health of, or monitoring the transfer of genes into, the plants. In a health-monitoring application, the plants are illuminated with full-spectrum visible and near infrared light and the scanner is used to acquire a reflected-light spectral signature known to be indicative of the health of the plants. In a gene-transfer- monitoring application, the plants are illuminated with blue or ultraviolet light and the scanner is used to capture fluorescence images from a green fluorescent protein (GFP) that is expressed as result of the gene transfer. The choice of wavelength of the illumination and the wavelength of the fluorescence to be monitored depends on the specific GFP.

UV reflection properties of plumage and skin of domesticated turkeys (Meleagris gallopavo f. dom.) as revealed by UV photography.

PubMed

Bartels, T; Lütgeharm, J-H; Wähner, M; Berk, J

2017-12-01

Reflection and fluorescence properties of feathered and non-feathered body regions of white- and bronze-colored fattening turkeys of various ages were examined by ultraviolet (UV) photography. The examinations were carried out on 20 white-feathered fattening turkeys (B.U.T. 6; 10 males, 10 females) and 20 bronze-feathered fattening turkeys (Grelier 708; 10 males, 10 females) over a period of 21 weeks. The turkeys were photographed once a wk under long-wave UV (UVA) radiation illumination (λ = 344-407 nm) using a digital camera. A bandpass filter was used for UV reflectography to filter out the visible components of the used light source. A longpass filter was used for UV fluorescence photography to avoid blurring in the image due to chromatic aberration as a result of UV illumination. We found that natal down feathers of white-feathered turkeys showed an intense yellowish-green fluorescence under UVA light. UVA fluorescence also was shown by the natal downs of the slightly melanized plumage areas of bronze turkeys. Vaned feathers of white fattening turkeys reflected UVA radiation. Freshly molted feathers were optically distinguishable from the previous feather generation due to their more intense UVA reflection. In bronze turkeys, both the bright end seams of the dark pennaceous feathers and rectrices and the bright banding of primary and secondary remiges reflected UVA radiation. Intense UVA fluorescence was recognizable in day-old chicks of both color variants on the scutellate scales of the legs and toes. In male turkeys of both color variants, UVA-reflecting parts were recognizable with increasing age on the featherless head region. The UVA-fluorescent and UVA-reflective characteristics of the plumage of fattening turkeys were closely related to the plumage color, the feather type, the molting state, and the age of the birds. Further research is needed regarding the UVA-reflecting properties of the turkey plumage and the effects of full-spectrum illumination, including the UVA spectrum, on the behavior and health of fattening turkeys. © 2017 Poultry Science Association Inc.

Multimodal confocal mosaics enable high sensitivity and specificity in screening of in situ squamous cell carcinoma

NASA Astrophysics Data System (ADS)

Grados Luyando, Maria del Carmen; Bar, Anna; Snavely, Nicholas; Jacques, Steven; Gareau, Daniel S.

2014-02-01

Screening cancer in excision margins with confocal microscopy may potentially save time and cost over the gold standard histopathology (H and E). However, diagnostic accuracy requires sufficient contrast and resolution to reveal pathological traits in a growing set of tumor types. Reflectance mode images structural details due to microscopic refractive index variation. Nuclear contrast with acridine orange fluorescence provides enhanced diagnostic value, but fails for in situ squamous cell carcinoma (SCC), where the cytoplasm is important to visualize. Combination of three modes [eosin (Eo) fluorescence, reflectance (R) and acridine orange (AO) fluorescence] enable imaging of cytoplasm, collagen and nuclei respectively. Toward rapid intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaics can image wide surgical margins (~1cm) with sub-cellular resolution and mimic the appearance of conventional H and E. Absorption contrast is achieved by alternating the excitation wavelength: 488nm (AO fluorescence) and 532nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H and E, enabling detection of the carcinoma in situ in the epidermal layer The sum mosaic Eo+R is false-colored pink to mimic eosins' appearance in H and E, while the AO mosaic is false-colored purple to mimic hematoxylins' appearance in H and E. In this study, mosaics of 10 Mohs surgical excisions containing SCC in situ and 5 containing only normal tissue were subdivided for digital presentation equivalent to 4X histology. Of the total 16 SCC in situ multimodal mosaics and 16 normal cases presented, two reviewers made 1 and 2 (respectively) type-2 errors (false positives) but otherwise scored perfectly when using the confocal images to screen for the presence of SCC in situ as compared to the gold standard histopathology. Limitations to precisely mimic H and E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.

Real-time monitoring the distribution of antibody-photo-absorber conjugates during photoimmunotherapy in vivo(Conference Presentation)

NASA Astrophysics Data System (ADS)

Tang, Qinggong; Lin, Jonathan; Nagaya, Tadanobu; Liu, Yi; Kobayashi, Hisataka; Chen, Yu

2017-02-01

Photo-immunotherapy (PIT) is an emerging low-side-effect cancer therapy based on monoclonal antibody (mAb) conjugated with a near-infrared (NIR) phthalocyanine dye IRDye700DX (IR700 is not only fluorescent which can be used as an imaging agent, but also phototoxic) that induces rapid cell death after exposure to NIR light. PIT induces highly-selective cancer cell death while leaving most of tumor blood vessels unharmed, leading to an effect termed super-enhanced permeability and retention (SUPR), which significantly improve the effectiveness of anti-cancer drug. Currently, the therapeutic effects of PIT were monitored using IR700 fluorescent signal based on macroscopic fluorescence reflectance imager, which lacks the resolution and depth information to reveal the intra-tumor heterogeneity of mAb-IR700 distribution. We developed a minimally-invasive two-channel fluorescence fiber imaging system by combining the traditional fluorescence imaging microscope with two imaging fiber bundles ( 0.85 mm) to monitor mAb-IR700 distribution and therapeutic effects during PIT at different intra-tumor locations (e.g. tumor periphery vs. tumor rim) in situ and in real time simutaneously, thereby enabling evaluation of the therapeutic effects in vivo and optimization of treatment regimens accordingly. Experiments were carried out on ten mice. The average fluorescence intensity recovery after PIT in tumor rim is 91.50% while 100.63% in tumor periphery. Significantly higher fluorescence redistribution (P=0.0371) in tumor periphery than tumor rim after PIT treatment were observed. In order to verify the results, two-photon microscopy combining with micro-prism was also used to record the mAb-IR700 distribution at different depth locations of the tumor during PIT.

Recovery of Degraded-Beyond-Recognition 19th Century Daguerreotypes with Rapid High Dynamic Range Elemental X-ray Fluorescence Imaging of Mercury L Emission.

PubMed

Kozachuk, Madalena S; Sham, Tsun-Kong; Martin, Ronald R; Nelson, Andrew J; Coulthard, Ian; McElhone, John P

2018-06-22

A daguerreotype image, the first commercialized photographic process, is composed of silver-mercury, and often silver-mercury-gold amalgam particles on the surface of a silver-coated copper plate. Specular and diffuse reflectance of light from these image particles produces the range of gray tones that typify these 19 th century images. By mapping the mercury distribution with rapid-scanning, synchrotron-based micro-X-ray fluorescence (μ-XRF) imaging, full portraits, which to the naked eye are obscured entirely by extensive corrosion, can be retrieved in a non-invasive, non-contact, and non-destructive manner. This work furthers the chemical understanding regarding the production of these images and suggests that mercury is retained in the image particles despite surface degradation. Most importantly, μ-XRF imaging provides curators with an image recovery method for degraded daguerreotypes, even if the artifact's condition is beyond traditional conservation treatments.

Visible and infrared reflectance imaging spectroscopy of paintings: pigment mapping and improved infrared reflectography

NASA Astrophysics Data System (ADS)

Delaney, John K.; Zeibel, Jason G.; Thoury, Mathieu; Littleton, Roy; Morales, Kathryn M.; Palmer, Michael; de la Rie, E. René

2009-07-01

Reflectance imaging spectroscopy, the collection of images in narrow spectral bands, has been developed for remote sensing of the Earth. In this paper we present findings on the use of imaging spectroscopy to identify and map artist pigments as well as to improve the visualization of preparatory sketches. Two novel hyperspectral cameras, one operating from the visible to near-infrared (VNIR) and the other in the shortwave infrared (SWIR), have been used to collect diffuse reflectance spectral image cubes on a variety of paintings. The resulting image cubes (VNIR 417 to 973 nm, 240 bands, and SWIR 970 to 1650 nm, 85 bands) were calibrated to reflectance and the resulting spectra compared with results from a fiber optics reflectance spectrometer (350 to 2500 nm). The results show good agreement between the spectra acquired with the hyperspectral cameras and those from the fiber reflectance spectrometer. For example, the primary blue pigments and their distribution in Picasso's Harlequin Musician (1924) are identified from the reflectance spectra and agree with results from X-ray fluorescence data and dispersed sample analysis. False color infrared reflectograms, obtained from the SWIR hyperspectral images, of extensively reworked paintings such as Picasso's The Tragedy (1903) are found to give improved visualization of changes made by the artist. These results show that including the NIR and SWIR spectral regions along with the visible provides for a more robust identification and mapping of artist pigments than using visible imaging spectroscopy alone.

Multispectral fluorescence imaging of human ovarian and Fallopian tube tissue for early stage cancer detection

NASA Astrophysics Data System (ADS)

Tate, Tyler; Baggett, Brenda; Rice, Photini; Watson, Jennifer; Orsinger, Gabe; Nymeyer, Ariel C.; Welge, Weston A.; Keenan, Molly; Saboda, Kathylynn; Roe, Denise J.; Hatch, Kenneth; Chambers, Setsuko; Black, John; Utzinger, Urs; Barton, Jennifer

2015-03-01

With early detection, five year survival rates for ovarian cancer are over 90%, yet no effective early screening method exists. Emerging consensus suggests that perhaps over 50% of the most lethal form of the disease, high grade serous ovarian cancer, originates in the Fallopian tube. Cancer changes molecular concentrations of various endogenous fluorophores. Using specific excitation wavelengths and emissions bands on a Multispectral Fluorescence Imaging (MFI) system, spatial and spectral data over a wide field of view can be collected from endogenous fluorophores. Wavelength specific reflectance images provide additional information to normalize for tissue geometry and blood absorption. Ratiometric combination of the images may create high contrast between neighboring normal and abnormal tissue. Twenty-six women undergoing oophorectomy or debulking surgery consented the use of surgical discard tissue samples for MFI imaging. Forty-nine pieces of ovarian tissue and thirty-two pieces of Fallopian tube tissue were collected and imaged with excitation wavelengths between 280 nm and 550 nm. After imaging, each tissue sample was fixed, sectioned and HE stained for pathological evaluation. Comparison of mean intensity values between normal, benign, and cancerous tissue demonstrate a general trend of increased fluorescence of benign tissue and decreased fluorescence of cancerous tissue when compared to normal tissue. The predictive capabilities of the mean intensity measurements are tested using multinomial logistic regression and quadratic discriminant analysis. Adaption of the system for in vivo Fallopian tube and ovary endoscopic imaging is possible and is briefly described.

Active substrates improving sensitivity in biomedical fluorescence microscopy

NASA Astrophysics Data System (ADS)

Le Moal, E.; Leveque-Fort, S.; Fort, E.; Lacharme, J.-P.; Fontaine-Aupart, M.-P.; Ricolleau, C.

2005-08-01

Fluorescence is widely used as a spectroscopic tool or for biomedical imaging, in particular for DNA chips. In some cases, detection of very low molecular concentrations and precise localization of biomarkers are limited by the weakness of the fluorescence signal. We present a new method based on sample substrates that improve fluorescence detection sensitivity. These active substrates consist in glass slides covered with metal (gold or silver) and dielectric (alumina) films and can directly be used with common microscope set-up. Fluorescence enhancement affects both excitation and decay rates and is strongly dependant on the distance to the metal surface. Furthermore, fluorescence collection is improved since fluorophore emission lobes are advantageously modified close to a reflective surface. Finally, additional improvements are achieved by structuring the metallic layer. Substrates morphology was mapped by Atomic Force Microscopy (AFM). Substrates optical properties were studied using mono- and bi-photonic fluorescence microscopy with time resolution. An original set-up was implemented for spatial radiation pattern's measurement. Detection improvement was then tested on commercial devices. Several biomedical applications are presented. Enhancement by two orders of magnitude are achieved for DNA chips and signal-to-noise ratio is greatly increased for cells imaging.

Multiphoton imaging for assessing renal disposition in acute kidney injury

NASA Astrophysics Data System (ADS)

Liu, Xin; Liang, Xiaowen; Wang, Haolu; Roberts, Darren M.; Roberts, Michael S.

2016-11-01

Estimation of renal function and drug renal disposition in acute kidney injury (AKI), is important for appropriate dosing of drugs and adjustment of therapeutic strategies, but is challenging due to fluctuations in kidney function. Multiphoton microscopy has been shown to be a useful tool in studying drug disposition in liver and can reflect dynamic changes of liver function. We extend this imaging technique to investigate glomerular filtration rate (GFR) and tubular transporter functional change in various animal models of AKI, which mimic a broad range of causes of AKI such as hypoxia (renal ischemia- reperfusion), therapeutic drugs (e.g. cisplatin), rhabdomyolysis (e.g. glycerol-induced) and sepsis (e.g. LPSinduced). The MPM images revealed acute injury of tubular cells as indicated by reduced autofluorescence and cellular vacuolation in AKI groups compared to control group. In control animal, systemically injected FITC-labelled inulin was rapidly cleared from glomerulus, while the clearance of FITC-inulin was significantly delayed in most of animals in AKI group, which may reflect the reduced GFR in AKI. Following intravenous injection, rhodamine 123, a fluorescent substrate of p-glycoprotein (one of tubular transporter), was excreted into urine in proximal tubule via p-glycoprotein; in response to AKI, rhodamine 123 was retained in tubular cells as revealed by slower decay of fluorescence intensity, indicating P-gp transporter dysfunction in AKI. Thus, real-time changes in GFR and transporter function can be imaged in rodent kidney with AKI using multiphoton excitation of exogenously injected fluorescent markers.

Metabolic microscopy of head and neck cancer organoids

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Skala, Melissa C.

2016-03-01

Studies for head and neck cancer have primarily relied on cell lines or in vivo animal studies. However, a technique that combines the benefits of high-throughput in vitro studies with a complex, physiologically relevant microenvironment would be advantageous for understanding drug effects. Organoids provide a unique platform that fulfills these goals. Organoids are generated from excised and digested tumor tissue and are grown in culture. Fluorescence microscopy provides high-resolution images on a similar spatial scale as organoids. In particular, autofluorescence imaging of the metabolic cofactors NAD(P)H and FAD can provide insight into response to anti-cancer treatment. The optical redox ratio reflects relative amounts of NAD(P)H and FAD, and the fluorescence lifetime reflects enzyme activity of NAD(P)H and FAD. This study optimizes and characterizes the generation and culture of organoids grown from head and neck cancer tissue. Additionally, organoids were treated for 24 hours with a standard chemotherapy, and metabolic response in the organoids was measured using optical metabolic imaging. Ultimately, combining head and neck cancer organoids with optical metabolic imaging could be applied to test drug sensitivity for drug development studies as well as treatment planning for cancer patients.

Instrument and method for X-ray diffraction, fluorescence, and crystal texture analysis without sample preparation

NASA Technical Reports Server (NTRS)

Gendreau, Keith (Inventor); Martins, Jose Vanderlei (Inventor); Arzoumanian, Zaven (Inventor)

2010-01-01

An X-ray diffraction and X-ray fluorescence instrument for analyzing samples having no sample preparation includes a X-ray source configured to output a collimated X-ray beam comprising a continuum spectrum of X-rays to a predetermined coordinate and a photon-counting X-ray imaging spectrometer disposed to receive X-rays output from an unprepared sample disposed at the predetermined coordinate upon exposure of the unprepared sample to the collimated X-ray beam. The X-ray source and the photon-counting X-ray imaging spectrometer are arranged in a reflection geometry relative to the predetermined coordinate.

Fluorescence molecular imaging system with a novel mouse surface extraction method and a rotary scanning scheme

NASA Astrophysics Data System (ADS)

Zhao, Yue; Zhu, Dianwen; Baikejiang, Reheman; Li, Changqing

2015-03-01

We have developed a new fluorescence molecular tomography (FMT) imaging system, in which we utilized a phase shifting method to extract the mouse surface geometry optically and a rotary laser scanning approach to excite fluorescence molecules and acquire fluorescent measurements on the whole mouse body. Nine fringe patterns with a phase shifting of 2π/9 are projected onto the mouse surface by a projector. The fringe patterns are captured using a webcam to calculate a phase map that is converted to the geometry of the mouse surface with our algorithms. We used a DigiWarp approach to warp a finite element mesh of a standard digital mouse to the measured mouse surface thus the tedious and time-consuming procedure from a point cloud to mesh is avoided. Experimental results indicated that the proposed method is accurate with errors less than 0.5 mm. In the FMT imaging system, the mouse is placed inside a conical mirror and scanned with a line pattern laser that is mounted on a rotation stage. After being reflected by the conical mirror, the emitted fluorescence photons travel through central hole of the rotation stage and the band pass filters in a motorized filter wheel, and are collected by a CCD camera. Phantom experimental results of the proposed new FMT imaging system can reconstruct the target accurately.

Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching.

PubMed Central

Sund, S E; Axelrod, D

2000-01-01

Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics. PMID:10969025

Laser induced fluorescence as a diagnostic tool integrated into a scanning fiber endoscope for mouse imaging

NASA Astrophysics Data System (ADS)

Brown, Christopher M.; Maggio-Price, Lillian; Seibel, Eric J.

2007-02-01

Scanning fiber endoscope (SFE) technology has shown promise as a minimally invasive optical imaging tool. To date, it is capable of capturing full-color 500-line images, at 15 Hz frame rate in vivo, as a 1.6 mm diameter endoscope. The SFE uses a singlemode optical fiber actuated at mechanical resonance to scan a light spot over tissue while backscattered or fluorescent light at each pixel is detected in time series using several multimode optical fibers. We are extending the capability of the SFE from a RGB reflectance imaging device to a diagnostic tool by imaging laser induced fluorescence (LIF) in tissue, allowing for correlation of endogenous fluorescence to tissue state. Design of the SFE for diagnostic imaging is guided by a comparison of single point spectra acquired from an inflammatory bowel disease (IBD) model to tissue histology evaluated by a pathologist. LIF spectra were acquired by illuminating tissue with a 405 nm light source and detecting intrinsic fluorescence with a multimode optical fiber. The IBD model used in this study was mdr1a-/- mice, where IBD was modulated by infection with Helicobacter bilis. IBD lesions in the mouse model ranged from mild to marked hyperplasia and dysplasia, from the distal colon to the cecum. A principle components analysis (PCA) was conducted on single point spectra of control and IBD tissue. PCA allowed for differentiation between healthy and dysplastic tissue, indicating that emission wavelengths from 620 - 650 nm were best able to differentiate diseased tissue and inflammation from normal healthy tissue.

Darkfield Adapter for Whole Slide Imaging: Adapting a Darkfield Internal Reflection Illumination System to Extend WSI Applications

PubMed Central

Kawano, Yoshihiro; Higgins, Christopher; Yamamoto, Yasuhito; Nyhus, Julie; Bernard, Amy; Dong, Hong-Wei; Karten, Harvey J.; Schilling, Tobias

2013-01-01

We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining. PMID:23520500

Simultaneous optical and electrical recording of a single ion-channel.

PubMed

Ide, Toru; Takeuchi, Yuko; Aoki, Takaaki; Yanagida, Toshio

2002-10-01

In recent years, the single-molecule imaging technique has proven to be a valuable tool in solving many basic problems in biophysics. The technique used to measure single-molecule functions was initially developed to study electrophysiological properties of channel proteins. However, the technology to visualize single channels at work has not received as much attention. In this study, we have for the first time, simultaneously measured the optical and electrical properties of single-channel proteins. The large conductance calcium-activated potassium channel (BK-channel) labeled with fluorescent dye molecules was incorporated into a planar bilayer membrane and the fluorescent image captured with a total internal reflection fluorescence microscope simultaneously with single-channel current recording. This innovative technology will greatly advance the study of channel proteins as well as signal transduction processes that involve ion permeation processes.

Breast Cancer Treatment in the Era of Molecular Imaging

PubMed Central

Edelhauser, Gundula; Funovics, Martin

2008-01-01

Summary Molecular imaging employs molecularly targeted probes to visualize and often quantify distinct disease-specific markers and pathways. Modalities like intravital confocal or multiphoton microscopy, near-infrared fluorescence combined with endoscopy, surface reflectance imaging, or fluorescence-mediated tomography, and radionuclide imaging with positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are increasingly used for small animal high-throughput screening, drug development and testing, and monitoring gene therapy experiments. In the clinical treatment of breast cancer, PET and SPECT as well as magnetic resonance-based molecular imaging are already established for the staging of distant disease and intrathoracic nodal status, for patient selection regarding receptor-directed treatments, and to gain early information about treatment efficacy. In the near future, reporter gene imaging during gene therapy and further spatial and qualitative characterization of the disease can become clinically possible with radionuclide and optical methods. Ultimately, it may be expected that every level of breast cancer treatment will be affected by molecular imaging, including screening. PMID:21048912

Super-resolution and super-localization microscopy: A novel tool for imaging chemical and biological processes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Bin

2015-01-01

Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes.This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the singlemore » molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM).« less

Chip-based wide field-of-view nanoscopy

NASA Astrophysics Data System (ADS)

Diekmann, Robin; Helle, Øystein I.; Øie, Cristina I.; McCourt, Peter; Huser, Thomas R.; Schüttpelz, Mark; Ahluwalia, Balpreet S.

2017-04-01

Present optical nanoscopy techniques use a complex microscope for imaging and a simple glass slide to hold the sample. Here, we demonstrate the inverse: the use of a complex, but mass-producible optical chip, which hosts the sample and provides a waveguide for the illumination source, and a standard low-cost microscope to acquire super-resolved images via two different approaches. Waveguides composed of a material with high refractive-index contrast provide a strong evanescent field that is used for single-molecule switching and fluorescence excitation, thus enabling chip-based single-molecule localization microscopy. Additionally, multimode interference patterns induce spatial fluorescence intensity variations that enable fluctuation-based super-resolution imaging. As chip-based nanoscopy separates the illumination and detection light paths, total-internal-reflection fluorescence excitation is possible over a large field of view, with up to 0.5 mm × 0.5 mm being demonstrated. Using multicolour chip-based nanoscopy, we visualize fenestrations in liver sinusoidal endothelial cells.

A Quantitative Microscopy Technique for Determining the Number of Specific Proteins in Cellular Compartments

PubMed Central

Mutch, Sarah A.; Gadd, Jennifer C.; Fujimoto, Bryant S.; Kensel-Hammes, Patricia; Schiro, Perry G.; Bajjalieh, Sandra M.; Chiu, Daniel T.

2013-01-01

This protocol describes a method to determine both the average number and variance of proteins in the few to tens of copies in isolated cellular compartments, such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number but lack information on the variance or are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling the cellular compartment with fluorescent primary-secondary antibody complexes, TIRF (total internal reflection fluorescence) microscopy imaging of the cellular compartment, digital image analysis, and deconvolution of the fluorescence intensity data. A minimum of 2.5 days is required to complete the labeling, imaging, and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes. PMID:22094731

Measurement of Zeta-Potential at Microchannel Wall by a Nanoscale Laser Induced Fluorescence Imaging

NASA Astrophysics Data System (ADS)

Kazoe, Yutaka; Sato, Yohei

A nanoscale laser induced fluorescence imaging was proposed by using fluorescent dye and the evanescent wave with total internal reflection of a laser beam. The present study focused on the two-dimensional measurement of zeta-potential at the microchannel wall, which is an electrostatic potential at the wall surface and a dominant parameter of electroosmotic flow. The evanescent wave, which decays exponentially from the wall, was used as an excitation light of the fluorescent dye. The fluorescent intensity detected by a CCD camera is closely related to the zeta-potential. Two kinds of fluorescent dye solution at different ionic concentrations were injected into a T-shaped microchannel, and formed a mixing flow field in the junction area. The two-dimensional distribution of zeta-potential at the microchannel wall in the pressure-driven flow field was measured. The obtained zeta-potential distribution has a transverse gradient toward the mixing flow field and was changed by the difference in the averaged velocity of pressure-driven flow. To understand the ion motion in the mixing flow field, the three-dimensional flow structure was analyzed by the velocity measurement using micron-resolution particle image velocimetry and the numerical simulation. It is concluded that the two-dimensional distribution of zeta-potential at the microchannel wall was dependent on the ion motion in the flow field, which was governed by the convection and molecular diffusion.

Optimal parameters for near infrared fluorescence imaging of amyloid plaques in Alzheimer’s disease mouse models

PubMed Central

Raymond, S B; Kumar, A T N; Boas, D A; Bacskai, B J

2012-01-01

Amyloid-β plaques are an Alzheimer’s disease biomarker which present unique challenges for near-infrared fluorescence tomography because of size (<50 μm diameter) and distribution. We used high-resolution simulations of fluorescence in a digital Alzheimer’s disease mouse model to investigate the optimal fluorophore and imaging parameters for near-infrared fluorescence tomography of amyloid plaques. Fluorescence was simulated for amyloid-targeted probes with emission at 630 and 800 nm, plaque-to-background ratios from 1–1000, amyloid burden from 0–10%, and for transmission and reflection measurement geometries. Fluorophores with high plaque-to-background contrast ratios and 800 nm emission performed significantly better than current amyloid imaging probes. We tested idealized fluorophores in transmission and full-angle tomographic measurement schemes (900 source–detector pairs), with and without anatomical priors. Transmission reconstructions demonstrated strong linear correlation with increasing amyloid burden, but underestimated fluorescence yield and suffered from localization artifacts. Full-angle measurements did not improve upon the transmission reconstruction qualitatively or in semi-quantitative measures of accuracy; anatomical and initial-value priors did improve reconstruction localization and accuracy for both transmission and full-angle schemes. Region-based reconstructions, in which the unknowns were reduced to a few distinct anatomical regions, produced highly accurate yield estimates for cortex, hippocampus and brain regions, even with a reduced number of measurements (144 source–detector pairs). PMID:19794239

Molecular Hydrogen Fluorescence in IC 63

NASA Technical Reports Server (NTRS)

Andersson, B-G

2005-01-01

This grant has supported the acquisition, reduction and analysis of data targeting the structure and excitation of molecular hydrogen in the reflection nebula IC 63 and in particular the fluorescent emission seen in the UV. In addition to manpower for analyzing the FUSE data, the grant supported the (attempted) acquisition of supporting ground-based data. We proposed for and received observing time for two sets of ground based, data; narrow band imaging ([S II], [O III) at KPNO (July 2002; Observer: Burgh) and imaging spectro-photometry of several of the near-infrared rotation-vibration lines of H2 at the IRTF (October 2003; Observer: Andersson). Unfortunately, both of these runs were failures, primarily because of bad weather, and did not result in any useful data. We combined the FUSE observations with rocket borne observations of the star responsible for exciting the H2 fluorescence in IC 63: gamma Cas, and with archival HUT observations of IC 63, covering the long-wavelength part of the molecular hydrogen fluorescence.

Non-destructive Determination of Shikimic Acid Concentration in Transgenic Maize Exhibiting Glyphosate Tolerance Using Chlorophyll Fluorescence and Hyperspectral Imaging

PubMed Central

Feng, Xuping; Yu, Chenliang; Chen, Yue; Peng, Jiyun; Ye, Lanhan; Shen, Tingting; Wen, Haiyong; He, Yong

2018-01-01

The development of transgenic glyphosate-tolerant crops has revolutionized weed control in crops in many regions of the world. The early, non-destructive identification of superior plant phenotypes is an important stage in plant breeding programs. Here, glyphosate-tolerant transgenic maize and its parental wild-type control were studied at 2, 4, 6, and 8 days after glyphosate treatment. Visible and near-infrared hyperspectral imaging and chlorophyll fluorescence imaging techniques were applied to monitor the performance of plants. In our research, transgenic maize, which was highly tolerant to glyphosate, was phenotyped using these high-throughput non-destructive methods to validate low levels of shikimic acid accumulation and high photochemical efficiency of photosystem II as reflected by maximum quantum yield and non-photochemical quenching in response to glyphosate. For hyperspectral imaging analysis, the combination of spectroscopy and chemometric methods was used to predict shikimic acid concentration. Our results indicated that a partial least-squares regression model, built on optimal wavelengths, effectively predicted shikimic acid concentrations, with a coefficient of determination value of 0.79 for the calibration set, and 0.82 for the prediction set. Moreover, shikimic acid concentration estimates from hyperspectral images were visualized on the prediction maps by spectral features, which could help in developing a simple multispectral imaging instrument for non-destructive phenotyping. Specific physiological effects of glyphosate affected the photochemical processes of maize, which induced substantial changes in chlorophyll fluorescence characteristics. A new data-driven method, combining mean fluorescence parameters and featuring a screening approach, provided a satisfactory relationship between fluorescence parameters and shikimic acid content. The glyphosate-tolerant transgenic plants can be identified with the developed discrimination model established on important wavelengths or sensitive fluorescence parameters 6 days after glyphosate treatment. The overall results indicated that both hyperspectral imaging and chlorophyll fluorescence imaging techniques could provide useful tools for stress phenotyping in maize breeding programs and could enable the detection and evaluation of superior genotypes, such as glyphosate tolerance, with a non-destructive high-throughput technique. PMID:29686693

Non-destructive Determination of Shikimic Acid Concentration in Transgenic Maize Exhibiting Glyphosate Tolerance Using Chlorophyll Fluorescence and Hyperspectral Imaging.

PubMed

Feng, Xuping; Yu, Chenliang; Chen, Yue; Peng, Jiyun; Ye, Lanhan; Shen, Tingting; Wen, Haiyong; He, Yong

2018-01-01

The development of transgenic glyphosate-tolerant crops has revolutionized weed control in crops in many regions of the world. The early, non-destructive identification of superior plant phenotypes is an important stage in plant breeding programs. Here, glyphosate-tolerant transgenic maize and its parental wild-type control were studied at 2, 4, 6, and 8 days after glyphosate treatment. Visible and near-infrared hyperspectral imaging and chlorophyll fluorescence imaging techniques were applied to monitor the performance of plants. In our research, transgenic maize, which was highly tolerant to glyphosate, was phenotyped using these high-throughput non-destructive methods to validate low levels of shikimic acid accumulation and high photochemical efficiency of photosystem II as reflected by maximum quantum yield and non-photochemical quenching in response to glyphosate. For hyperspectral imaging analysis, the combination of spectroscopy and chemometric methods was used to predict shikimic acid concentration. Our results indicated that a partial least-squares regression model, built on optimal wavelengths, effectively predicted shikimic acid concentrations, with a coefficient of determination value of 0.79 for the calibration set, and 0.82 for the prediction set. Moreover, shikimic acid concentration estimates from hyperspectral images were visualized on the prediction maps by spectral features, which could help in developing a simple multispectral imaging instrument for non-destructive phenotyping. Specific physiological effects of glyphosate affected the photochemical processes of maize, which induced substantial changes in chlorophyll fluorescence characteristics. A new data-driven method, combining mean fluorescence parameters and featuring a screening approach, provided a satisfactory relationship between fluorescence parameters and shikimic acid content. The glyphosate-tolerant transgenic plants can be identified with the developed discrimination model established on important wavelengths or sensitive fluorescence parameters 6 days after glyphosate treatment. The overall results indicated that both hyperspectral imaging and chlorophyll fluorescence imaging techniques could provide useful tools for stress phenotyping in maize breeding programs and could enable the detection and evaluation of superior genotypes, such as glyphosate tolerance, with a non-destructive high-throughput technique.

Comparison of Near-Infrared Imaging Camera Systems for Intracranial Tumor Detection.

PubMed

Cho, Steve S; Zeh, Ryan; Pierce, John T; Salinas, Ryan; Singhal, Sunil; Lee, John Y K

2018-04-01

Distinguishing neoplasm from normal brain parenchyma intraoperatively is critical for the neurosurgeon. 5-Aminolevulinic acid (5-ALA) has been shown to improve gross total resection and progression-free survival but has limited availability in the USA. Near-infrared (NIR) fluorescence has advantages over visible light fluorescence with greater tissue penetration and reduced background fluorescence. In order to prepare for the increasing number of NIR fluorophores that may be used in molecular imaging trials, we chose to compare a state-of-the-art, neurosurgical microscope (System 1) to one of the commercially available NIR visualization platforms (System 2). Serial dilutions of indocyanine green (ICG) were imaged with both systems in the same environment. Each system's sensitivity and dynamic range for NIR fluorescence were documented and analyzed. In addition, brain tumors from six patients were imaged with both systems and analyzed. In vitro, System 2 demonstrated greater ICG sensitivity and detection range (System 1 1.5-251 μg/l versus System 2 0.99-503 μg/l). Similarly, in vivo, System 2 demonstrated signal-to-background ratio (SBR) of 2.6 ± 0.63 before dura opening, 5.0 ± 1.7 after dura opening, and 6.1 ± 1.9 after tumor exposure. In contrast, System 1 could not easily detect ICG fluorescence prior to dura opening with SBR of 1.2 ± 0.15. After the dura was reflected, SBR increased to 1.4 ± 0.19 and upon exposure of the tumor SBR increased to 1.8 ± 0.26. Dedicated NIR imaging platforms can outperform conventional microscopes in intraoperative NIR detection. Future microscopes with improved NIR detection capabilities could enhance the use of NIR fluorescence to detect neoplasm and improve patient outcome.

Imaging of single retinal ganglion cell with differential interference contrast microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Oh, Juyeong; Kim, Yu Jeong; Kim, Chul-Ki; Lee, Taik Jin; Seo, Mina; Lee, Seok; Woo, Deok Ha; Jun, Seong Chan; Park, Ki-Ho; Kim, Seok Hwan; Kim, Jae Hun

2017-02-01

Glaucoma is a progressive optic neuropathy, characterized by the selective loss of retinal ganglion cells (RGCs). Therefore, monitoring the change of number or morphology of RGC is essential for the early detection as well as investigation of pathophysiology of glaucoma. Since RGC layer is transparent and hyporeflective, the direct optical visualization of RGCs has not been successful so far. Therefore, glaucoma evaluation mostly depends on indirect diagnostic methods such as the evaluation of optic disc morphology or retinal nerve fiber layer thickness measurement by optical coherence tomography. We have previously demonstrated single photoreceptor cell imaging with differential interference contrast (DIC) microscopy. Herein, we successfully visualized single RGC using DIC microscopy. Since RGC layer is much less reflective than photoreceptor layer, various techniques including the control of light wavelength and bandwidth using a tunable band pass filter were adopted to reduce the chromatic aberration in z-axis for higher and clearer resolution. To verify that the imaged cells were the RGCs, the flat-mounted retina of Sprague-Dawley rat, in which the RGCs were retrogradely labeled with fluorescence, was observed by both fluorescence and DIC microscopies for direct comparison. We have confirmed that the cell images obtained by fluorescence microscopy were perfectly matched with cell images by DIC microscopy. As conclusion, we have visualized single RGC with DIC microscopy, and confirmed with fluorescence microscopy.

Relationship between leaf optical properties, chlorophyll fluorescence and pigment changes in senescing Acer saccharum leaves.

PubMed

Junker, Laura Verena; Ensminger, Ingo

2016-06-01

The ability of plants to sequester carbon is highly variable over the course of the year and reflects seasonal variation in photosynthetic efficiency. This seasonal variation is most prominent during autumn, when leaves of deciduous tree species such as sugar maple (Acer saccharum Marsh.) undergo senescence, which is associated with downregulation of photosynthesis and a change of leaf color. The remote sensing of leaf color by spectral reflectance measurements and digital repeat images is increasingly used to improve models of growing season length and seasonal variation in carbon sequestration. Vegetation indices derived from spectral reflectance measurements and digital repeat images might not adequately reflect photosynthetic efficiency of red-senescing tree species during autumn due to the changes in foliar pigment content associated with autumn phenology. In this study, we aimed to assess how effectively several widely used vegetation indices capture autumn phenology and reflect the changes in physiology and photosynthetic pigments during autumn. Chlorophyll fluorescence and pigment content of green, yellow, orange and red leaves were measured to represent leaf senescence during autumn and used as a reference to validate and compare vegetation indices derived from leaf-level spectral reflectance measurements and color analysis of digital images. Vegetation indices varied in their suitability to track the decrease of photosynthetic efficiency and chlorophyll content despite increasing anthocyanin content. Commonly used spectral reflectance indices such as the normalized difference vegetation index and photochemical reflectance index showed major constraints arising from a limited representation of gradual decreases in chlorophyll content and an influence of high foliar anthocyanin levels. The excess green index and green-red vegetation index were more suitable to assess the process of senescence. Similarly, digital image analysis revealed that vegetation indices such as Hue and normalized difference index are superior compared with the often-used green chromatic coordinate. We conclude that indices based on red and green color information generally represent autumn phenology most efficiently. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Imaging, scattering, and spectroscopic systems for biomedical optics: Tools for bench top and clinical applications

NASA Astrophysics Data System (ADS)

Cottrell, William J.

Optical advances have had a profound impact on biology and medicine. The capabilities range from sensing biological analytes to whole animal and subcellular imaging and clinical therapies. The work presented in this thesis describes three independent and multifunctional optical systems, which explore clinical therapy at the tissue level, biological structure at the cell/organelle level, and the function of underlying fundamental cellular processes. First, we present a portable clinical instrument for delivering delta-aminolevulinic acid photodynamic therapy (ALA-PDT) while performing noninvasive spectroscopic monitoring in vivo. Using an off-surface probe, the instrument delivered the treatment beam to a user-defined field on the skin and performed reflectance and fluorescence spectroscopies at two regions within this field. The instrument was used to monitor photosensitizer fluorescence photobleaching, fluorescent photoproduct kinetics, and blood oxygen saturation during a clinical ALA-PDT trial on superficial basal cell carcinoma (sBCC). Protoporphyrin IX and photoproduct fluorescence excited by the 632.8 nm PDT treatment laser was collected between 665 and 775 nm. During a series of brief treatment interruptions at programmable time points, white-light reflectance spectra between 475 and 775 nm were acquired. Fluorescence spectra were corrected for the effects of absorption and scattering, informed by the reflectance measurements, and then decomposed into known fluorophore contributions in real time using a robust singular-value decomposition fitting routine. Reflectance spectra additionally provided information on hemoglobin oxygen saturation. We next describe the incorporation of this instrument into clinical trials at Roswell Park Cancer Institute (Buffalo, NY). In this trial we examined the effects of light irradiance on photodynamic efficiency and pain. The rate of singlet-oxygen production depends on the product of irradiance and photosensitizer and oxygen concentrations. High irradiance and/or photosensitizer levels cause inefficient treatment from oxygen depletion in preclinical models. This trial established the irradiance-dependence of patient tolerability to ALA-PDT of sBCC and a pain-threshold irradiance, below which patients did not experience significant pain or require anesthetic. The irradiance-dependence of sensitizer photobleaching was also used to determine an optimal irradiance that maximized treatment efficiency. The optimal fluence at a single low irradiance is yet to be determined. We additionally report the design, construction, and initial characterization of two optical systems used for cellular scattering measurements: a forward scattering white-light spectroscopy system used to characterize lysosomal refractive index and a multifunctional scattering and fluorescence microscope that exploited an angle-resolved forward-scattering geometry. The multifunctional scattering and fluorescence microscope employed brightfield, Fourier-filtered darkfield, direct imaging of the Fourier plane, angle-resolved scattering, and white-light scattering spectroscopy while preserving a fluorescence imaging channel. Lastly, we report on the development of a microscope-based system used for high-powered, focal laser photolysis. This system was used with cell-permeable caged messenger molecules and analyte specific fluorophores to provide local stimulation of intact cells and subsequent analyte monitoring. This provided a high-precision, non-invasive means for studying Ca2+ dynamics between cell types and between sub-cellular regions within a single cell type. The resulting studies compared the mechanisms underlying the Ca2+ signal globalization in these individual exocrine cell types and under regional messenger release.

Design and demonstration of multimodal optical scanning microscopy for confocal and two-photon imaging

NASA Astrophysics Data System (ADS)

Chun, Wanhee; Do, Dukho; Gweon, Dae-Gab

2013-01-01

We developed a multimodal microscopy based on an optical scanning system in order to obtain diverse optical information of the same area of a sample. Multimodal imaging researches have mostly depended on a commercial microscope platform, easy to use but restrictive to extend imaging modalities. In this work, the beam scanning optics, especially including a relay lens, was customized to transfer broadband (400-1000 nm) lights to a sample without any optical error or loss. The customized scanning optics guarantees the best performances of imaging techniques utilizing the lights within the design wavelength. Confocal reflection, confocal fluorescence, and two-photon excitation fluorescence images were obtained, through respective implemented imaging channels, to demonstrate imaging feasibility for near-UV, visible, near-IR continuous light, and pulsed light in the scanning optics. The imaging performances for spatial resolution and image contrast were verified experimentally; the results were satisfactory in comparison with theoretical results. The advantages of customization, containing low cost, outstanding combining ability and diverse applications, will contribute to vitalize multimodal imaging researches.

Spatial and temporal patterns of solar-induced chlorophyll fluorescence from a Finnish boreal landscape: Comparisons from the ground up to space

NASA Astrophysics Data System (ADS)

Drolet, G.; Nichol, C. J.; Wade, T. J.; Porcar-Castell, A.; Nikinmaa, E.; Middleton, E.; Ong, L.; Vesala, T.; Levula, J.; Moncrieff, J. B.

2010-12-01

Remote sensing of the solar-induced chlorophyll fluorescence (F) by vegetation has the potential to provide important information about carbon uptake dynamics in terrestrial ecosystems. Because of the strong physiological link between F and the photosynthetic status, accurate and timely estimates of F over large areas could significantly improve the understanding and predictions of how terrestrial ecosystems respond to climate change. In the past few decades, a number of different techniques and models aimed at retrieving F from remotely sensed measurements of vegetation reflectance were developed and in this study, we took advantage of these new developments to look at the spatial and temporal patterns of F in boreal coniferous forests. The results we present here are part of a larger research project aimed at improving reflectance-based estimates of photosynthesis efficiency and carbon uptake using space-based observations of boreal vegetation. During the summer of 2010, we continuously measured Scots pine (Pinus sylvestris) canopy reflectance using a tower-based spectrometer system (USB-2000+, Ocean Optics, USA) and leaf-level fluorescence using an automated multi channel chlorophyll fluorescence system (MONI-PAM, Heinz Walz GmbH, Germany). These measurements allowed studying the temporal dynamics of canopy-level F and testing methods for extracting F from canopy reflectance. During an intensive airborne campaign in July 2010, we used the University of Edinburgh’s research aircraft equipped with a dual field-of-view spectrometer system (FieldSpec Pro, Analytical Spectral Devices, USA) to repeatedly measure vegetation hyperspectral reflectance over a large area of boreal forest which encompassed the forest canopy sampled by the tower-based system. Airborne- and tower-based estimates of F where correlated to enable studying the spatial and temporal patterns of chlorophyll fluorescence and photosynthetic status over a larger extent of this boreal landscape in Finland. During the airborne campaign, EO-1 Hyperion satellites images encompassing the study region were acquired near-concomitantly with the airborne transects. These satellite images were used, along with the airborne measurements, to study the effect of increasing spatial scale on retrieving F. We further used the airborne- and satellite-based retrievals of F to look at the impact of a 76-year old record heat wave which occurred during the airborne campaign, on the photosynthetic status of boreal coniferous ecosystems over that region.

Quantitative Whole Body Biodistribution of Fluorescent-Labeled Agents by Non-Invasive Tomographic Imaging

PubMed Central

Vasquez, Kristine O.; Casavant, Chelsea; Peterson, Jeffrey D.

2011-01-01

When small molecules or proteins are injected into live animals, their physical and chemical properties will significantly affect pharmacokinetics, tissue penetration, and the ultimate routes of metabolism and clearance. Fluorescence molecular tomography (FMT) offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the major organ systems of living animals, in a manner analogous to traditional approaches with radiolabeled agents. This approach is best used with biotherapeutics (therapeutic antibodies, or other large proteins) or large-scaffold drug-delivery vectors, that are minimally affected by low-level fluorophore conjugation. Application to small molecule drugs should take into account the significant impact of fluorophore labeling on size and physicochemical properties, however, the presents studies show that this technique is readily applied to small molecule agents developed for far-red (FR) or near infrared (NIR) imaging. Quantification by non-invasive FMT correlated well with both fluorescence from tissue homogenates as well as with planar (2D) fluorescence reflectance imaging of excised intact organs (r2 = 0.996 and 0.969, respectively). Dynamic FMT imaging (multiple times from 0 to 24 h) performed in live mice after the injection of four different FR/NIR-labeled agents, including immunoglobulin, 20–50 nm nanoparticles, a large vascular imaging agent, and a small molecule integrin antagonist, showed clear differences in the percentage of injected dose per gram of tissue (%ID/g) in liver, kidney, and bladder signal. Nanoparticles and IgG1 favored liver over kidney signal, the small molecule integrin-binding agent favored rapid kidney and bladder clearance, and the vascular agent, showed both liver and kidney clearance. Further assessment of the volume of distribution of these agents by fluorescent volume added information regarding their biodistribution and highlighted the relatively poor extravasation into tissue by IgG1. These studies demonstrate the ability of quantitative FMT imaging of FR/NIR agents to non-invasively visualize and quantify the biodistribution of different agents over time. PMID:21731618

Indocyanine green fluorescence angiography for quantitative evaluation of in situ parathyroid gland perfusion and function after total thyroidectomy.

PubMed

Lang, Brian Hung-Hin; Wong, Carlos K H; Hung, Hing Tsun; Wong, Kai Pun; Mak, Ka Lun; Au, Kin Bun

2017-01-01

Because the fluorescent light intensity on an indocyanine green fluorescence angiography reflects the blood perfusion within a focused area, the fluorescent light intensity in the remaining in situ parathyroid glands may predict postoperative hypocalcemia risk after total thyroidectomy. Seventy patients underwent intraoperative indocyanine green fluorescence angiography after total thyroidectomy. Any parathyroid glands with a vascular pedicle was left in situ while any parathyroid glands without pedicle or inadvertently removed was autotransplanted. After total thyroidectomy, an intravenous 2.5 mg indocyanine green fluorescence angiography was given and real-time fluorescent images of the thyroid bed were recorded using the SPY imaging system (Novadaq, Ontario, Canada). The fluorescent light intensity of each indocyanine green fluorescence angiography as well as the average and greatest fluorescent light intensity in each patient were calculated. Postoperative hypocalcemia was defined as adjusted calcium <2.00 mmol/L within 24 hours. The fluorescent light intensity between discolored and normal-looking indocyanine green fluorescence angiographies was similar (P = .479). No patients with a greatest fluorescent light intensity >150% developed postoperative hypocalcemia while 9 (81.8%) patients with a greatest fluorescent light intensity ≤150% did. Similarly, no patients with an average fluorescent light intensity >109% developed PH while 9 (30%) with an average fluorescent light intensity ≤109% did. The greatest fluorescent light intensity was more predictive than day-0 postoperative hypocalcemia (P = .027) and % PTH drop day-0 to 1 (P < .001). Indocyanine green fluorescence angiography is a promising operative adjunct in determining residual parathyroid glands function and predicting postoperative hypocalcemia risk after total thyroidectomy. Copyright © 2016 Elsevier Inc. All rights reserved.

Low-dose intrathecal fluorescein for diagnosis of cerebrospinal fluid rhinorrhea using the scanning fiber endoscope in the human nasal cavities

NASA Astrophysics Data System (ADS)

Hou, Vivian W.; Davis, Calvin G.; Davis, Greg E.; Seibel, Eric J.

2016-03-01

Intrathecal fluorescein (ITF) enhances detection of cerebrospinal fluid rhinorrhea (CSFR). Clinically administered doses fall in the range of 0.1ml to 0.5ml of 5% to 10% fluorescein (1.3×10-3M to 1.3×10-2M). Though uncommon, significant morbidities associated with high doses of fluorescein have been reported. High concentrations are necessary for white light visual assessment; in contrast, fluorescent imaging enhances signal contrast and requires lower ITF concentrations for visualization. The ultrathin and flexible, multimodal scanning fiber endoscope (SFE) can visualize nanomolar concentrations of fluorescein as pseudocolor over reflectance, video-rate imaging. The application of the SFE for CSFR detection was assessed in a cadaver study. Briefly, 10μM (1×10-5M) fluorescein, 100X-1000X less than the standard clinical dose, was injected intra-cranially into the epidural space through an orbital roof puncture. The resulting rhinorrhea was assessed with a conventional, rigid ENT scope and second with the SFE in both video reflectance and multimodal fluorescent imaging modes. Neither system could visualize the 10μM ITF during white light imaging however the nanomolar sensitive SFE visualized the rhinorrhea during fluorescent imaging. Despite the low concentration used, a target-to-background ratio of 5.6 +/- 2.7 was achieved. To demonstrate SFE guidance of CSFR detection and repair, de-identified patient computed tomography (CT) scans were used to generate 3D printed phantoms. Cases were selected for unique anatomical features and overall clinical difficulty as determined by an experienced ENT clinician (GED). The sensitivity and minimally invasive nature of the SFE provide a unique platform for enhancing diagnosis and monitoring interventions in surgical endoscopic approaches into the sinuses.

Classification of corn kernels contaminated with aflatoxins using fluorescence and reflectance hyperspectral image analysis

USDA-ARS?s Scientific Manuscript database

Aflatoxins are secondary metabolites produced by certain fungal species of the Aspergillus genus. Aflatoxin contamination remains a problem in agricultural products due to its toxic and carcinogenic properties. Conventional chemical methods for aflatoxin detection are time-consuming and destructive....

A targeted molecular probe for colorectal cancer imaging

NASA Astrophysics Data System (ADS)

Attramadal, T.; Bjerke, R.; Indrevoll, B.; Moestue, S.; Rogstad, A.; Bendiksen, R.; Healey, A.; Johannesen, E.

2008-02-01

Colorectal cancer is a major cause of cancer death. Morbidity, mortality and healthcare costs can be reduced if the disease can be detected at an early stage. Screening is a viable approach as there is a clear link to risk factors such as age. We have developed a fluorescent contrast agent for use during colonoscopy. The agent is administered intravenously and is targeted to an early stage molecular marker for colorectal cancer. The agent consists of a targeting section comprising a peptide, and a fluorescent reporter molecule. Clinical imaging of the agent is to be performed with a far red fluorescence imaging channel (635 nm excitation/660-700 nm emission) as an adjunct to white light colonoscopy. Preclinical proof of mechanism results are presented. The compound has a K d of ~3nM. Two human xenograft tumour models were used. Tumour cells were implanted and grown subcutaneously in nude mice. Imaging using a fluorescence reflectance imaging system and quantitative biodistribution studies were performed. Substances tested include the targeted agent, and a scrambled sequence of the peptide (no binding) used as a negative control. Competition studies were also performed by co-administration of 180 times excess unlabelled peptide. Positive imaging contrast was shown in the tumours, with a clear relationship to expression levels (confirmed with quantitative biodistribution data). There was a significant difference between the positive and negative control substances, and a significant reduction in contrast in the competition experiment.

Three-dimensional image formation in fiber-optical second-harmonic-generation microscopy.

PubMed

Gu, Min; Fu, Ling

2006-02-06

Three-dimensional (3-D) image formation in fiber-optical second-harmonic-generation microscopy is revealed to be purely coherent and therefore can be described by a 3-D coherent transfer function (CTF) that exhibits the same spatial frequency passband as that of fiber-optical reflection-mode non-fluorescence microscopy. When the numerical aperture of the fiber is much larger than the angle of convergence of the illumination on the fiber aperture, the performance of fiber-optical second-harmonic-generation microscopy behaves as confocal second-harmonic-generation microscopy. The dependence of axial resolution on fiber coupling parameters shows an improvement of approximately 7%, compared with that in fiber-optical two-photon fluorescence microscopy.

Simultaneous multicolor detection system of the single-molecular microbial antigen by total internal reflection fluorescence microscopy with fluorescent nanocrystal quantum dots

NASA Astrophysics Data System (ADS)

Hoshino, Akiyoshi; Fujioka, Kouki; Yamamoto, Mayu; Manabe, Noriyoshi; Yasuhara, Masato; Suzuki, Kazuo; Yamamoto, Kenji

2005-11-01

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystals QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

ELKS, a Protein Structurally Related to the Active Zone-associated Protein CAST, Is Expressed in Pancreatic β Cells and Functions in Insulin Exocytosis: Interaction of ELKS with Exocytotic Machinery Analyzed by Total Internal Reflection Fluorescence MicroscopyV⃞

PubMed Central

Ohara-Imaizumi, Mica; Ohtsuka, Toshihisa; Matsushima, Satsuki; Akimoto, Yoshihiro; Nishiwaki, Chiyono; Nakamichi, Yoko; Kikuta, Toshiteru; Nagai, Shintaro; Kawakami, Hayato; Watanabe, Takashi; Nagamatsu, Shinya

2005-01-01

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic β cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic β cells. PMID:15888548

FLORIS: phase A status of the fluorescence imaging spectrometer of the Earth Explorer mission candidate FLEX

NASA Astrophysics Data System (ADS)

Kraft, S.; Bézy, J.-L.; Del Bello, U.; Berlich, R.; Drusch, M.; Franco, R.; Gabriele, A.; Harnisch, B.; Meynart, R.; Silvestrin, P.

2013-10-01

The Fluorescence Explorer (FLEX) mission is currently subject to feasibility (Phase A) study as one of the two candidates of ESA's 8th Earth Explorer opportunity mission. The FLuORescence Imaging Spectrometer (FLORIS) will be an imaging grating spectrometer onboard of a medium sized satellite flying in tandem with Sentinel-3 in a Sun synchronous orbit at a height of about 815 km. FLORIS will observe vegetation fluorescence and reflectance within a spectral range between 500 nm and 780 nm. It will thereby cover the photochemical reflection features between 500 nm and 600 nm, the Chlorophyll absorption band between 600 and 677 nm, and the red-edge in the region from 697 nm to 755 nm being located between the Oxygen A and B absorption bands. By this measurement approach, it is expected that the full spectrum and amount of the vegetation fluorescence radiance can be retrieved, and that atmospheric corrections can efficiently be applied. FLORIS will measure Earth reflected spectral radiance at a relatively high spectral resolution of ~0.3 nm around the Oxygen absorption bands. Other spectral band areas with less pronounced absorption features will be measured at medium spectral resolution between 0.5 and 2 nm. FLORIS will provide imagery at 300 m resolution on ground with a swath width of 150 km. This will allow achieving global revisit times of less than one month so as to monitor seasonal variations of the vegetation cycles. The mission life time is expected to be at least 4 years. The fluorescence retrieval will make use of information coming from OLCI and SLSTR, which are onboard of Sentinel-3, to monitor temperature, to detect thin clouds and to derive vegetation reflectance and information on the aerosol content also outside the FLORIS spectral range. In order to mitigate the technological and programmatic risk of this Explorer mission candidate, ESA has initiated two comprehensive bread-boarding activities, in which the most critical technologies and instrument performance shall be investigated and demonstrated. The breadboards will include representative optics and dispersive elements in a configuration, which is expected to be very close to the instrument flight configuration. This approach follows the guideline to reach, before it goes into the implementation phase, a technology readiness level of at least 5. It thereby requires a demonstration of predicted performance in a configuration, where the basic technological components are integrated with reasonably realistic supporting elements such that it can be tested in a simulated environment. We will report, within the limits of the competitive nature of the industrial studies, on the currently running or planned preparatory activities. We will present the mission configuration, the imposed instrument requirements and the identified instrument concepts as derived by the Phase A studies.

Quantitative Light-induced Fluorescence-Digital as an oral hygiene evaluation tool to assess plaque accumulation and enamel demineralization in orthodontics.

PubMed

Miller, Cara C; Burnside, Girvan; Higham, Susan M; Flannigan, Norah L

2016-11-01

  To assess the use of Quantitative Light-induced Fluorescence-Digital as an oral hygiene evaluation tool during orthodontic treatment.   In this prospective, randomized clinical trial, 33 patients undergoing fixed orthodontic appliance treatment were randomly allocated to receive oral hygiene reinforcement at four consecutive appointments using either white light (WL) or Quantitative Light-induced Fluorescence-Digital (QLF) images, taken with a device, as visual aids. Oral hygiene was recorded assessing the QLF images for demineralization, by fluorescence loss (ΔF), and plaque coverage (ΔR30). A debriefing questionnaire ascertained patient perspectives.   There were no significant differences in demineralization (P  =  .56) or plaque accumulation (P  =  .82) between the WL and QLF groups from T0 to T4. There was no significant reduction in demineralization, ΔF, in the WL, or the QLF group from T0-T4 (P > .05); however, there was a significant reduction in ΔR30 plaque scores (P < .05). All the participants found being shown the images helpful, with 100% of the QLF group reflecting that it would be useful to have oral hygiene reinforcement for the full duration of treatment compared with 81% of the WL group (OR 2.3; P < .05).   Quantitative Light-induced Fluorescence-Digital can be used to detect and monitor demineralization and plaque during orthodontics. Oral hygiene reinforcement at consecutive appointments using WL or QLF images as visual aids is effective in reducing plaque coverage. In terms of clinical benefits, QLF and WL images are of similar effectiveness; however, patients preferred the QLF images.

Fully integrated high-speed intravascular optical coherence tomography/near-infrared fluorescence structural/molecular imaging in vivo using a clinically available near-infrared fluorescence-emitting indocyanine green to detect inflamed lipid-rich atheromata in coronary-sized vessels.

PubMed

Lee, Sunki; Lee, Min Woo; Cho, Han Saem; Song, Joon Woo; Nam, Hyeong Soo; Oh, Dong Joo; Park, Kyeongsoon; Oh, Wang-Yuhl; Yoo, Hongki; Kim, Jin Won

2014-08-01

Lipid-rich inflamed coronary plaques are prone to rupture. The purpose of this study was to assess lipid-rich inflamed plaques in vivo using fully integrated high-speed optical coherence tomography (OCT)/near-infrared fluorescence (NIRF) molecular imaging with a Food and Drug Administration-approved indocyanine green (ICG). An integrated high-speed intravascular OCT/NIRF imaging catheter and a dual-modal OCT/NIRF system were constructed based on a clinical OCT platform. For imaging lipid-rich inflamed plaques, the Food and Drug Administration-approved NIRF-emitting ICG (2.25 mg/kg) or saline was injected intravenously into rabbit models with experimental atheromata induced by balloon injury and 12- to 14-week high-cholesterol diets. Twenty minutes after injection, in vivo OCT/NIRF imaging of the infrarenal aorta and iliac arteries was acquired only under contrast flushing through catheter (pullback speed up to ≤20 mm/s). NIRF signals were strongly detected in the OCT-visualized atheromata of the ICG-injected rabbits. The in vivo NIRF target-to-background ratio was significantly larger in the ICG-injected rabbits than in the saline-injected controls (P<0.01). Ex vivo peak plaque target-to-background ratios were significantly higher in ICG-injected rabbits than in controls (P<0.01) on fluorescence reflectance imaging, which correlated well with the in vivo target-to-background ratios (P<0.01; r=0.85) without significant bias (0.41). Cellular ICG uptake, correlative fluorescence microscopy, and histopathology also corroborated the in vivo imaging findings. Integrated OCT/NIRF structural/molecular imaging with a Food and Drug Administration -approved ICG accurately identified lipid-rich inflamed atheromata in coronary-sized vessels. This highly translatable dual-modal imaging approach could enhance our capabilities to detect high-risk coronary plaques. © 2014 American Heart Association, Inc.

Widefield and total internal reflection fluorescent structured illumination microscopy with scanning galvo mirrors

NASA Astrophysics Data System (ADS)

Chen, Youhua; Cao, Ruizhi; Liu, Wenjie; Zhu, Dazhao; Zhang, Zhiming; Kuang, Cuifang; Liu, Xu

2018-04-01

We present an alternative approach to realize structured illumination microscopy (SIM), which is capable for live cell imaging. The prototype utilizes two sets of scanning galvo mirrors, a polarization converter and a piezo-platform to generate a fast shifted, s-polarization interfered and periodic variable illumination patterns. By changing the angle of the scanning galvanometer, we can change the position of the spots at the pupil plane of the objective lens arbitrarily, making it easy to switch between widefield and total internal reflection fluorescent-SIM mode and adapting the penetration depth in the sample. Also, a twofold resolution improvement is achieved in our experiments. The prototype offers more flexibility of pattern period and illumination orientation changing than previous systems.

Diffusion of photoacid generators by laser scanning confocal microscopy

NASA Astrophysics Data System (ADS)

Zhang, Ping L.; Webber, Stephen E.; Mendenhall, J.; Byers, Jeffrey D.; Chao, Keith K.

1998-06-01

Diffusion of the photogenerated acid during the period of time between exposure and development can cause contrast loss and ultimately loss of the latent image. This is especially relevant for chemically amplified photoresists that require a post-exposure baking step, which in turn facilitates acid diffusion due to the high temperature normally employed. It is thus important to develop techniques with good spatial resolution to monitor the photogeneration of acid. More precisely, we need techniques that provide two distinct types of information: spatial resolution on various length scales within the surface layer and also sufficient depth resolution so that one can observe the transition from very surface layer to bulk structure in the polymer blend coated on silicon substrate. Herein laser scanning confocal microscopy is used to evaluate the resist for the first time. We report the use of the confocal microscopy to map the pag/dye distribution in PHS matrices, with both reflectance images and fluorescence images. A laser beam is focused onto a small 3D volume element, termed a voxel. It is typically 200 nm X 200 nm laterally and 800 nm axially. The illuminated voxel is viewed such that only signals emanating from this voxel are detected, i.e., signal from outside the probed voxel is not detected. By adjusting the vertical position of the laser focal point, the voxel can be moved to the designated lateral plane to produce an image. Contrast caused by topology difference between the exposed and unexposed area can be eliminated. Bis-p-butylphenyl iodonium triflat (7% of polyhydroxystyrene) is used as photoacid generators. 5% - 18% (by weight, PHS Mn equals 13 k) resist in PGMEA solution is spin cast onto the treated quartz disk with thickness of 1.4 micrometers , 5 micrometers space/10 micrometers pitch chrome mask is used to generate the pattern with mercury DUV illumination. Fluoresceinamine, the pH-sensitive dye, is also used to enhance the contrast of fluorescence image. The typical PEB temperature is 90 degree(s)C for 90 seconds. 488 nm is used as the excitation wavelength. Both reflectance and fluorescence images (> 510 nm) are processed by using Adobe Photoshop. It was found that the reflectance is more sensitive to the change of the refractive index of the resist while the fluorescence is more sensitive to the distribution of the PAG/dye. The NIH Image software is used for acid exchange rate calculation. Second Fick's Law is applied to analyze the image change. The diffusion coefficient for this PAG in PHS during PEB is smaller than 8.8 X 10-13 cm2/s.

Macro-/micro-environment-sensitive chemosensing and biological imaging.

PubMed

Yang, Zhigang; Cao, Jianfang; He, Yanxia; Yang, Jung Ho; Kim, Taeyoung; Peng, Xiaojun; Kim, Jong Seung

2014-07-07

Environment-related parameters, including viscosity, polarity, temperature, hypoxia, and pH, play pivotal roles in controlling the physical or chemical behaviors of local molecules. In particular, in a biological environment, such factors predominantly determine the biological properties of the local environment or reflect corresponding status alterations. Abnormal changes in these factors would cause cellular malfunction or become a hallmark of the occurrence of severe diseases. Therefore, in recent years, they have increasingly attracted research interest from the fields of chemistry and biological chemistry. With the emergence of fluorescence sensing and imaging technology, several fluorescent chemosensors have been designed to respond to such parameters and to further map their distributions and variations in vitro/in vivo. In this work, we have reviewed a number of various environment-responsive chemosensors related to fluorescent recognition of viscosity, polarity, temperature, hypoxia, and pH that have been reported thus far.

Hybrid reflecting objectives for functional multiphoton microscopy in turbid media

PubMed Central

Vučinić, Dejan; Bartol, Thomas M.; Sejnowski, Terrence J.

2010-01-01

Most multiphoton imaging of biological specimens is performed using microscope objectives optimized for high image quality under wide-field illumination. We present a class of objectives designed de novo without regard for these traditional constraints, driven exclusively by the needs of fast multiphoton imaging in turbid media: the delivery of femtosecond pulses without dispersion and the efficient collection of fluorescence. We model the performance of one such design optimized for a typical brain-imaging setup and show that it can greatly outperform objectives commonly used for this task. PMID:16880851

In vivo cellular imaging with microscopes enabled by MEMS scanners

NASA Astrophysics Data System (ADS)

Ra, Hyejun

High-resolution optical imaging plays an important role in medical diagnosis and biomedical research. Confocal microscopy is a widely used imaging method for obtaining cellular and sub-cellular images of biological tissue in reflectance and fluorescence modes. Its characteristic optical sectioning capability also enables three-dimensional (3-D) image reconstruction. However, its use has mostly been limited to excised tissues due to the requirement of high numerical aperture (NA) lenses for cellular resolution. Microscope miniaturization can enable in vivo imaging to make possible early cancer diagnosis and biological studies in the innate environment. In this dissertation, microscope miniaturization for in vivo cellular imaging is presented. The dual-axes confocal (DAC) architecture overcomes limitations of the conventional single-axis confocal (SAC) architecture to allow for miniaturization with high resolution. A microelectromechanical systems (MEMS) scanner is the central imaging component that is key in miniaturization of the DAC architecture. The design, fabrication, and characterization of the two-dimensional (2-D) MEMS scanner are presented. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer and is actuated by self-aligned vertical electrostatic combdrives. The imaging performance of the MEMS scanner in a DAC configuration is shown in a breadboard microscope setup, where reflectance and fluorescence imaging is demonstrated. Then, the MEMS scanner is integrated into a miniature DAC microscope. The whole imaging system is integrated into a portable unit for research in small animal models of human biology and disease. In vivo 3-D imaging is demonstrated on mouse skin models showing gene transfer and siRNA silencing. The siRNA silencing process is sequentially imaged in one mouse over time.

Single-molecule imaging of cytoplasmic dynein in vivo.

PubMed

Ananthanarayanan, Vaishnavi; Tolić, Iva M

2015-01-01

While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior. Copyright © 2015 Elsevier Inc. All rights reserved.

Brightness-compensated 3-D optical flow algorithm for monitoring cochlear motion patterns

NASA Astrophysics Data System (ADS)

von Tiedemann, Miriam; Fridberger, Anders; Ulfendahl, Mats; de Monvel, Jacques Boutet

2010-09-01

A method for three-dimensional motion analysis designed for live cell imaging by fluorescence confocal microscopy is described. The approach is based on optical flow computation and takes into account brightness variations in the image scene that are not due to motion, such as photobleaching or fluorescence variations that may reflect changes in cellular physiology. The 3-D optical flow algorithm allowed almost perfect motion estimation on noise-free artificial sequences, and performed with a relative error of <10% on noisy images typical of real experiments. The method was applied to a series of 3-D confocal image stacks from an in vitro preparation of the guinea pig cochlea. The complex motions caused by slow pressure changes in the cochlear compartments were quantified. At the surface of the hearing organ, the largest motion component was the transverse one (normal to the surface), but significant radial and longitudinal displacements were also present. The outer hair cell displayed larger radial motion at their basolateral membrane than at their apical surface. These movements reflect mechanical interactions between different cellular structures, which may be important for communicating sound-evoked vibrations to the sensory cells. A better understanding of these interactions is important for testing realistic models of cochlear mechanics.

Brightness-compensated 3-D optical flow algorithm for monitoring cochlear motion patterns.

PubMed

von Tiedemann, Miriam; Fridberger, Anders; Ulfendahl, Mats; de Monvel, Jacques Boutet

2010-01-01

A method for three-dimensional motion analysis designed for live cell imaging by fluorescence confocal microscopy is described. The approach is based on optical flow computation and takes into account brightness variations in the image scene that are not due to motion, such as photobleaching or fluorescence variations that may reflect changes in cellular physiology. The 3-D optical flow algorithm allowed almost perfect motion estimation on noise-free artificial sequences, and performed with a relative error of <10% on noisy images typical of real experiments. The method was applied to a series of 3-D confocal image stacks from an in vitro preparation of the guinea pig cochlea. The complex motions caused by slow pressure changes in the cochlear compartments were quantified. At the surface of the hearing organ, the largest motion component was the transverse one (normal to the surface), but significant radial and longitudinal displacements were also present. The outer hair cell displayed larger radial motion at their basolateral membrane than at their apical surface. These movements reflect mechanical interactions between different cellular structures, which may be important for communicating sound-evoked vibrations to the sensory cells. A better understanding of these interactions is important for testing realistic models of cochlear mechanics.

Chlorophyll fluorescence lifetime imaging provides new insight into the chlorosis induced by plant virus infection.

PubMed

Lei, Rong; Jiang, Hongshan; Hu, Fan; Yan, Jin; Zhu, Shuifang

2017-02-01

Leaf chlorosis induced by plant virus infection has a short fluorescence lifetime, which reflects damaged photosynthetic complexes and degraded chloroplasts. Plant viruses often induce chlorosis and necrosis, which are intimately related to photosynthetic functions. Chlorophyll fluorescence lifetime measurement is a valuable noninvasive tool for analyzing photosynthetic processes and is a sensitive indicator of the environment surrounding the fluorescent molecules. In this study, our central goal was to explore the effect of viral infection on photosynthesis by employing chlorophyll fluorescence lifetime imaging (FLIM), steady-state fluorescence, non-photochemical quenching (NPQ), transmission electron microscopy (TEM), and pigment analysis. The data indicated that the chlorophyll fluorescence lifetime of chlorotic leaves was significantly shorter than that of healthy control leaves, and the fitted short lifetime component of chlorophyll fluorescence of chlorotic leaves was dominant. This dominant short lifetime component may result from damage to the structure of thylakoid, which was confirmed by TEM. The NPQ value of chlorotic leaves was slightly higher than that of healthy green leaves, which can be explained by increased neoxanthin, lutein and violaxanthin content relative to chlorophyll a. The difference in NPQ is slight, but FLIM can provide simple and direct characterization of PSII structure and photosynthetic function. Therefore, this technique shows great potential as a simple and rapid method for studying mechanisms of plant virus infection.

Simultaneous Visualization of Hydrogen Peroxide and Water Concentrations Using Photofragmentation Laser-Induced Fluorescence.

PubMed

Larsson, Kajsa; Aldén, Marcus; Bood, Joakim

2017-09-01

A concept based on photofragmentation laser-induced fluorescence (PFLIF) is for the first time demonstrated for simultaneous detection of hydrogen peroxide (H 2 O 2 ) and water (H 2 O) vapor in various mixtures containing the two constituents in a bath of argon gas. A photolysis laser pulse at 248 nm dissociates H 2 O 2 into OH fragments, whereupon a probe pulse, delayed 100 ns and tuned to an absorption line in the A 2 Σ + (v = 1) ← X 2 Π(v = 0) band of OH near 282 nm, induces fluorescence. The total OH fluorescence reflects the H 2 O 2 concentration, while its spectral shape is utilized to determine the H 2 O concentration via a model predicting the ratio between the fluorescence intensities of the A 2 Σ + (v = 1) → X 2 Π(v = 1) and the A 2 Σ + (v = 0) → X 2 Π(v = 0) bands. The H 2 O detection scheme requires that the bath gas has a collisional cross-section with OH(A) that is significantly lower than that of H 2 O, which is the case for argon. Spectrally dispersed OH fluorescence spectra were recorded for five different H 2 O 2 /H 2 O/Ar mixtures; the H 2 O 2 concentration in the range of 30-500 ppm and the H 2 O concentration in the range of 0-3%. Fluorescence intensity ratios predicted by the model for these mixtures agree very well with corresponding experimental data, which thus validates the model. The concept was also demonstrated for two-dimensional imaging, using two intensified charge-coupled device (CCD) cameras for signal detection. Water content was here sensed through the different temporal characteristics of the two fluorescence bands by triggering the two cameras so that one captures the total OH fluorescence while the other one captures only the early part, which mainly stems from A 2 Σ + (v = 1) → X 2 Π(v = 1) fluorescence. Hence, the H 2 O 2 concentration is reflected by the image of the camera recording the total OH fluorescence, whereas H 2 O concentration is extracted from the ratio between the two camera images. Quantification of the concentrations was carried out based on calibration measurements performed in known mixtures of H 2 O 2 (30-500 ppm) and H 2 O (0-3%) in bulk argon. The detection limits for single-shot imaging are estimated to be 20 ppm for H 2 O 2 and 0.05% for H 2 O. The authors believe that the concept provides a valuable asset in, for example, pharmaceutical or aseptic food packaging applications, where H 2 O 2 /H 2 O vapor is routinely used for sterilization.

Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

PubMed

Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2015-03-01

High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Indocyanine Green Enables Near-Infrared Fluorescence Imaging of Lipid-Rich, Inflamed Atherosclerotic Plaques

PubMed Central

Vinegoni, Claudio; Botnaru, Ion; Aikawa, Elena; Calfon, Marcella A.; Iwamoto, Yoshiko; Folco, Eduardo J.; Ntziachristos, Vasilis; Weissleder, Ralph; Libby, Peter; Jaffer, Farouc A.

2011-01-01

New high-resolution molecular and structural imaging strategies are needed to visualize high-risk plaques that are likely to cause acute myocardial infarction, because current diagnostic methods do not reliably identify at-risk subjects. While molecular imaging agents are available for lower-resolution detection of atherosclerosis in large arteries, a lack of imaging agents coupled to high-resolution modalities has limited molecular imaging of atherosclerosis in the smaller coronary arteries [AU: ok? YES]. Here, we have demonstrated that indocyanine green (ICG), an FDA-approved near-infrared fluorescence (NIRF) emitting compound, targets atheromas within 20 minutes of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. In vivo NIRF sensing was achieved with an intravascular wire in the aortae, a vessel of comparable caliber to human coronary arteries. Ex vivo fluorescence reflectance imaging studies showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG, compared to atheroma-bearing rabbits injected with saline. In vitro studies using human macrophages established that ICG preferentially targets lipid-loaded macrophages. In an early clinical study of human atheroma specimens from four patients, we found that ICG colocalized with plaque macrophages and lipids. The atheroma-targeting capability of ICG has the potential to accelerate the clinical development of NIRF molecular imaging of high-risk plaques in humans. PMID:21613624

High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging and reflectance confocal microscopy.

PubMed

Manfredini, Marco; Arginelli, Federica; Dunsby, Christopher; French, Paul; Talbot, Clifford; König, Karsten; Pellacani, Giovanni; Ponti, Giovanni; Seidenari, Stefania

2013-02-01

The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher's exact test and Spearman's rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPT-FLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPT-FLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). According to our data, both methods are suitable to image BCC's features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view. © 2012 John Wiley & Sons A/S.

Trimodal color-fluorescence-polarization endoscopy aided by a tumor selective molecular probe accurately detects flat lesions in colitis-associated cancer

NASA Astrophysics Data System (ADS)

Charanya, Tauseef; York, Timothy; Bloch, Sharon; Sudlow, Gail; Liang, Kexian; Garcia, Missael; Akers, Walter J.; Rubin, Deborah; Gruev, Viktor; Achilefu, Samuel

2014-12-01

Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real-time detection of these lesions. Using azoxymethane-dextran sodium sulfate (AOM-DSS) treated mice, which recapitulates human CAC and dysplasia, we show that an octapeptide labeled with a near-infrared (NIR) fluorescent dye selectively identified all precancerous and cancerous lesions. A new thermoresponsive sol-gel formulation allowed topical application of the molecular probe during endoscopy. This method yielded high contrast-to-noise ratios (CNR) between adenomatous tumors (20.6±1.65) and flat lesions (12.1±1.03) and surrounding uninvolved colon tissue versus CNR of inflamed tissues (1.62±0.41). Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy shows a high depolarization contrast in both adenomatous tumors and flat lesions in CAC, reflecting compromised structural integrity of these tissues. Together, the real-time polarization imaging provides real-time validation of suspicious colon tissue highlighted by molecular fluorescence endoscopy.

Recovering fluorophore concentration profiles from confocal images near lateral refractive index step changes.

PubMed

Jonášová, Eleonóra Parelius; Bjørkøy, Astrid; Stokke, Bjørn Torger

2016-12-01

Optical aberrations due to refractive index mismatches occur in various types of microscopy due to refractive differences between the sample and the immersion fluid or within the sample. We study the effects of lateral refractive index differences by fluorescence confocal laser scanning microscopy due to glass or polydimethylsiloxane cuboids and glass cylinders immersed in aqueous fluorescent solution, thereby mimicking realistic imaging situations in the proximity of these materials. The reduction in fluorescence intensity near the embedded objects was found to depend on the geometry and the refractive index difference between the object and the surrounding solution. The observed fluorescence intensity gradients do not reflect the fluorophore concentration in the solution. It is suggested to apply a Gaussian fit or smoothing to the observed fluorescence intensity gradient and use this as a basis to recover the fluorophore concentration in the proximity of the refractive index step change. The method requires that the reference and sample objects have the same geometry and refractive index. The best results were obtained when the sample objects were also used for reference since small differences such as uneven surfaces will result in a different extent of aberration.

Multimodal coherent anti-Stokes Raman scattering microscopy reveals microglia-associated myelin and axonal dysfunction in multiple sclerosis-like lesions in mice

PubMed Central

Imitola, Jaime; Côté, Daniel; Rasmussen, Stine; Xie, X. Sunney; Liu, Yingru; Chitnis, Tanuja; Sidman, Richard L.; Lin, Charles. P.; Khoury, Samia J.

2011-01-01

Myelin loss and axonal degeneration predominate in many neurological disorders; however, methods to visualize them simultaneously in live tissue are unavailable. We describe a new imaging strategy combining video rate reflectance and fluorescence confocal imaging with coherent anti-Stokes Raman scattering (CARS) microscopy tuned to CH2 vibration of myelin lipids, applied in live tissue of animals with chronic experimental autoimmune encephalomyelitis (EAE). Our method allows monitoring over time of demyelination and neurodegeneration in brain slices with high spatial resolution and signal-to-noise ratio. Local areas of severe loss of lipid signal indicative of demyelination and loss of the reflectance signal from axons were seen in the corpus callosum and spinal cord of EAE animals. Even in myelinated areas of EAE mice, the intensity of myelin lipid signals is significantly reduced. Using heterozygous knock-in mice in which green fluorescent protein replaces the CX3CR1 coding sequence that labels central nervous system microglia, we find areas of activated microglia colocalized with areas of altered reflectance and CARS signals reflecting axonal injury and demyelination. Our data demonstrate the use of multimodal CARS microscopy for characterization of demyelinating and neurodegenerative pathology in a mouse model of multiple sclerosis, and further confirm the critical role of microglia in chronic inflammatory neurodegeneration. PMID:21361672

History of Chandra X-Ray Observatory

NASA Image and Video Library

2001-01-10

This Chandra image, the first x-ray image ever made of Venus, shows a half crescent due to the relative orientation of the Sun, Earth, and Venus. The x-rays are produced by fluorescent radiation from oxygen and other atoms in the atmosphere between 120 and 140 kilometers above the surface of the planet. In contrast, the optical light from Venus is caused by the reflection from clouds 50 to 70 kilometers above the surface.

Reconstructed Solar-Induced Fluorescence: A Machine Learning Vegetation Product Based on MODIS Surface Reflectance to Reproduce GOME-2 Solar-Induced Fluorescence

NASA Astrophysics Data System (ADS)

Gentine, P.; Alemohammad, S. H.

2018-04-01

Solar-induced fluorescence (SIF) observations from space have resulted in major advancements in estimating gross primary productivity (GPP). However, current SIF observations remain spatially coarse, infrequent, and noisy. Here we develop a machine learning approach using surface reflectances from Moderate Resolution Imaging Spectroradiometer (MODIS) channels to reproduce SIF normalized by clear sky surface irradiance from the Global Ozone Monitoring Experiment-2 (GOME-2). The resulting product is a proxy for ecosystem photosynthetically active radiation absorbed by chlorophyll (fAPARCh). Multiplying this new product with a MODIS estimate of photosynthetically active radiation provides a new MODIS-only reconstruction of SIF called Reconstructed SIF (RSIF). RSIF exhibits much higher seasonal and interannual correlation than the original SIF when compared with eddy covariance estimates of GPP and two reference global GPP products, especially in dry and cold regions. RSIF also reproduces intense productivity regions such as the U.S. Corn Belt contrary to typical vegetation indices and similarly to SIF.

3D mesoscopic fluorescence tomography for imaging micro-distribution of antibody-photon absorber conjugates during near infrared photoimmunotherapy in vivo.

PubMed

Tang, Qinggong; Nagaya, Tadanobu; Liu, Yi; Horng, Hannah; Lin, Jonathan; Sato, Kazuhide; Kobayashi, Hisataka; Chen, Yu

2018-06-10

As a novel low-side-effect cancer therapy, photo-immunotherapy (PIT) is based on conjugating monoclonal antibody (mAb) with a near-infrared (NIR) phthalocyanine dye IRDye700DX (IR 700). IR700 is not only fluorescent to be used as an imaging agent, but also phototoxic. When illuminating with NIR light, PIT can induce highly-selective cancer cell death while leaving most of tumor blood vessels unharmed, leading to an effect termed super-enhanced permeability and retention (SUPR), which can significantly improve the effectiveness of anti-cancer drug. Currently, the therapeutic effects of PIT are monitored using 2D macroscopic fluorescence reflectance imager, which lacks the resolution and depth information to reveal the 3D distribution of mAb-IR700. In the study, we applied a multi-modal optical imaging approach including high-resolution optical coherence tomography (OCT) and high-sensitivity fluorescence laminar optical tomography (FLOT), to provide 3D tumor micro-structure and micro-distribution of mAb-IR700 in the tumor simultaneously during PIT in situ and in vivo. The multi-wavelength FLOT can also provide the blood vessels morphology of the tumor. Thus, the 3D FLOT reconstructed images allow us to evaluate the IR700 fluorescence distribution change with respect to the blood vessels and at different tumor locations/depths non-invasively, thereby enabling evaluation of the therapeutic effects in vivo and optimization of treatment regimens accordingly. The mAb-IR700 can access more tumor areas after PIT treatment, which can be explained by increased vascular permeability immediately after NIR-PIT. Two-photon microscopy was also used to record the mAb-IR700 on the tumor surface near the blood vessels to verify the results. Published by Elsevier B.V.

Automatic neutron dosimetry system based on fluorescent nuclear track detector technology.

PubMed

Akselrod, M S; Fomenko, V V; Bartz, J A; Haslett, T L

2014-10-01

For the first time, the authors are describing an automatic fluorescent nuclear track detector (FNTD) reader for neutron dosimetry. FNTD is a luminescent integrating type of detector made of aluminium oxide crystals that does not require electronics or batteries during irradiation. Non-destructive optical readout of the detector is performed using a confocal laser scanning fluorescence imaging with near-diffraction limited resolution. The fully automatic table-top reader allows one to load up to 216 detectors on a tray, read their engraved IDs using a CCD camera and optical character recognition, scan and process simultaneously two types of images in fluorescent and reflected laser light contrast to eliminate false-positive tracks related to surface and volume crystal imperfections. The FNTD dosimetry system allows one to measure neutron doses from 0.1 mSv to 20 Sv and covers neutron energies from thermal to 20 MeV. The reader is characterised by a robust, compact optical design, fast data processing electronics and user-friendly software. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Imaging the photoinduced charge injection in CdS/TiO2 nanoparticles by the sequential fluorescence mapping method

NASA Astrophysics Data System (ADS)

Frederice, Rafael; Lencione, Diego; Gehlen, Marcelo H.

2017-03-01

The combination of a sensitizer and TiO2 nanoparticles forming a photocatalytic material is a central issue in many fields of applied photochemistry. The charge injection of emissive sensitizers into the conduction band of the semiconductor TiO2 may form a photoactive region that becomes dark, or it has a very low emission signal due to the generation of sensitizer radicals. However, by sequential coupling of a selected photoredox dye, such as resazurin, the dark region may become fluorescent at the interfaces where the charge injection has taken place due to the concomitant formation of fluorescent resorufin by cascade electron transfer. Using this strategy and a total internal reflection fluorescence microscopy (TIRFM) image, the charge injection in TiO2/CdS and SiO2/TiO2/CdS nanoparticles is investigated The method allows the charge injection efficiency of the excited CdS into TiO2 to be evaluated qualitatively, explaining the differences observed for these photocatalytic materials in H2 generation.

Imaging the photoinduced charge injection in CdS/TiO2 nanoparticles by the sequential fluorescence mapping method.

PubMed

Frederice, Rafael; Lencione, Diego; Gehlen, Marcelo H

2017-02-10

The combination of a sensitizer and TiO 2 nanoparticles forming a photocatalytic material is a central issue in many fields of applied photochemistry. The charge injection of emissive sensitizers into the conduction band of the semiconductor TiO 2 may form a photoactive region that becomes dark, or it has a very low emission signal due to the generation of sensitizer radicals. However, by sequential coupling of a selected photoredox dye, such as resazurin, the dark region may become fluorescent at the interfaces where the charge injection has taken place due to the concomitant formation of fluorescent resorufin by cascade electron transfer. Using this strategy and a total internal reflection fluorescence microscopy (TIRFM) image, the charge injection in TiO 2 /CdS and SiO 2 /TiO 2 /CdS nanoparticles is investigated The method allows the charge injection efficiency of the excited CdS into TiO 2 to be evaluated qualitatively, explaining the differences observed for these photocatalytic materials in H 2 generation.

Sensitivity and accuracy of hybrid fluorescence-mediated tomography in deep tissue regions.

PubMed

Rosenhain, Stefanie; Al Rawashdeh, Wa'el; Kiessling, Fabian; Gremse, Felix

2017-09-01

Fluorescence-mediated tomography (FMT) enables noninvasive assessment of the three-dimensional distribution of near-infrared fluorescence in mice. The combination with micro-computed tomography (µCT) provides anatomical data, enabling improved fluorescence reconstruction and image analysis. The aim of our study was to assess sensitivity and accuracy of µCT-FMT under realistic in vivo conditions in deeply-seated regions. Accordingly, we acquired fluorescence reflectance images (FRI) and µCT-FMT scans of mice which were prepared with rectal insertions with different amounts of fluorescent dye. Default and high-sensitivity scans were acquired and background signal was analyzed for three FMT channels (670 nm, 745 nm, and 790 nm). Analysis was performed for the original and an improved FMT reconstruction using the µCT data. While FRI and the original FMT reconstruction could detect 100 pmol, the improved FMT reconstruction could detect 10 pmol and significantly improved signal localization. By using a finer sampling grid and increasing the exposure time, the sensitivity could be further improved to detect 0.5 pmol. Background signal was highest in the 670 nm channel and most prominent in the gastro-intestinal tract and in organs with high relative amounts of blood. In conclusion, we show that µCT-FMT allows sensitive and accurate assessment of fluorescence in deep tissue regions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gold nanorods for cell imaging with confocal reflectance microscopy and two-photon fluorescence microscopy

NASA Astrophysics Data System (ADS)

Chen, Ji-Yao; Wang, Pei-Nan

2010-02-01

Gold nanorods have unique optical properties as their two photon absorption cross sections are very high and their spectral positions of extinction bands can be controlled by their aspect ratio only, so that gold nanorods have been considered as agents for cell imaging. Two-photon photoluminescence imaging could be used to detect the cellular gold nanorods with the high power femto-second (fs) infrared laser, but may cause the photothermal effect melting the rods. The 3-D distribution of gold nanorods in living cells also can be measured by confocal reflectance microscopy with a very low laser power, and thus the cell damaging can be avoided. In this work, these two methods were comparatively studied in living rat basophilic leukemia (RBL-2H3) cells.

Two-photon microscopy of fungal keratitis-affected rabbit cornea ex vivo using moxifloxacin as a labeling agent.

PubMed

Lee, Jun Ho; Le, Viet-Hoan; Lee, Seunghun; Park, Jin Hyoung; Lee, Jin Ah; Tchah, Hungwon; Kim, Sungjee; Kim, Myoung Joon; Kim, Ki Hean

2018-05-19

Two-photon microscopy (TPM) is a three dimensional (3D) microscopic technique based on nonlinear two-photon fluorescence, which has been tested as an alternative to reflectance confocal microscopy (RCM) for detecting fungal keratitis via optical imaging. Although TPM provided images with better contrast than RCM for fungal keratitis, its imaging speed was relatively low because of weak intrinsic signal. Moxifloxacin, a Food and Drug Administration (FDA)-approved antibiotic, was recently used as a cell-labeling agent for TPM. In this study, moxifloxacin was used to label fungal cells for TPM imaging of fungal keratitis models. Fungal cell suspensions and ex vivo fungal keratitis-affected rabbit corneas were prepared using two types of fungal pathogens, Aspergillus fumigatus and Candida albicans, and TPM imaging was performed both with and without moxifloxacin treatment. Fungal cells with enhanced fluorescence were clearly visible by TPM of moxifloxacin-treated fungal cell suspensions. TPM of moxifloxacin-treated fungal keratitis rabbit corneas revealed both the infecting fungal cells and corneal cells similar to those observed in TPM without moxifloxacin treatment, albeit with approximately 10-times enhanced fluorescence. Fungal cells were distinguished from corneal cells on the basis of their distinct morphologies. Thus, TPM with moxifloxacin labeling might be useful for the detection of fungal keratitis at the improved imaging speed. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Wide-field and high-resolution optical imaging for early detection of oral neoplasia

NASA Astrophysics Data System (ADS)

Pierce, Mark C.; Schwarz, Richard A.; Rosbach, Kelsey; Roblyer, Darren; Muldoon, Tim; Williams, Michelle D.; El-Naggar, Adel K.; Gillenwater, Ann M.; Richards-Kortum, Rebecca

2010-02-01

Current procedures for oral cancer screening typically involve visual inspection of the entire tissue surface at risk under white light illumination. However, pre-cancerous lesions can be difficult to distinguish from many benign conditions when viewed under these conditions. We have developed wide-field (macroscopic) imaging system which additionally images in cross-polarized white light, narrowband reflectance, and fluorescence imaging modes to reduce specular glare, enhance vascular contrast, and detect disease-related alterations in tissue autofluorescence. We have also developed a portable system to enable high-resolution (microscopic) evaluation of cellular features within the oral mucosa in situ. This system is a wide-field epi-fluorescence microscope coupled to a 1 mm diameter, flexible fiber-optic imaging bundle. Proflavine solution was used to specifically label cell nuclei, enabling the characteristic differences in N/C ratio and nuclear distribution between normal, dysplastic, and cancerous oral mucosa to be quantified. This paper discusses the technical design and performance characteristics of these complementary imaging systems. We will also present data from ongoing clinical studies aimed at evaluating diagnostic performance of these systems for detection of oral neoplasia.

Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner

PubMed Central

Liu, Jonathan T. C.; Mandella, Michael J.; Ra, Hyejun; Wong, Larry K.; Solgaard, Olav; Kino, Gordon S.; Piyawattanametha, Wibool; Contag, Christopher H.; Wang, Thomas D.

2007-01-01

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5–7 μm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 × 100 μm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy. PMID:17215937

Study of noninvasive detection of latent fingerprints using UV laser

NASA Astrophysics Data System (ADS)

Li, Hong-xia; Cao, Jing; Niu, Jie-qing; Huang, Yun-gang; Mao, Lin-jie; Chen, Jing-rong

2011-06-01

Latent fingerprints present a considerable challenge in forensics, and noninvasive procedure that captures a digital image of the latent fingerprints is significant in the field of criminal investigation. The capability of photography technologies using 266nm UV Nd:YAG solid state laser as excitation light source to provide detailed images of unprocessed latent fingerprints is demonstrated. Unprocessed latent fingerprints were developed on various non-absorbent and absorbing substrates. According to the special absorption, reflection, scattering and fluorescence characterization of the various residues in fingerprints (fatty acid ester, protein, and carbosylic acid salts etc) to the UV light to weaken or eliminate the background disturbance and increase the brightness contrast of fingerprints with the background, and using 266nm UV laser as excitation light source, fresh and old latent fingerprints on the surface of four types of non-absorbent objects as magazine cover, glass, back of cellphone, wood desktop paintwork and two types of absorbing objects as manila envelope, notebook paper were noninvasive detected and appeared through reflection photography and fluorescence photography technologies, and the results meet the fingerprint identification requirements in forensic science.

Direct method for imaging elemental distribution profiles with long-period x-ray standing waves

NASA Astrophysics Data System (ADS)

Kohli, Vaibhav; Bedzyk, Michael J.; Fenter, Paul

2010-02-01

A model-independent Fourier-inversion method for imaging elemental profiles from multilayer and total-external reflection x-ray standing wave (XSW) data is developed for the purpose of understanding the assembly of atoms, ions, and molecules at well-defined interfaces in complex environments. The direct-method formalism is derived for the case of a long-period XSW generated by low-angle specular reflection in an attenuating overlayer medium. It is validated through comparison with simulated and experimental data to directly obtain an elemental distribution contained within the overlayer. We demonstrate this formalism by extracting the one-dimensional profile of Ti normal to the surface for a TiO2/Si/Mo trilayer deposited on a Si substrate using the TiKα fluorescence yield measured in air and under an aqueous electrolyte. The model-independent results demonstrate reduced coherent fractions for the in situ results associated with an incoherency of the x-ray beam (which are attributed to fluorescence excitation by diffusely or incoherently scattered x-rays). The uniqueness and limitations of the approach are discussed.

Combined FLIM and reflectance confocal microscopy for epithelial imaging

NASA Astrophysics Data System (ADS)

Jabbour, Joey M.; Cheng, Shuna; Shrestha, Sebina; Malik, Bilal; Jo, Javier A.; Applegate, Brian; Maitland, Kristen C.

2012-03-01

Current methods for detection of oral cancer lack the ability to delineate between normal and precancerous tissue with adequate sensitivity and specificity. The usual diagnostic mechanism involves visual inspection and palpation followed by tissue biopsy and histopathology, a process both invasive and time-intensive. A more sensitive and objective screening method can greatly facilitate the overall process of detection of early cancer. To this end, we present a multimodal imaging system with fluorescence lifetime imaging (FLIM) for wide field of view guidance and reflectance confocal microscopy for sub-cellular resolution imaging of epithelial tissue. Moving from a 12 x 12 mm2 field of view with 157 ìm lateral resolution using FLIM to 275 x 200 μm2 with lateral resolution of 2.2 μm using confocal microscopy, hamster cheek pouch model is imaged both in vivo and ex vivo. The results indicate that our dual modality imaging system can identify and distinguish between different tissue features, and, therefore, can potentially serve as a guide in early oral cancer detection..

Total internal reflection fluorescence anisotropy imaging microscopy: setup, calibration, and data processing for protein polymerization measurements in living cells

NASA Astrophysics Data System (ADS)

Ströhl, Florian; Wong, Hovy H. W.; Holt, Christine E.; Kaminski, Clemens F.

2018-01-01

Fluorescence anisotropy imaging microscopy (FAIM) measures the depolarization properties of fluorophores to deduce molecular changes in their environment. For successful FAIM, several design principles have to be considered and a thorough system-specific calibration protocol is paramount. One important calibration parameter is the G factor, which describes the system-induced errors for different polarization states of light. The determination and calibration of the G factor is discussed in detail in this article. We present a novel measurement strategy, which is particularly suitable for FAIM with high numerical aperture objectives operating in TIRF illumination mode. The method makes use of evanescent fields that excite the sample with a polarization direction perpendicular to the image plane. Furthermore, we have developed an ImageJ/Fiji plugin, AniCalc, for FAIM data processing. We demonstrate the capabilities of our TIRF-FAIM system by measuring β -actin polymerization in human embryonic kidney cells and in retinal neurons.

Atmospheric scanning electron microscope for correlative microscopy.

PubMed

Morrison, Ian E G; Dennison, Clare L; Nishiyama, Hidetoshi; Suga, Mitsuo; Sato, Chikara; Yarwood, Andrew; O'Toole, Peter J

2012-01-01

The JEOL ClairScope is the first truly correlative scanning electron and optical microscope. An inverted scanning electron microscope (SEM) column allows electron images of wet samples to be obtained in ambient conditions in a biological culture dish, via a silicon nitride film window in the base. A standard inverted optical microscope positioned above the dish holder can be used to take reflected light and epifluorescence images of the same sample, under atmospheric conditions that permit biochemical modifications. For SEM, the open dish allows successive staining operations to be performed without moving the holder. The standard optical color camera used for fluorescence imaging can be exchanged for a high-sensitivity monochrome camera to detect low-intensity fluorescence signals, and also cathodoluminescence emission from nanophosphor particles. If these particles are applied to the sample at a suitable density, they can greatly assist the task of perfecting the correlation between the optical and electron images. Copyright © 2012 Elsevier Inc. All rights reserved.

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

PubMed Central

Brunstein, Maia; Teremetz, Maxime; Hérault, Karine; Tourain, Christophe; Oheim, Martin

2014-01-01

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2). PMID:24606927

Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

PubMed Central

Bellomo, G; Vairetti, M; Stivala, L; Mirabelli, F; Richelmi, P; Orrenius, S

1992-01-01

The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury. Images PMID:1584774

Biological applications of near-field scanning optical microscopy

NASA Astrophysics Data System (ADS)

Moers, Marco H. P.; Ruiter, A. G. T.; Jalocha, Alain; van Hulst, Niko F.; Kalle, W. H. J.; Wiegant, J. C. A. G.; Raap, A. K.

1995-09-01

Near-field Scanning Optical Microscopy (NSOM) is a true optical microscopic technique allowing fluorescence, absorption, reflection and polarization contrast with the additional advantage of nanometer lateral resolution, unlimited by diffraction and operation at ambient conditions. NSOM based on metal coated adiabatically tapered fibers, combined with shear force feedback and operated in illumination mode, has proven to be the most powerful NSOM arrangement, because of its true localization of the optical interaction, its various optical contrast possibilities and its sensitivity down to the single molecular level. In this paper applications of `aperture' NSOM to Fluorescence In Situ Hybridization of human metaphase chromosomes are presented, where the localized fluorescence allows to identify specific DNA sequences. All images are accompanied by the simultaneously acquired force image, enabling direct comparison of the optical contrast with the sample topography on nanometer scale, far beyond the diffraction limit. Thus the unique combination of high resolution, specific optical contrast and ambient operation offers many new direction possibilities in biological studies.

Non-invasive, photonics-based diagnostic, imaging, monitoring, and light delivery techniques for the recognition, quantification and treatment of malignant and chronic inflammatory conditions

NASA Astrophysics Data System (ADS)

Davies, N.; Davies-Shaw, D.; Shaw, J. D.

2007-02-01

We report firsthand on innovative developments in non-invasive, biophotonic techniques for a wide range of diagnostic, imaging and treatment options, including the recognition and quantification of cancerous, pre-cancerous cells and chronic inflammatory conditions. These techniques have benefited from the ability to target the affected site by both monochromatic light and broad multiple wavelength spectra. The employment of such wavelength or color-specific properties embraces the fluorescence stimulation of various photosensitizing drugs, and the instigation and detection of identified fluorescence signatures attendant upon laser induced fluorescence (LIF) phenomena as transmitted and propagated by precancerous, cancerous and normal tissue. In terms of tumor imaging and therapeutic and treatment options, we have exploited the abilities of various wavelengths to penetrate to different depths, through different types of tissues, and have explored quantifiable absorption and reflection characteristics upon which diagnostic assumptions can be reliably based and formulated. These biophotonic-based diagnostic, sensing and imaging techniques have also benefited from, and have been further enhanced by, the integrated ability to provide various power levels to be employed at various stages in the procedure. Applications are myriad, including non-invasive, non destructive diagnosis of in vivo cell characteristics and functions; light-based tissue analysis; real-time monitoring and mapping of brain function and of tumor growth; real time monitoring of the surgical completeness of tumor removal during laser-imaged/guided brain resection; diagnostic procedures based on fluorescence life-time monitoring, the monitoring of chronic inflammatory conditions (including rheumatoid arthritis), and continuous blood glucose monitoring in the control of diabetes.

Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles

PubMed Central

Guggenheim, Emily J.; Khan, Abdullah; Pike, Jeremy; Chang, Lynne; Lynch, Iseult; Rappoport, Joshua Z.

2016-01-01

The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the <100 nm size of NPs. Techniques such as super-resolution light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP within fluorescently labelled cellular compartments. NP signal can be observed using RCM, R-SIM and TEM and a direct comparison is presented. Each of these techniques has its own benefits and limitations; RCM and R-SIM provide novel complementary information while the combination of modalities provides a unique opportunity to gain additional information regarding NP uptake. The use of multiple imaging methods therefore greatly enhances the range of NPs that can be studied under label-free conditions. PMID:27695038

Fluorescence imaging spectrometer optical design

NASA Astrophysics Data System (ADS)

Taiti, A.; Coppo, P.; Battistelli, E.

2015-09-01

The optical design of the FLuORescence Imaging Spectrometer (FLORIS) studied for the Fluorescence Explorer (FLEX) mission is discussed. FLEX is a candidate for the ESA's 8th Earth Explorer opportunity mission. FLORIS is a pushbroom hyperspectral imager foreseen to be embarked on board of a medium size satellite, flying in tandem with Sentinel-3 in a Sun synchronous orbit at a height of about 815 km. FLORIS will observe the vegetation fluorescence and reflectance within a spectral range between 500 and 780 nm. Multi-frames acquisitions on matrix detectors during the satellite movement will allow the production of 2D Earth scene images in two different spectral channels, called HR and LR with spectral resolution of 0.3 and 2 nm respectively. A common fore optics is foreseen to enhance by design the spatial co-registration between the two spectral channels, which have the same ground spatial sampling (300 m) and swath (150 km). An overlapped spectral range between the two channels is also introduced to simplify the spectral coregistration. A compact opto-mechanical solution with all spherical and plane optical elements is proposed, and the most significant design rationales are described. The instrument optical architecture foresees a dual Babinet scrambler, a dioptric telescope and two grating spectrometers (HR and LR), each consisting of a modified Offner configuration. The developed design is robust, stable vs temperature, easy to align, showing very high optical quality along the whole field of view. The system gives also excellent correction for transverse chromatic aberration and distortions (keystone and smile).

Integrating fluorescence and interactance measurements to improve apple maturity assessment

NASA Astrophysics Data System (ADS)

Noh, Hyun Kwon; Lu, Renfu

2006-10-01

Fluorescence and reflectance (or interactance) are promising techniques for measuring fruit quality and condition. Our previous research showed that a hyperspectral imaging technique integrating fluorescence and reflectance could improve predictions of selected quality parameters compared to single sensing techniques. The objective of this research was to use a low cost spectrometer for rapid acquisition of fluorescence and interactance spectra from apples and develop an algorithm integrating the two types of data for predicting skin and flesh color, fruit firmness, starch index, soluble solids content, and titratable acid. Experiments were performed to measure UV light induced transient fluorescence and interactance spectra from 'Golden Delicious' apples that were harvested over a period of four weeks during the 2005 harvest season. Standard destructive tests were performed to measure maturity parameters from the apples. Principal component (PC) analysis was applied to the interactance and fluorescence data. A back-propagation feedforward neural network with the inputs of PC data was used to predict individual maturity parameters. Interactance mode was consistently better than fluorescence mode in predicting the maturity parameters. Integrating interactance and fluorescence improved predictions of all parameters except flesh chroma; values of the correlation coefficient for firmness, soluble solids content, starch index, and skin and flesh hue were 0.77, 0.77, 0.89, 0.99, and 0.96 respectively, with the corresponding standard errors of 6.93 N, 0.90%, 0.97 g/L, 0.013 rad, and 0.013 rad. These results represented 4.1% to 23.5% improvements in terms of standard error, in comparison with the better results from the two single sensing methods. Integrating interactance and fluorescence can better assess apple maturity and quality.

Multispectral processing of combined visible and x-ray fluorescence imagery in the Archimedes palimpsest

NASA Astrophysics Data System (ADS)

Walvoord, Derek; Bright, Allison; Easton, Roger L., Jr.

2008-02-01

The Archimedes palimpsest is one of the most significant early texts in the history of science that has survived to the present day. It includes the oldest known copies of text from seven treatises by Archimedes, along with pages from other important historical writings. In the 13th century, the original texts were erased and overwritten by a Christian prayer book, which was used in religious services probably into the 19th century. Since 2001, much of the text from treatises of Archimedes has been transcribed from images taken in reflected visible light and visible fluorescence generated by exposure of the parchment to ultraviolet light. However, these techniques do not work well on all pages of the manuscript, including the badly stained colophon, four pages of the manuscript obscured by icons painted during the first half of the 20th century, and some pages of non-Archimedes texts. Much of the text on the colophon and overpainted pages has been recovered from X-ray fluorescence (XRF) imagery. In this work, the XRF images of one of the other pages were combined with the bands of optical images to create hyperspectral image cubes and processed using standard statistical classification techniques developed for environmental remote sensing to test if this improved the recovery of the original text.

Selective Removal of Demineralization Using Near Infrared Cross Polarization Reflectance and a Carbon Dioxide Laser.

PubMed

Chan, Kenneth H; Fried, Daniel

2012-02-09

Lasers can ablate/remove tissue in a non-contact mode of operation and a pulsed laser beam does not interfere with the ability to image the tooth surface, therefore lasers are ideally suited for integration with imaging devices for image-guided ablation. Laser energy can be rapidly and efficiently delivered to tooth surfaces using a digitally controlled laser beam scanning system for precise and selective laser ablation with minimal loss of healthy tissues. Under the appropriate irradiation conditions such laser energy can induce beneficial chemical and morphological changes in the walls of the drilled cavity that can increase resistance to further dental decay and produce surfaces with enhanced adhesive properties to restorative materials. Previous studies have shown that images acquired using near-IR transillumination, optical coherence tomography and fluorescence can be used to guide the laser for selective removal of demineralized enamel. Recent studies have shown that NIR reflectance measurements at 1470-nm can be used to obtain images of enamel demineralization with very high contrast. The purpose of this study was to demonstrate that image guided ablation of occlusal lesions can be successfully carried out using a NIR reflectance imaging system coupled with a carbon dioxide laser operating at 9.3-μm with high pulse repetition rates.

Spectral reflectance of the ocular fundus as a diagnostic marker for cerebral malaria

NASA Astrophysics Data System (ADS)

Liu, Xun; Rice, David A.; Khoobehi, Bahram

2012-03-01

The challenge of correctly identifying malaria infection continues to impede our efforts to control this disease. Recent studies report highly specific retinal changes in severe malaria patients; these retinal changes may represent a very useful diagnostic indicator for this disease. To further explore the ocular manifestations of malaria, we used hyperspectral imaging to study retinal changes caused by Plasmodium berghei ANKA parasitization in a mouse model. We collected the spectral reflectance of the ocular fundus from hyperspectral images of the mouse eye. The blood oxygen sensitive spectral region was normalized for variances in illumination, and used to calculate relative values that correspond to oxygenated hemoglobin levels. Oxygen hemoglobin levels are markedly lower in parasitized mice, indicating that hemoglobin digestion by P. berghei may be detected using spectral reflectance. Furthermore, the ocular reflectance of parasitized mice was abnormally elevated between 660nm and 750nm, suggesting fluorescence in this region. While the source of this fluorescence is not yet clear, its presence correlates strongly with P. Berghei parasitization, and may indicate the presence of hemozoin deposits in the retinal vasculature. The pathology of severe malaria still presents many questions for clinicians and scientists, and our understanding of cerebral malaria has been generally confined to clinical observation and postmortem examination. As the retina represents a portion of the central nervous system that can be easily examined noninvasively, our technique may provide the basis for an automated tool to detect and examine severe malaria via retinal changes.

Possibility for new PolyCO imaging: stroboscopic imaging based on vibrating capillary optics

NASA Astrophysics Data System (ADS)

Liedl, A.; Dabagov, S. B.; Della Ventura, G.; Hampai, D.; Polese, C.

2015-08-01

Polycapillary lenses are well known optical devices for radiation and charged particles. These lenses consist of thousands channels through which the signal is transmitted by total external reflection phenomenon. Their application have made possible technical improvements in different fields such as imaging, fluorescence analysis, channeling studies etc. In particular, the application of this optics coupled with conventional sources such as X-ray tubes has opened a new season for potential applications of desktop instrumentations. For instance, the usage of such lenses has enhanced the spatial coherence and the brilliance over the sample allowing better resolution and contrast for imaging purposes. In addiction, improved focusing power and confocal configuration of other lenses has improved the resolution, from both the energy and the spatial points of view, in fluorescence mapping. A recent work has addressed the behavior of the transmitted radiation through a single capillary in vibrating regime. In this work a test of using a vibrating capillary for stroboscopic imaging is presented. A sample characterized by a known periodic event is studied with a synchronized vibrating capillary.

Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface.

PubMed Central

Iino, R; Koyama, I; Kusumi, A

2001-01-01

Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions). PMID:11371443

Design and validation of a near-infrared fluorescence endoscope for detection of early esophageal malignancy using a targeted imaging probe

NASA Astrophysics Data System (ADS)

Waterhouse, Dale J.; Joseph, James; Neves, Andre A.; di Pietro, Massimiliano; Brindle, Kevin M.; Fitzgerald, Rebecca C.; Bohndiek, Sarah E.

2016-03-01

Barrett's esophagus is a condition that predisposes patients to esophageal cancer. Early detection of cancer in these patients can be curative, but is confounded by a lack of contrast in white light endoscopy (WLE). Application of fluorescently-labeled lectins to the esophagus during endoscopy can more accurately delineate dysplasia emerging within Barrett's than WLE1, but strong tissue autofluorescence has limited sensitivity and dynamic range of this approach. To overcome this challenge, we synthesized a near-infrared (NIR) fluorescent lectin and have constructed a clinically translatable endoscope for simultaneous WLE and NIR imaging. An imaging fiber bundle, shielded from patient contact using a disposable catheter, relays collected light into an optical path that splits the WL reflectance and NIR emission onto two cameras for simultaneous video-rate recording. The captured images are co-registered and the honeycomb artifact arising from the fiber bundle is removed using interpolation between image points derived from individual fibers. A minimum detectable concentration of 110 nM was determined using a dilution series of IRDye800CW-lectin in black well plates. We have demonstrated the ability to use our endoscope to distinguish between different tissue types in ex vivo mouse stomachs. Future work using human ex vivo tissue specimens will determine safe illumination limits and sensitivity for dysplasia and adenocarcinoma in Barrett's esophagus, prior to commencing clinical trials.

Single Molecule Visualization of Protein-DNA Complexes: Watching Machines at Work

NASA Astrophysics Data System (ADS)

Kowalczykowski, Stephen

2013-03-01

We can now watch individual proteins acting on single molecules of DNA. Such imaging provides unprecedented interrogation of fundamental biophysical processes. Visualization is achieved through the application of two complementary procedures. In one, single DNA molecules are attached to a polystyrene bead and are then captured by an optical trap. The DNA, a worm-like coil, is extended either by the force of solution flow in a micro-fabricated channel, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Protein action is visualized by fluorescent reporters: fluorescent dyes that bind double-stranded DNA (dsDNA), fluorescent biosensors for single-stranded DNA (ssDNA), or fluorescently-tagged proteins. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Using these approaches, we imaged the search for DNA sequence homology conducted by the RecA-ssDNA filament. The manner by which RecA protein finds a single homologous sequence in the genome had remained undefined for almost 30 years. Single-molecule imaging revealed that the search occurs through a mechanism termed ``intersegmental contact sampling,'' in which the randomly coiled structure of DNA is essential for reiterative sampling of DNA sequence identity: an example of parallel processing. In addition, the assembly of RecA filaments on single molecules of single-stranded DNA was visualized. Filament assembly requires nucleation of a protein dimer on DNA, and subsequent growth occurs via monomer addition. Furthermore, we discovered a class of proteins that catalyzed both nucleation and growth of filaments, revealing how the cell controls assembly of this protein-DNA complex.

Complementary standoff chemical imaging to map and identify artist materials in an early Italian Renaissance panel painting.

PubMed

Dooley, Kathryn A; Conover, Damon M; Glinsman, Lisha Deming; Delaney, John K

2014-12-08

Two imaging modalities based on molecular and elemental spectroscopy were used to characterize a painting by Cosimo Tura. Visible-to-near-infrared (400-1680 nm) reflectance imaging spectroscopy (RIS) and X-ray fluorescence (XRF) imaging spectroscopy were employed to identify pigments and determine their spatial distribution with higher confidence than from either technique alone. For example, Mary's red robe was modeled through the distribution of an insect-derived red lake (RIS map) and lead white (XRF lead map), rather than a layer of red lake on vermilion. The RIS image cube was also used to isolate the preparatory design by mapping the reflectance spectra associated with it. In conjunction with results from an earlier RIS study (1650-2500 nm) to map and identify the binding media, a more thorough understanding was gained of the materials and techniques used in the painting. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The use of near-infrared photography to image fired bullets and cartridge cases.

PubMed

Stein, Darrell; Yu, Jorn Chi Chung

2013-09-01

An imaging technique that is capable of reducing glare, reflection, and shadows can greatly assist the process of toolmarks comparison. In this work, a camera with near-infrared (near-IR) photographic capabilities was fitted with an IR filter, mounted to a stereomicroscope, and used to capture images of toolmarks on fired bullets and cartridge cases. Fluorescent, white light-emitting diode (LED), and halogen light sources were compared for use with the camera. Test-fired bullets and cartridge cases from different makes and models of firearms were photographed under either near-IR or visible light. With visual comparisons, near-IR images and visible light images were comparable. The use of near-IR photography did not reveal more details and could not effectively eliminate reflections and glare associated with visible light photography. Near-IR photography showed little advantages in manual examination of fired evidence when it was compared with visible light (regular) photography. © 2013 American Academy of Forensic Sciences.

Three-dimensional fluorescence-enhanced optical tomography using a hand-held probe based imaging system

PubMed Central

Ge, Jiajia; Zhu, Banghe; Regalado, Steven; Godavarty, Anuradha

2008-01-01

Hand-held based optical imaging systems are a recent development towards diagnostic imaging of breast cancer. To date, all the hand-held based optical imagers are used to perform only surface mapping and target localization, but are not capable of demonstrating tomographic imaging. Herein, a novel hand-held probe based optical imager is developed towards three-dimensional (3-D) optical tomography studies. The unique features of this optical imager, which primarily consists of a hand-held probe and an intensified charge coupled device detector, are its ability to; (i) image large tissue areas (5×10 sq. cm) in a single scan, (ii) perform simultaneous multiple point illumination and collection, thus reducing the overall imaging time; and (iii) adapt to varying tissue curvatures, from a flexible probe head design. Experimental studies are performed in the frequency domain on large slab phantoms (∼650 ml) using fluorescence target(s) under perfect uptake (1:0) contrast ratios, and varying target depths (1–2 cm) and X-Y locations. The effect of implementing simultaneous over sequential multiple point illumination towards 3-D tomography is experimentally demonstrated. The feasibility of 3-D optical tomography studies has been demonstrated for the first time using a hand-held based optical imager. Preliminary fluorescence-enhanced optical tomography studies are able to reconstruct 0.45 ml target(s) located at different target depths (1–2 cm). However, the depth recovery was limited as the actual target depth increased, since only reflectance measurements were acquired. Extensive tomography studies are currently carried out to determine the resolution and performance limits of the imager on flat and curved phantoms. PMID:18697559

Three-dimensional fluorescence-enhanced optical tomography using a hand-held probe based imaging system.

PubMed

Ge, Jiajia; Zhu, Banghe; Regalado, Steven; Godavarty, Anuradha

2008-07-01

Hand-held based optical imaging systems are a recent development towards diagnostic imaging of breast cancer. To date, all the hand-held based optical imagers are used to perform only surface mapping and target localization, but are not capable of demonstrating tomographic imaging. Herein, a novel hand-held probe based optical imager is developed towards three-dimensional (3-D) optical tomography studies. The unique features of this optical imager, which primarily consists of a hand-held probe and an intensified charge coupled device detector, are its ability to; (i) image large tissue areas (5 x 10 sq. cm) in a single scan, (ii) perform simultaneous multiple point illumination and collection, thus reducing the overall imaging time; and (iii) adapt to varying tissue curvatures, from a flexible probe head design. Experimental studies are performed in the frequency domain on large slab phantoms (approximately 650 ml) using fluorescence target(s) under perfect uptake (1:0) contrast ratios, and varying target depths (1-2 cm) and X-Y locations. The effect of implementing simultaneous over sequential multiple point illumination towards 3-D tomography is experimentally demonstrated. The feasibility of 3-D optical tomography studies has been demonstrated for the first time using a hand-held based optical imager. Preliminary fluorescence-enhanced optical tomography studies are able to reconstruct 0.45 ml target(s) located at different target depths (1-2 cm). However, the depth recovery was limited as the actual target depth increased, since only reflectance measurements were acquired. Extensive tomography studies are currently carried out to determine the resolution and performance limits of the imager on flat and curved phantoms.

Linking Physiological Responses, Chlorophyll Fluorescence and Hyperspectral Imagery to Detect Salinity Stress Using the Physiological Reflectance Index in the Coastal Shrub, Myrica cerifera

DTIC Science & Technology

2008-01-01

rainforests under various precipitation and substrate conditions (Asner et al., 2005). AMODIS-derived PRI has also been correlated to ecosystem-level...carbon uptake in an Amazon forest measured with spaceborne imaging spectroscopy. Proceedings of the National Academy of Sciences of the United States of

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part II. Combined Evanescent-Wave Excitation and Supercritical-Angle Fluorescence Detection Improves Optical Sectioning

PubMed Central

Brunstein, Maia; Hérault, Karine; Oheim, Martin

2014-01-01

Azimuthal beam scanning makes evanescent-wave (EW) excitation isotropic, thereby producing total internal reflection fluorescence (TIRF) images that are evenly lit. However, beam spinning does not fundamentally address the problem of propagating excitation light that is contaminating objective-type TIRF. Far-field excitation depends more on the specific objective than on cell scattering. As a consequence, the excitation impurities in objective-type TIRF are only weakly affected by changes of azimuthal or polar beam angle. These are the main results of the first part of this study (Eliminating unwanted far-field excitation in objective-type TIRF. Pt.1. Identifying sources of nonevanescent excitation light). This second part focuses on exactly where up beam in the illumination system stray light is generated that gives rise to nonevanescent components in TIRF. Using dark-field imaging of scattered excitation light we pinpoint the objective, intermediate lenses and, particularly, the beam scanner as the major sources of stray excitation. We study how adhesion-molecule coating and astrocytes or BON cells grown on the coverslip surface modify the dark-field signal. On flat and weakly scattering cells, most background comes from stray reflections produced far from the sample plane, in the beam scanner and the objective lens. On thick, optically dense cells roughly half of the scatter is generated by the sample itself. We finally show that combining objective-type EW excitation with supercritical-angle fluorescence (SAF) detection efficiently rejects the fluorescence originating from deeper sample regions. We demonstrate that SAF improves the surface selectivity of TIRF, even at shallow penetration depths. The coplanar microscopy scheme presented here merges the benefits of beam spinning EW excitation and SAF detection and provides the conditions for quantitative wide-field imaging of fluorophore dynamics at or near the plasma membrane. PMID:24606929

Optics clustered to output unique solutions: A multi-laser facility for combined single molecule and ensemble microscopy

NASA Astrophysics Data System (ADS)

Clarke, David T.; Botchway, Stanley W.; Coles, Benjamin C.; Needham, Sarah R.; Roberts, Selene K.; Rolfe, Daniel J.; Tynan, Christopher J.; Ward, Andrew D.; Webb, Stephen E. D.; Yadav, Rahul; Zanetti-Domingues, Laura; Martin-Fernandez, Marisa L.

2011-09-01

Optics clustered to output unique solutions (OCTOPUS) is a microscopy platform that combines single molecule and ensemble imaging methodologies. A novel aspect of OCTOPUS is its laser excitation system, which consists of a central core of interlocked continuous wave and pulsed laser sources, launched into optical fibres and linked via laser combiners. Fibres are plugged into wall-mounted patch panels that reach microscopy end-stations in adjacent rooms. This allows multiple tailor-made combinations of laser colours and time characteristics to be shared by different end-stations minimising the need for laser duplications. This setup brings significant benefits in terms of cost effectiveness, ease of operation, and user safety. The modular nature of OCTOPUS also facilitates the addition of new techniques as required, allowing the use of existing lasers in new microscopes while retaining the ability to run the established parts of the facility. To date, techniques interlinked are multi-photon/multicolour confocal fluorescence lifetime imaging for several modalities of fluorescence resonance energy transfer (FRET) and time-resolved anisotropy, total internal reflection fluorescence, single molecule imaging of single pair FRET, single molecule fluorescence polarisation, particle tracking, and optical tweezers. Here, we use a well-studied system, the epidermal growth factor receptor network, to illustrate how OCTOPUS can aid in the investigation of complex biological phenomena.

Quantifying the assembly of multicomponent molecular machines by single-molecule total internal reflection fluorescence microscopy

PubMed Central

Boehm, Elizabeth M.; Subramanyam, Shyamal; Ghoneim, Mohamed; Washington, M. Todd; Spies, Maria

2016-01-01

Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary, quaternary, etc.) complexes using multi-color setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches has become today. PMID:27793278

Reflective Filters Design for Self-Filtering Narrowband Ultraviolet Imaging Experiment Wide-Field Surveys (NUVIEWS) Project

NASA Technical Reports Server (NTRS)

Park, Jung- Ho; Kim, Jongmin; Zukic, Muamer; Torr, Douglas G.

1994-01-01

We report the design of multilayer reflective filters for the self-filtering cameras of the NUVIEWS project. Wide angle self-filtering cameras were designed to image the C IV (154.9 nm) line emission, and H2 Lyman band fluorescence (centered at 161 nm) over a 20 deg x 30 deg field of view. A key element of the filter design includes the development of pi-multilayers optimized to provide maximum reflectance at 154.9 nm and 161 nm for the respective cameras without significant spectral sensitivity to the large cone angle of the incident radiation. We applied self-filtering concepts to design NUVIEWS telescope filters that are composed of three reflective mirrors and one folding mirror. The filters with narrowband widths of 6 and 8 rim at 154.9 and 161 nm, respectively, have net throughputs of more than 50 % with average blocking of out-of-band wavelengths better than 3 x 10(exp -4)%.

A novel multimodal optical imaging system for early detection of oral cancer

PubMed Central

Malik, Bilal H.; Jabbour, Joey M.; Cheng, Shuna; Cuenca, Rodrigo; Cheng, Yi-Shing Lisa; Wright, John M.; Jo, Javier A.; Maitland, Kristen C.

2015-01-01

Objectives Several imaging techniques have been advocated as clinical adjuncts to improve identification of suspicious oral lesions. However, these have not yet shown superior sensitivity or specificity over conventional oral examination techniques. We developed a multimodal, multi-scale optical imaging system that combines macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM) for early detection of oral cancer. We tested our system on excised human oral tissues. Study Design A total of four tissue specimen were imaged. These specimens were diagnosed as one each: clinically normal, oral lichen planus, gingival hyperplasia, and superficially-invasive squamous cell carcinoma (SCC). The optical and fluorescence lifetime properties of each specimen were recorded. Results Both quantitative and qualitative differences between normal, benign and SCC lesions can be resolved with FLIM-RCM imaging. The results demonstrate that an integrated approach based on these two methods can potentially enable rapid screening and evaluation of large areas of oral epithelial tissue. Conclusions Early results from ongoing studies of imaging human oral cavity illustrate the synergistic combination of the two modalities. An adjunct device based on such optical characterization of oral mucosa can potentially be used to detect oral carcinogenesis in early stages. PMID:26725720

Protocol for Biomarker Ratio Imaging Microscopy with Specific Application to Ductal Carcinoma In situ of the Breast

PubMed Central

Clark, Andrea J.; Petty, Howard R.

2016-01-01

This protocol describes the methods and steps involved in performing biomarker ratio imaging microscopy (BRIM) using formalin fixed paraffin-embedded (FFPE) samples of human breast tissue. The technique is based on the acquisition of two fluorescence images of the same microscopic field using two biomarkers and immunohistochemical tools. The biomarkers are selected such that one biomarker correlates with breast cancer aggressiveness while the second biomarker anti-correlates with aggressiveness. When the former image is divided by the latter image, a computed ratio image is formed that reflects the aggressiveness of tumor cells while increasing contrast and eliminating path-length and other artifacts from the image. For example, the aggressiveness of epithelial cells may be assessed by computing ratio images of N-cadherin and E-cadherin images or CD44 and CD24 images, which specifically reflect the mesenchymal or stem cell nature of the constituent cells, respectively. This methodology is illustrated for tissue samples of ductal carcinoma in situ (DCIS) and invasive breast cancer. This tool should be useful in tissue studies of experimental cancer as well as the management of cancer patients. PMID:27857940

Characterization of malaria infected blood cells by scanning confocal laser and acoustic vector contrast microscopy

NASA Astrophysics Data System (ADS)

Ahmed Mohamed, E. T.; Schubert, S.; Gilberger, T. W.; Kamanyi, A., Jr.; Wannemacher, R.; Grill, W.

2006-03-01

Acoustic and optical multiple contrast microscopy has been employed in order to explore characterizable parameters of red blood cells, including cells infected by the parasite Plasmodium falciparum, in order to investigate cellular modifications caused by the infection and to identify possible detection schemes for disease monitoring. Imaging schemes were based on fluorescence, optical transmission, optical reflection, and amplitude and phase of ultrasound reflected from the cells. Contrast variations observed in acoustic microscopy, but not in optical microscopy, were tentatively ascribed to changes caused by the infection.

Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA

NASA Astrophysics Data System (ADS)

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-10-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ˜40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.

Sub-micrometer Geometrically Encoded Fluorescent Barcodes Self-Assembled from DNA

PubMed Central

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-01-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here we use DNA-origami technology to construct sub-micrometer nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be unambiguously decoded using epifluorescence or total internal reflection fluorescence (TIRF) microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ~40 nm. One species of the barcodes was used to tag yeast surface receptors, suggesting their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments. PMID:23000997

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

PubMed Central

Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

2015-01-01

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

Expanding Imaging Capabilities for Microfluidics: Applicability of Darkfield Internal Reflection Illumination (DIRI) to Observations in Microfluidics

PubMed Central

Kawano, Yoshihiro; Otsuka, Chino; Sanzo, James; Higgins, Christopher; Nirei, Tatsuo; Schilling, Tobias; Ishikawa, Takuji

2015-01-01

Microfluidics is used increasingly for engineering and biomedical applications due to recent advances in microfabrication technologies. Visualization of bubbles, tracer particles, and cells in a microfluidic device is important for designing a device and analyzing results. However, with conventional methods, it is difficult to observe the channel geometry and such particles simultaneously. To overcome this limitation, we developed a Darkfield Internal Reflection Illumination (DIRI) system that improved the drawbacks of a conventional darkfield illuminator. This study was performed to investigate its utility in the field of microfluidics. The results showed that the developed system could clearly visualize both microbubbles and the channel wall by utilizing brightfield and DIRI illumination simultaneously. The methodology is useful not only for static phenomena, such as clogging, but also for dynamic phenomena, such as the detection of bubbles flowing in a channel. The system was also applied to simultaneous fluorescence and DIRI imaging. Fluorescent tracer beads and channel walls were observed clearly, which may be an advantage for future microparticle image velocimetry (μPIV) analysis, especially near a wall. Two types of cell stained with different colors, and the channel wall, can be recognized using the combined confocal and DIRI system. Whole-slide imaging was also conducted successfully using this system. The tiling function significantly expands the observing area of microfluidics. The developed system will be useful for a wide variety of engineering and biomedical applications for the growing field of microfluidics. PMID:25748425

Expanding imaging capabilities for microfluidics: applicability of darkfield internal reflection illumination (DIRI) to observations in microfluidics.

PubMed

Kawano, Yoshihiro; Otsuka, Chino; Sanzo, James; Higgins, Christopher; Nirei, Tatsuo; Schilling, Tobias; Ishikawa, Takuji

2015-01-01

Microfluidics is used increasingly for engineering and biomedical applications due to recent advances in microfabrication technologies. Visualization of bubbles, tracer particles, and cells in a microfluidic device is important for designing a device and analyzing results. However, with conventional methods, it is difficult to observe the channel geometry and such particles simultaneously. To overcome this limitation, we developed a Darkfield Internal Reflection Illumination (DIRI) system that improved the drawbacks of a conventional darkfield illuminator. This study was performed to investigate its utility in the field of microfluidics. The results showed that the developed system could clearly visualize both microbubbles and the channel wall by utilizing brightfield and DIRI illumination simultaneously. The methodology is useful not only for static phenomena, such as clogging, but also for dynamic phenomena, such as the detection of bubbles flowing in a channel. The system was also applied to simultaneous fluorescence and DIRI imaging. Fluorescent tracer beads and channel walls were observed clearly, which may be an advantage for future microparticle image velocimetry (μPIV) analysis, especially near a wall. Two types of cell stained with different colors, and the channel wall, can be recognized using the combined confocal and DIRI system. Whole-slide imaging was also conducted successfully using this system. The tiling function significantly expands the observing area of microfluidics. The developed system will be useful for a wide variety of engineering and biomedical applications for the growing field of microfluidics.

Multimodal microscopy of immune cells and melanoma for longitudinal studies

NASA Astrophysics Data System (ADS)

Entenberg, David; Aranda, Iana; Li, Yongbiao; Toledo-Crow, Ricardo; Schaer, David; Li, Yanyun

2006-02-01

Intravital microscopy of cancer is a well established tool that provides direct visualization of the tumor cycle. It traditionally involves one of several strategies: invasive subcutaneous (SC) implantation of tumors followed by surgical opening of skin flaps for imaging, techniques utilizing skin fold chambers and implanted optical windows or intradermal injections under 200μm from the skin surface. All of these techniques allow the use of fluorescent proteins as markers for biologically significant constituents. However, observation methods utilizing skin-flaps, skin-fold chambers and optical windows are invasive and tend to alter the immune environment of the tissue and/or limit the duration of studies that can be performed. If implanted correctly, intradermally injected tumors can be minimally invasive, will not require biopsies or surgical intervention to observe and are accessible for direct transdermal imaging with a number of in vivo modalities. We present our work in the development of a small animal intravital microscopy workstation that allows the acquisition of different contrast imaging modalities: reflectance confocal, wide field epifluorescence, multiphoton and second harmonic generation (SHG). The images are acquired pair-wise simultaneously and sequentially in time. The aim of our instrumentation is to gather all information generated by the single probing beam via the reflected or back-scattered signal, SHG signal and various fluorescence signals. Additionally, we also present our development of a microscopic tissue navigation technique to mark, label and track sites of interest. This technique enables us to revisit sites periodically and record, with different imaging contrasts, their biological changes over time.

Terahertz analysis of an East Asian historical mural painting

NASA Astrophysics Data System (ADS)

Fukunaga, K.; Hosako, I.; Kohdzuma, Y.; Koezuka, T.; Kim, M.-J.; Ikari, T.; Du, X.

2010-05-01

Terahertz (THz) spectroscopy and THz and imaging techniques are expected to have great potential for the non-invasive analysis of artworks. We have applied THz imaging to analyse the historic mural painting of a Lamaism temple by using a transportable time-domain THz imaging system; such an attempt is the first in the world. The reflection image revealed that there are two orange colours in the painting, although they appear the same to the naked eye. THz imaging can also estimate the depth of cracks. The colours were examined by X-ray fluorescence and Raman spectroscopy, and the results were found to be in good agreement. This work proved that THz imaging can contribute to the non-invasive analysis of cultural heritage.

[Rapid Identification of Epicarpium Citri Grandis via Infrared Spectroscopy and Fluorescence Spectrum Imaging Technology Combined with Neural Network].

PubMed

Pan, Sha-sha; Huang, Fu-rong; Xiao, Chi; Xian, Rui-yi; Ma, Zhi-guo

2015-10-01

To explore rapid reliable methods for detection of Epicarpium citri grandis (ECG), the experiment using Fourier Transform Attenuated Total Reflection Infrared Spectroscopy (FTIR/ATR) and Fluorescence Spectrum Imaging Technology combined with Multilayer Perceptron (MLP) Neural Network pattern recognition, for the identification of ECG, and the two methods are compared. Infrared spectra and fluorescence spectral images of 118 samples, 81 ECG and 37 other kinds of ECG, are collected. According to the differences in tspectrum, the spectra data in the 550-1 800 cm(-1) wavenumber range and 400-720 nm wavelength are regarded as the study objects of discriminant analysis. Then principal component analysis (PCA) is applied to reduce the dimension of spectroscopic data of ECG and MLP Neural Network is used in combination to classify them. During the experiment were compared the effects of different methods of data preprocessing on the model: multiplicative scatter correction (MSC), standard normal variable correction (SNV), first-order derivative(FD), second-order derivative(SD) and Savitzky-Golay (SG). The results showed that: after the infrared spectra data via the Savitzky-Golay (SG) pretreatment through the MLP Neural Network with the hidden layer function as sigmoid, we can get the best discrimination of ECG, the correct percent of training set and testing set are both 100%. Using fluorescence spectral imaging technology, corrected by the multiple scattering (MSC) results in the pretreatment is the most ideal. After data preprocessing, the three layers of the MLP Neural Network of the hidden layer function as sigmoid function can get 100% correct percent of training set and 96.7% correct percent of testing set. It was shown that the FTIR/ATR and fluorescent spectral imaging technology combined with MLP Neural Network can be used for the identification study of ECG and has the advantages of rapid, reliable effect.

A surface science compatible epifluorescence microscope for inspection of samples under ultra high vacuum and cryogenic conditions.

PubMed

Marquardt, Christian; Paulheim, Alexander; Rohbohm, Nils; Merkel, Rudolf; Sokolowski, Moritz

2017-08-01

We modified an epi-illumination light microscope and mounted it on an ultra high vacuum chamber for investigating samples used in a surface science experiment. For easy access and bake out, all optical components are placed outside the vacuum and the sample is imaged through a glass window. The microscope can be operated in reflection brightfield or epifluorescence mode to image the sample surface or fluorescent dye molecules adsorbed on it. The homemade sample mounting was made compatible for the use under the microscope; sample temperatures as low as 6 K can be achieved. The performance of the microscope is demonstrated on two model samples: Brightfield-images of a well-prepared Ag(100) surface show a macroscopic corrugation of the surface, although low energy electron diffraction data indicate a highly ordered crystalline surface. The surface shows macroscopic protrusions with flat regions, about 20-200 μm in diameter, in between. Fluorescence images of diluted 3,4,9,10-perylene tetracarboxylicacid dianhydride (PTCDA) molecules adsorbed on an ultrathin epitaxial KCl film on the Ag(100) surface show a shading effect at surface protrusions due to an inclined angle of incidence of the PTCDA beam during deposition. For some preparations, the distribution of the fluorescence intensity is inhomogeneous and shows a dense network of bright patches about 5 μm in diameter related to the macroscopic corrugation of the surface. We propose that such a light microscope can aid many surface science experiments, especially those dealing with epitaxial growth or fluorescent materials.

Compact multi-band fluorescent microscope with an electrically tunable lens for autofocusing

PubMed Central

Wang, Zhaojun; Lei, Ming; Yao, Baoli; Cai, Yanan; Liang, Yansheng; Yang, Yanlong; Yang, Xibin; Li, Hui; Xiong, Daxi

2015-01-01

Autofocusing is a routine technique in redressing focus drift that occurs in time-lapse microscopic image acquisition. To date, most automatic microscopes are designed on the distance detection scheme to fulfill the autofocusing operation, which may suffer from the low contrast of the reflected signal due to the refractive index mismatch at the water/glass interface. To achieve high autofocusing speed with minimal motion artifacts, we developed a compact multi-band fluorescent microscope with an electrically tunable lens (ETL) device for autofocusing. A modified searching algorithm based on equidistant scanning and curve fitting is proposed, which no longer requires a single-peak focus curve and then efficiently restrains the impact of external disturbance. This technique enables us to achieve an autofocusing time of down to 170 ms and the reproductivity of over 97%. The imaging head of the microscope has dimensions of 12 cm × 12 cm × 6 cm. This portable instrument can easily fit inside standard incubators for real-time imaging of living specimens. PMID:26601001

Estimating the leaf nitrogen content of paddy rice by using the combined reflectance and laser-induced fluorescence spectra.

PubMed

Yang, Jian; Du, Lin; Sun, Jia; Zhang, Zhenbing; Chen, Biwu; Shi, Shuo; Gong, Wei; Song, Shalei

2016-08-22

Paddy rice is one of the most important crops in China, and leaf nitrogen content (LNC) serves as a significant indictor for monitoring crop status. A reliable method is needed for precise and fast quantification of LNC. Laser-induced fluorescence (LIF) technology and reflectance spectra of crops are widely used to monitor leaf biochemical content. However, comparison between the fluorescence and reflectance spectra has been rarely investigated in the monitoring of LNC. In this study, the performance of the fluorescence and reflectance spectra for LNC estimation was discussed based on principal component analysis (PCA) and back-propagation neural network (BPNN). The combination of fluorescence and reflectance spectra was also proposed to monitor paddy rice LNC. The fluorescence and reflectance spectra exhibited a high degree of multi-collinearity. About 95.38%, and 97.76% of the total variance included in the spectra were efficiently extracted by using the first three PCs in PCA. The BPNN was implemented for LNC prediction based on new variables calculated using PCA. The experimental results demonstrated that the fluorescence spectra (R² = 0.810, 0.804 for 2014 and 2015, respectively) are superior to the reflectance spectra (R² = 0.721, 0.671 for 2014 and 2015, respectively) for estimating LNC based on the PCA-BPNN model. The proposed combination of fluorescence and reflectance spectra can greatly improve the accuracy of LNC estimation (R² = 0.912, 0.890 for 2014 and 2015, respectively).

The role of ultraviolet-A reflectance and ultraviolet-A induced fluorescence in the appearance of budgerigar plumage: insights from spectrofluorometry and reflectance spectrophotometry.

PubMed Central

Pearn, Sophie M; Bennett, Andrew T D; Cuthill, Innes C

2003-01-01

Fluorescence has so far been found in 52 parrot species when illuminated with ultraviolet-A (UVA) 'black' lamps, and two attempts have been made to determine whether such fluorescence plays any role in sexual signalling. However, the contribution of the reflectance versus fluorescence to the total radiance from feathers, even in the most studied species to date (budgerigars), is unclear. Nor has the plumage of this study species been systematically assessed to determine the distribution of fluorescent patches. We therefore used spectrofluorometry to determine which areas of budgerigars fluoresce and the excitation and emission spectra involved; this is the first time that such a technique has been applied to avian plumage. We found that both the yellow crown and (normally hidden) white downy chest feathers exhibit strong UVA-induced fluorescence, with peak emissions at 527 nm and 436 nm, respectively. Conversely, the bright-green chest and dark-blue tail feathers do not fluoresce. When comparing reflectance spectra (400-700 nm) from the yellow crown using illuminants with a proportion of UVA comparable to daylight, and illuminants with all UVA removed, no measurable difference resulting from fluorescence was found. This suggests that under normal daylight the contribution of fluorescence to radiance is probably trivial. Furthermore, these spectra revealed that males had fluorescent crowns with substantially higher reflectance than those of females, in both the UV waveband and at longer wavelengths. Reflectance spectrophotometry was also performed on a number of live wild-type male budgerigars to investigate the chromatic contrast between the different plumage areas. This showed that many plumage regions are highly UV-reflective. Overall our results suggest that rapid surveys using UVA black lamps may overestimate the contribution of fluorescence to plumage coloration, and that any signalling role of fluorescence emissions, at least from the yellow crown of budgerigars, may not be as important as previously thought. PMID:12737665

The Seasonal Cycle of Satellite Chlorophyll Fluorescence Observations and its Relationship to Vegetation Phenology and Ecosystem Atmosphere Carbon Exchange

NASA Technical Reports Server (NTRS)

Joiner, J.; Yoshida, Y.; Vasilkov, A. P.; Schaefer, K.; Jung, M.; Guanter, L.; Zhang, Y; Garrity, S.; Middleton, E. M.; Huemmrich, K. F.;

Accurate three-dimensional virtual reconstruction of surgical field using calibrated trajectories of an image-guided medical robot

PubMed Central

Gong, Yuanzheng; Hu, Danying; Hannaford, Blake; Seibel, Eric J.

2014-01-01

Abstract. Brain tumor margin removal is challenging because diseased tissue is often visually indistinguishable from healthy tissue. Leaving residual tumor leads to decreased survival, and removing normal tissue causes life-long neurological deficits. Thus, a surgical robotics system with a high degree of dexterity, accurate navigation, and highly precise resection is an ideal candidate for image-guided removal of fluorescently labeled brain tumor cells. To image, we developed a scanning fiber endoscope (SFE) which acquires concurrent reflectance and fluorescence wide-field images at a high resolution. This miniature flexible endoscope was affixed to the arm of a RAVEN II surgical robot providing programmable motion with feedback control using stereo-pair surveillance cameras. To verify the accuracy of the three-dimensional (3-D) reconstructed surgical field, a multimodal physical-sized model of debulked brain tumor was used to obtain the 3-D locations of residual tumor for robotic path planning to remove fluorescent cells. Such reconstruction is repeated intraoperatively during margin clean-up so the algorithm efficiency and accuracy are important to the robotically assisted surgery. Experimental results indicate that the time for creating this 3-D surface can be reduced to one-third by using known trajectories of a robot arm, and the error from the reconstructed phantom is within 0.67 mm in average compared to the model design. PMID:26158071

Multiparametric evaluation of hindlimb ischemia using time-series indocyanine green fluorescence imaging.

PubMed

Guang, Huizhi; Cai, Chuangjian; Zuo, Simin; Cai, Wenjuan; Zhang, Jiulou; Luo, Jianwen

2017-03-01

Peripheral arterial disease (PAD) can further cause lower limb ischemia. Quantitative evaluation of the vascular perfusion in the ischemic limb contributes to diagnosis of PAD and preclinical development of new drug. In vivo time-series indocyanine green (ICG) fluorescence imaging can noninvasively monitor blood flow and has a deep tissue penetration. The perfusion rate estimated from the time-series ICG images is not enough for the evaluation of hindlimb ischemia. The information relevant to the vascular density is also important, because angiogenesis is an essential mechanism for post-ischemic recovery. In this paper, a multiparametric evaluation method is proposed for simultaneous estimation of multiple vascular perfusion parameters, including not only the perfusion rate but also the vascular perfusion density and the time-varying ICG concentration in veins. The target method is based on a mathematical model of ICG pharmacokinetics in the mouse hindlimb. The regression analysis performed on the time-series ICG images obtained from a dynamic reflectance fluorescence imaging system. The results demonstrate that the estimated multiple parameters are effective to quantitatively evaluate the vascular perfusion and distinguish hypo-perfused tissues from well-perfused tissues in the mouse hindlimb. The proposed multiparametric evaluation method could be useful for PAD diagnosis. The estimated perfusion rate and vascular perfusion density maps (left) and the time-varying ICG concentration in veins of the ankle region (right) of the normal and ischemic hindlimbs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging

PubMed Central

Spagnol, Stephen T.; Dahl, Kris Noel

2016-01-01

The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes. PMID:26765322

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability.

PubMed

Hasan, Md Mehedi; Alam, Mohammad Wajih; Wahid, Khan A; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size.

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability

PubMed Central

Hasan, Md. Mehedi; Wahid, Khan A.; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size. PMID:27977709

Visualization of the post-Golgi vesicle-mediated transportation of TGF-β receptor II by quasi-TIRFM.

PubMed

Luo, Wangxi; Xia, Tie; Xu, Li; Chen, Ye-Guang; Fang, Xiaohong

2014-10-01

Transforming growth factor β receptor II (Tβ RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live-cell fluorescence imaging techniques, in particular quasi-total internal reflection fluorescence microscopy, to imaging fluorescent protein-tagged Tβ RII and monitoring its secretion process. We observed punctuate-like Tβ RII-containing post-Golgi vesicles formed in MCF7 cells. Single-particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 μm/s. When stimulated by TGF-β ligand, these receptor-containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post-Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tβ RII-containing vesicles provide new information on intracellular Tβ RII transportation. It also renders Tβ RII a good model system for studying post-Golgi vesicle-trafficking and protein transportation. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A portable near-infrared fluorescence image overlay device for surgical navigation (Conference Presentation)

NASA Astrophysics Data System (ADS)

McWade, Melanie A.

2016-03-01

A rise in the use of near-infrared (NIR) fluorescent dyes or intrinsic fluorescent markers for surgical guidance and tissue diagnosis has triggered the development of NIR fluorescence imaging systems. Because NIR wavelengths are invisible to the naked eye, instrumentation must allow surgeons to visualize areas of high fluorescence. Current NIR fluorescence imaging systems have limited ease-of-use because they display fluorescent information on remote display monitors that require surgeons to divert attention away from the patient to identify the location of tissue fluorescence. Furthermore, some systems lack simultaneous visible light imaging which provides valuable spatial context to fluorescence images. We have developed a novel, portable NIR fluorescence imaging approach for intraoperative surgical guidance that provides information for surgical navigation within the clinician's line of sight. The system utilizes a NIR CMOS detector to collect excited NIR fluorescence from the surgical field. Tissues with NIR fluorescence are overlaid with visible light to provide information on tissue margins directly on the surgical field. In vitro studies have shown this versatile imaging system can be applied to applications with both extrinsic NIR contrast agents such as indocyanine green and weaker sources of biological fluorescence such as parathyroid gland tissue. This non-invasive, portable NIR fluorescence imaging system overlays an image directly on tissue, potentially allowing surgical decisions to be made quicker and with greater ease-of-use than current NIR fluorescence imaging systems.

Three-dimensional imaging of sulfides in silicate rocks at submicron resolution with multiphoton microscopy.

PubMed

Bénard, Antoine; Palle, Sabine; Doucet, Luc Serge; Ionov, Dmitri A

2011-12-01

We report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (∼120 μm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.

3D Actin Network Centerline Extraction with Multiple Active Contours

PubMed Central

Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

2013-01-01

Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we propose a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels. PMID:24316442

qF-SSOP: real-time optical property corrected fluorescence imaging

PubMed Central

Valdes, Pablo A.; Angelo, Joseph P.; Choi, Hak Soo; Gioux, Sylvain

2017-01-01

Fluorescence imaging is well suited to provide image guidance during resections in oncologic and vascular surgery. However, the distorting effects of tissue optical properties on the emitted fluorescence are poorly compensated for on even the most advanced fluorescence image guidance systems, leading to subjective and inaccurate estimates of tissue fluorophore concentrations. Here we present a novel fluorescence imaging technique that performs real-time (i.e., video rate) optical property corrected fluorescence imaging. We perform full field of view simultaneous imaging of tissue optical properties using Single Snapshot of Optical Properties (SSOP) and fluorescence detection. The estimated optical properties are used to correct the emitted fluorescence with a quantitative fluorescence model to provide quantitative fluorescence-Single Snapshot of Optical Properties (qF-SSOP) images with less than 5% error. The technique is rigorous, fast, and quantitative, enabling ease of integration into the surgical workflow with the potential to improve molecular guidance intraoperatively. PMID:28856038

Quantum dots versus organic fluorophores in fluorescent deep-tissue imaging--merits and demerits.

PubMed

Bakalova, Rumiana; Zhelev, Zhivko; Gadjeva, Veselina

2008-12-01

The use of fluorescence in deep-tissue imaging is rapidly expanding in last several years. The progress in fluorescent molecular probes and fluorescent imaging techniques gives an opportunity to detect single cells and even molecular targets in live organisms. The highly sensitive and high-speed fluorescent molecular sensors and detection devices allow the application of fluorescence in functional imaging. With the development of novel bright fluorophores based on nanotechnologies and 3D fluorescence scanners with high spatial and temporal resolution, the fluorescent imaging has a potential to become an alternative of the other non-invasive imaging techniques as magnetic resonance imaging, positron-emission tomography, X-ray, computing tomography. The fluorescent imaging has also a potential to give a real map of human anatomy and physiology. The current review outlines the advantages of fluorescent nanoparticles over conventional organic dyes in deep-tissue imaging in vivo and defines the major requirements to the "perfect fluorophore". The analysis proceeds from the basic principles of fluorescence and major characteristics of fluorophores, light-tissue interactions, and major limitations of fluorescent deep-tissue imaging. The article is addressed to a broad readership - from specialists in this field to university students.

X-ray Fluorescence Spectroscopy: the Potential of Astrophysics-developed Techniques

NASA Astrophysics Data System (ADS)

Elvis, M.; Allen, B.; Hong, J.; Grindlay, J.; Kraft, R.; Binzel, R. P.; Masterton, R.

2012-12-01

X-ray fluorescence from the surface of airless bodies has been studied since the Apollo X-ray fluorescence experiment mapped parts of the lunar surface in 1971-1972. That experiment used a collimated proportional counter with a resolving power of ~1 and a beam size of ~1degree. Filters separated only Mg, Al and SI lines. We review progress in X-ray detectors and imaging for astrophysics and show how these advances enable much more powerful use of X-ray fluorescence for the study of airless bodies. Astrophysics X-ray instrumentation has developed enormously since 1972. Low noise, high quantum efficiency, X-ray CCDs have flown on ASCA, XMM-Newton, the Chandra X-ray Observatory, Swift and Suzaku, and are the workhorses of X-ray astronomy. They normally span 0.5 to ~8 keV with an energy resolution of ~100 eV. New developments in silicon based detectors, especially individual pixel addressable devices, such as CMOS detectors, can withstand many orders of magnitude more radiation than conventional CCDs before degradation. The capability of high read rates provides dynamic range and temporal resolution. Additionally, the rapid read rates minimize shot noise from thermal dark current and optical light. CMOS detectors can therefore run at warmer temperatures and with ultra-thin optical blocking filters. Thin OBFs mean near unity quantum efficiency below 1 keV, thus maximizing response at the C and O lines.such as CMOS detectors, promise advances. X-ray imaging has advanced similarly far. Two types of imager are now available: specular reflection and coded apertures. X-ray mirrors have been flown on the Einstein Observatory, XMM-Newton, Chandra and others. However, as X-ray reflection only occurs at small (~1degree) incidence angles, which then requires long focal lengths (meters), mirrors are not usually practical for planetary missions. Moreover the field of view of X-ray mirrors is comparable to the incident angle, so can only image relatively small regions. More useful are coded-aperture imagers, which have flown on ART-P, Integral, and Swift. The shadow pattern from a 50% full mask allows the distribution of X-rays from a wide (10s of degrees) field of view to be imaged, but uniform emission presents difficulties. A version of a coded-aperture plus CCD detector for airless bodies study is being built for OSIRIS-REx as the student experiment REXIS. We will show the quality of the spectra that can be expected from this class of instrument.

Spectral Neugebauer-based color halftone prediction model accounting for paper fluorescence.

PubMed

Hersch, Roger David

2014-08-20

We present a spectral model for predicting the fluorescent emission and the total reflectance of color halftones printed on optically brightened paper. By relying on extended Neugebauer models, the proposed model accounts for the attenuation by the ink halftones of both the incident exciting light in the UV wavelength range and the emerging fluorescent emission in the visible wavelength range. The total reflectance is predicted by adding the predicted fluorescent emission relative to the incident light and the pure reflectance predicted with an ink-spreading enhanced Yule-Nielsen modified Neugebauer reflectance prediction model. The predicted fluorescent emission spectrum as a function of the amounts of cyan, magenta, and yellow inks is very accurate. It can be useful to paper and ink manufacturers who would like to study in detail the contribution of the fluorescent brighteners and the attenuation of the fluorescent emission by ink halftones.

Hyperspectral photoacoustic spectroscopy of highly-absorbing samples for diagnostic ocular imaging applications

NASA Astrophysics Data System (ADS)

Lim, Hoong-Ta; Murukeshan, Vadakke Matham

2017-01-01

Photoacoustic spectroscopy has been used to measure optical absorption coefficient and the application of tens of wavelength bands in photoacoustic spectroscopy was reported. Using optical methods, absorption-related information is, generally, derived from reflectance or transmittance values. Hence measurement accuracy is limited for highly absorbing samples where the reflectance or transmittance is too low to give reasonable signal-to-noise ratio. In this context, this paper proposes and illustrates a hyperspectral photoacoustic spectroscopy system to measure the absorption-related properties of highly absorbing samples directly. The normalized optical absorption coefficient spectrum of the highly absorbing iris is acquired using an optical absorption coefficient standard. The proposed concepts and the feasibility of the developed diagnostic medical imaging system are demonstrated using fluorescent microsphere suspensions and porcine eyes as test samples.

Quantitative optical imaging of paracetamol-induced metabolism changes in the liver

NASA Astrophysics Data System (ADS)

Liang, Xiaowen; Wang, Haolu; Liu, Xin; Roberts, Michael

2016-12-01

Paracetamol is the most readily available and widely used painkiller. However, its toxicity remains the most common cause of liver injury. The toxicity of paracetamol has been attributing to its toxic metabolite, which depletes cellular glutathione (GSH) stores and reacts within cells to increase oxidative stress, leading to mitochondrial dysfunction and cell necrosis. Multiphoton microscopy (MPM) and fluorescence lifetime imaging (FLIM) can provide quantitative imaging of biological tissues and organs in vivo and allow direct visualization of cellular events, which were used to monitor cellular metabolism in paracetamol-induced toxicity in this study. To better understand mechanisms of paracetamol induced liver injury, the redox ratio of NADH/FAD in liver cells were detected and quantified by MPM imaging to represent the relative rates of glycolysis and oxidative phosphorylation within cells. Compared to normal liver, average fluorescence lifetime of NADH and redox ratio of NADH/FAD in hepatocytes was significantly decreased after paracetamol overdose for 12 and 24 hrs, reflecting impaired metabolic activity. GSH levels of treatment groups were significantly lower than those of normal livers, with gradually decreasing from periportal to centrilobular zonation. This imaging technique has significant implications for investigating metabolic mechanisms of paracetamol toxicity.

Diffuse reflectance spectra measured in vivo in human tissues during Photofrin-mediated pleural photodynamic therapy

NASA Astrophysics Data System (ADS)

Finlay, Jarod C.; Zhu, Timothy C.; Dimofte, Andreea; Friedberg, Joseph S.; Hahn, Stephen M.

2006-02-01

Optimal delivery of light in photodynamic therapy (PDT) requires not only optimal placement and power of light sources, but knowledge of the dynamics of light propagation in the tissue being treated and in the surrounding normal tissue, and of their respective accumulations of sensitizer. In an effort to quantify both tissue optical properties and sensitizer distribution, we have measured fluorescence emission and diffuse reflectance spectra at the surface of a variety of tissue types in the thoracic cavities of human patients. The patients studied here were enrolled in Phase II clinical trials of Photofrin-mediated PDT for the treatment of non-small cell lung cancer and cancers with pleural effusion. Patients were given Photofrin at dose of 2 mg per kg body weight 24 hours prior to treatment. Each patient received surgical resection of the affected lung and pleura. Patients received intracavity PDT at 630nm to a dose of 30 J/cm2, as determined by isotropic detectors sutured to the cavity walls. We measured the diffuse reflectance spectra before and after PDT in various positions within the cavity, including tumor, diaphragm, pericardium, skin, and chest wall muscle in 5 patients. The measurements we acquired using a specially designed fiber optic-based probe consisting of one fluorescence excitation fiber, one white light delivery fiber, and 9 detection fibers spaced at distances from 0.36 to 7.8 mm from the source, all of which are imaged via a spectrograph onto a CCD, allowing measurement of radially-resolved diffuse reflectance and fluorescence spectra. The light sources for these two measurements (a 403-nm diode laser and a halogen lamp, respectively) were blocked by computer-controlled shutters, allowing sequential fluorescence, reflectance, and background acquisition. The diffuse reflectance was analyzed to determine the absorption and scattering spectra of the tissue and from these, the concentration and oxygenation of hemoglobin and the local drug uptake. The total hemoglobin concentration in normal tissues varied from 50 to 300 µM, and the oxygen saturation was generally above 60%. One tumor measured exhibited higher hemoglobin concentration and lower saturation.

Detection of plum pox virus infection in selection plum trees using spectral imaging

NASA Astrophysics Data System (ADS)

Angelova, Liliya; Stoev, Antoniy; Borisova, Ekaterina; Avramov, Latchezar

2016-01-01

Plum pox virus (PPV) is among the most studied viral diseases in the world in plants. It is considered to be one of the most devastating diseases of stone fruits in terms of agronomic impact and economic importance. Noninvasive, fast and reliable techniques are required for evaluation of the pathology in selection trees with economic impact. Such advanced tools for PPV detection could be optical techniques as light-induced fluorescence and diffuse reflectance spectroscopies. Specific regions in the electromagnetic spectra have been found to provide information about the physiological stress in plants, and consequently, diseased plants usually exhibit different spectral signature than non-stressed healthy plants in those specific ranges. In this study spectral reflectance and chlorophyll fluorescence were used for the identification of biotic stress caused by the pox virus on plum trees. The spectral responses of healthy and infected leaves from cultivars, which are widespread in Bulgaria were investigated. The two applied techniques revealed statistically significant differences between the spectral data of healthy plum leaves and those infected by PPV in the visible and near-infrared spectral ranges. Their application for biotic stress detection helps in monitoring diseases in plants using the different plant spectral properties in these spectral ranges. The strong relationship between the results indicates the applicability of diffuse reflectance and fluorescence techniques for conducting health condition assessments of vegetation and their importance for plant protection practices.

Use of digital micromirror devices as dynamic pinhole arrays for adaptive confocal fluorescence microscopy

NASA Astrophysics Data System (ADS)

Pozzi, Paolo; Wilding, Dean; Soloviev, Oleg; Vdovin, Gleb; Verhaegen, Michel

2018-02-01

In this work, we present a new confocal laser scanning microscope capable to perform sensorless wavefront optimization in real time. The device is a parallelized laser scanning microscope in which the excitation light is structured in a lattice of spots by a spatial light modulator, while a deformable mirror provides aberration correction and scanning. A binary DMD is positioned in an image plane of the detection optical path, acting as a dynamic array of reflective confocal pinholes, images by a high performance cmos camera. A second camera detects images of the light rejected by the pinholes for sensorless aberration correction.

In vivo fluorescence confocal microscopy: indocyanine green enhances the contrast of epidermal and dermal structures

NASA Astrophysics Data System (ADS)

Skvara, Hans; Kittler, Harald; Schmid, Johannes A.; Plut, Ulrike; Jonak, Constanze

2011-09-01

In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 μm. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future.

High-speed imaging of submerged jet: visualization analysis using proper orthogonality decomposition

NASA Astrophysics Data System (ADS)

Liu, Yingzheng; He, Chuangxin

2016-11-01

In the present study, the submerged jet at low Reynolds numbers was visualized using laser induced fluoresce and high-speed imaging in a water tank. Well-controlled calibration was made to determine linear dependency region of the fluoresce intensity on its concentration. Subsequently, the jet fluid issuing from a circular pipe was visualized using a high-speed camera. The animation sequence of the visualized jet flow field was supplied for the snapshot proper orthogonality decomposition (POD) analysis. Spatio-temporally varying structures superimposed in the unsteady fluid flow were identified, e.g., the axisymmetric mode and the helical mode, which were reflected from the dominant POD modes. The coefficients of the POD modes give strong indication of temporal and spectral features of the corresponding unsteady events. The reconstruction using the time-mean visualization and the selected POD modes was conducted to reveal the convective motion of the buried vortical structures. National Natural Science Foundation of China.

Targeted Near-Infrared Fluorescence Imaging of Atherosclerosis: Clinical and Intracoronary Evaluation of Indocyanine Green.

PubMed

Verjans, Johan W; Osborn, Eric A; Ughi, Giovanni J; Calfon Press, Marcella A; Hamidi, Ehsan; Antoniadis, Antonios P; Papafaklis, Michail I; Conrad, Mark F; Libby, Peter; Stone, Peter H; Cambria, Richard P; Tearney, Guillermo J; Jaffer, Farouc A

2016-09-01

This study sought to determine whether indocyanine green (ICG)-enhanced near-infrared fluorescence (NIRF) imaging can illuminate high-risk histologic plaque features of human carotid atherosclerosis, and in coronary atheroma of living swine, using intravascular NIRF-optical coherence tomography (OCT) imaging. New translatable imaging approaches are needed to identify high-risk biological signatures of atheroma. ICG is a U.S. Food and Drug Administration-approved NIRF imaging agent that experimentally targets plaque macrophages and lipid in areas of enhanced endothelial permeability. However, it is unknown whether ICG can target atheroma in patients. Eight patients were enrolled in the BRIGHT-CEA (Indocyanine Green Fluorescence Uptake in Human Carotid Artery Plaque) trial. Five patients were injected intravenously with ICG 99 ± 25 min before clinically indicated carotid endarterectomy. Three saline-injected endarterectomy patients served as control subjects. Excised plaques underwent analysis by intravascular NIRF-OCT, reflectance imaging, microscopy, and histopathology. Next, following ICG intravenous injection, in vivo intracoronary NIRF-OCT and intravascular ultrasound imaged 3 atheroma-bearing coronary arteries of a diabetic, cholesterol-fed swine. ICG was well tolerated; no adverse clinical events occurred up to 30 days post-injection. Multimodal NIRF imaging including intravascular NIRF-OCT revealed that ICG accumulated in all endarterectomy specimens. Plaques from saline-injected control patients exhibited minimal NIRF signal. In the swine experiment, intracoronary NIRF-OCT identified ICG uptake in all intravascular ultrasound-identified plaques in vivo. On detailed microscopic evaluation, ICG localized to plaque areas exhibiting impaired endothelial integrity, including disrupted fibrous caps, and within areas of neovascularization. Within human plaque areas of endothelial abnormality, ICG was spatially related to localized zones of plaque macrophages and lipid, and, notably, intraplaque hemorrhage. This study demonstrates that ICG targets human plaques exhibiting endothelial abnormalities and provides new insights into its targeting mechanisms in clinical and experimental atheroma. Intracoronary NIRF-OCT of ICG may offer a novel, clinically translatable approach to image pathobiological aspects of coronary atherosclerosis. (Indocyanine Green Fluorescence Uptake in Human Carotid Artery Plaque [BRIGHT-CEA]; NCT01873716). Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

In-vivo imaging of retinal nerve fiber layer vasculature: imaging - histology comparison

PubMed Central

Scoles, Drew; Gray, Daniel C; Hunter, Jennifer J; Wolfe, Robert; Gee, Bernard P; Geng, Ying; Masella, Benjamin D; Libby, Richard T; Russell, Stephen; Williams, David R; Merigan, William H

2009-01-01

Background Although it has been suggested that alterations of nerve fiber layer vasculature may be involved in the etiology of eye diseases, including glaucoma, it has not been possible to examine this vasculature in-vivo. This report describes a novel imaging method, fluorescence adaptive optics (FAO) scanning laser ophthalmoscopy (SLO), that makes possible for the first time in-vivo imaging of this vasculature in the living macaque, comparing in-vivo and ex-vivo imaging of this vascular bed. Methods We injected sodium fluorescein intravenously in two macaque monkeys while imaging the retina with an FAO-SLO. An argon laser provided the 488 nm excitation source for fluorescence imaging. Reflectance images, obtained simultaneously with near infrared light, permitted precise surface registration of individual frames of the fluorescence imaging. In-vivo imaging was then compared to ex-vivo confocal microscopy of the same tissue. Results Superficial focus (innermost retina) at all depths within the NFL revealed a vasculature with extremely long capillaries, thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, typical of the radial peripapillary capillaries (RPCs). However, at a deeper focus beneath the NFL, (toward outer retina) the polygonal pattern typical of the ganglion cell layer (inner) and outer retinal vasculature was seen. These distinguishing patterns were also seen on histological examination of the same retinas. Furthermore, the thickness of the RPC beds and the caliber of individual RPCs determined by imaging closely matched that measured in histological sections. Conclusion This robust method demonstrates in-vivo, high-resolution, confocal imaging of the vasculature through the full thickness of the NFL in the living macaque, in precise agreement with histology. FAO provides a new tool to examine possible primary or secondary role of the nerve fiber layer vasculature in retinal vascular disorders and other eye diseases, such as glaucoma. PMID:19698151

3D Volumetric Analysis of Fluid Inclusions Using Confocal Microscopy

NASA Astrophysics Data System (ADS)

Proussevitch, A.; Mulukutla, G.; Sahagian, D.; Bodnar, B.

2009-05-01

Fluid inclusions preserve valuable information regarding hydrothermal, metamorphic, and magmatic processes. The molar quantities of liquid and gaseous components in the inclusions can be estimated from their volumetric measurements at room temperatures combined with knowledge of the PVTX properties of the fluid and homogenization temperatures. Thus, accurate measurements of inclusion volumes and their two phase components are critical. One of the greatest advantages of the Laser Scanning Confocal Microscopy (LSCM) in application to fluid inclsion analsyis is that it is affordable for large numbers of samples, given the appropriate software analysis tools and methodology. Our present work is directed toward developing those tools and methods. For the last decade LSCM has been considered as a potential method for inclusion volume measurements. Nevertheless, the adequate and accurate measurement by LSCM has not yet been successful for fluid inclusions containing non-fluorescing fluids due to many technical challenges in image analysis despite the fact that the cost of collecting raw LSCM imagery has dramatically decreased in recent years. These problems mostly relate to image analysis methodology and software tools that are needed for pre-processing and image segmentation, which enable solid, liquid and gaseous components to be delineated. Other challenges involve image quality and contrast, which is controlled by fluorescence of the material (most aqueous fluid inclusions do not fluoresce at the appropriate laser wavelengths), material optical properties, and application of transmitted and/or reflected confocal illumination. In this work we have identified the key problems of image analysis and propose some potential solutions. For instance, we found that better contrast of pseudo-confocal transmitted light images could be overlayed with poor-contrast true-confocal reflected light images within the same stack of z-ordered slices. This approach allows one to narrow the interface boundaries between the phases before the application of segmentation routines. In turn, we found that an active contour segmentation technique works best for these types of geomaterials. The method was developed by adapting a medical software package implemented using the Insight Toolkit (ITK) set of algorithms developed for segmentation of anatomical structures. We have developed a manual analysis procedure with the potential of 2 micron resolution in 3D volume rendering that is specifically designed for application to fluid inclusion volume measurements.

Impact of surface grafting density of PEG macromolecules on dually fluorescent silica nanoparticles used for the in vivo imaging of subcutaneous tumors.

PubMed

Adumeau, Laurent; Genevois, Coralie; Roudier, Lydia; Schatz, Christophe; Couillaud, Franck; Mornet, Stéphane

2017-06-01

In the context of systematically administered nanomedicines, the physicochemistry of NP surfaces must be controlled as a prerequisite to improve blood circulation time, and passive and active targeting. In particular, there is a real need to develop NP stealth and labelling for both in vivo and microscopic fluorescence imaging in a mice model. We have synthesized NIR/red dually fluorescent silica nanoparticles of 19nm covalently covered by a PEG layer of different grafting density in the brush conformational regime by using a reductive amination reaction. These particles were characterized by TEM, DRIFT, DLS, TGA, ζ potential measurements, UV-vis and fluorescence spectroscopy. Prostate tumors were generated in mice by subcutaneous injection of RM1-CMV-Fluc cells. Tumor growth was monitored by BLI after a D-luciferin injection. Four samples of PEGylated fluorescent NPs were individually intravenously injected into 6 mice (N=6, total 24 mice). Nanoparticle distribution was investigated using in vivo fluorescence reflectance imaging (FRI) over 48h and microscopy imaging was employed to localize the NPs within tumors in vitro. Fluorescent NP accumulation, due to the enhanced permeability and retention (EPR) effect, increases gradually as a function of increased PEG surface grafting density with a huge difference observed for the highest density grafting. For the highest grafting density, a blood circulation time of up to 24h was observed with a strong reduction in uptake by the liver. In vivo experimental results suggest that the biodistribution of NPs is very sensitive to slight variations in surface grafting density when the NPs present a high curvature radius. This study underlines the need to compensate a high curvature radius with a PEG-saturated NP surface to improve blood circulation and accumulation within tumors through the EPR effect. Dually fluorescent NPs PEGylated to saturation display physical properties useful for assessing the susceptibility of tumors to the EPR effect. Control of the physicochemical features of nanoparticle surfaces to improve blood circulation times and monitoring of the EPR effect. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader. Copyright © 2017 Elsevier B.V. All rights reserved.

Claudin-4-targeted optical imaging detects pancreatic cancer and its precursor lesions.

PubMed

Neesse, Albrecht; Hahnenkamp, Anke; Griesmann, Heidi; Buchholz, Malte; Hahn, Stefan A; Maghnouj, Abdelouahid; Fendrich, Volker; Ring, Janine; Sipos, Bence; Tuveson, David A; Bremer, Christoph; Gress, Thomas M; Michl, Patrick

2013-07-01

Novel imaging methods based on specific molecular targets to detect both established neoplasms and their precursor lesions are highly desirable in cancer medicine. Previously, we identified claudin-4, an integral constituent of tight junctions, as highly expressed in various gastrointestinal tumours including pancreatic cancer. Here, we investigate the potential of targeting claudin-4 with a naturally occurring ligand to visualise pancreatic cancer and its precursor lesions in vitro and in vivo by near-infrared imaging approaches. A non-toxic C-terminal fragment of the claudin-4 ligand Clostridium perfringens enterotoxin (C-CPE) was labelled with a cyanine dye (Cy5.5). Binding of the optical tracer was analysed on claudin-4 positive and negative cells in vitro, and tumour xenografts in vivo. In addition, two genetically engineered mouse models for pancreatic intraepithelial neoplasia (PanIN) and pancreatic cancer were used for in vivo validation. Optical imaging studies were conducted using 2D planar fluorescence reflectance imaging (FRI) technology and 3D fluorescence-mediated tomography (FMT). In vitro, the peptide-dye conjugate showed high binding affinity to claudin-4 positive CAPAN1 cells, while claudin-4 negative HT1080 cells revealed little or no fluorescence. In vivo, claudin-4 positive tumour xenografts, endogenous pancreatic tumours, hepatic metastases, as well as preinvasive PanIN lesions, were visualised by FRI and FMT up to 48 h after injection showing a significantly higher average of fluorochrome concentration as compared with claudin-4 negative xenografts and normal pancreatic tissue. C-CPE-Cy5.5 combined with novel optical imaging methods enables non-invasive visualisation of claudin-4 positive murine pancreatic tumours and their precursor lesions, representing a promising modality for early diagnostic imaging.

Multispectral open-air intraoperative fluorescence imaging.

PubMed

Behrooz, Ali; Waterman, Peter; Vasquez, Kristine O; Meganck, Jeff; Peterson, Jeffrey D; Faqir, Ilias; Kempner, Joshua

2017-08-01

Intraoperative fluorescence imaging informs decisions regarding surgical margins by detecting and localizing signals from fluorescent reporters, labeling targets such as malignant tissues. This guidance reduces the likelihood of undetected malignant tissue remaining after resection, eliminating the need for additional treatment or surgery. The primary challenges in performing open-air intraoperative fluorescence imaging come from the weak intensity of the fluorescence signal in the presence of strong surgical and ambient illumination, and the auto-fluorescence of non-target components, such as tissue, especially in the visible spectral window (400-650 nm). In this work, a multispectral open-air fluorescence imaging system is presented for translational image-guided intraoperative applications, which overcomes these challenges. The system is capable of imaging weak fluorescence signals with nanomolar sensitivity in the presence of surgical illumination. This is done using synchronized fluorescence excitation and image acquisition with real-time background subtraction. Additionally, the system uses a liquid crystal tunable filter for acquisition of multispectral images that are used to spectrally unmix target fluorescence from non-target auto-fluorescence. Results are validated by preclinical studies on murine models and translational canine oncology models.

Spectroscopic identification of individual fluorophores using photoluminescence excitation spectra.

PubMed

Czerski, J; Colomb, W; Cannataro, F; Sarkar, S K

2018-01-25

The identity of a fluorophore can be ambiguous if other fluorophores or nonspecific fluorescent impurities have overlapping emission spectra. The presence of overlapping spectra makes it difficult to differentiate fluorescent species using discrete detection channels and unmixing of spectra. The unique absorption and emission signatures of fluorophores provide an opportunity for spectroscopic identification. However, absorption spectroscopy may be affected by scattering, whereas fluorescence emission spectroscopy suffers from signal loss by gratings or other dispersive optics. Photoluminescence excitation spectra, where excitation is varied and emission is detected at a fixed wavelength, allows hyperspectral imaging with a single emission filter for high signal-to-background ratio without any moving optics on the emission side. We report a high throughput method for measuring the photoluminescence excitation spectra of individual fluorophores using a tunable supercontinuum laser and prism-type total internal reflection fluorescence microscope. We used the system to measure and sort the photoluminescence excitation spectra of individual Alexa dyes, fluorescent nanodiamonds (FNDs), and fluorescent polystyrene beads. We used a Gaussian mixture model with maximum likelihood estimation to objectively separate the spectra. Finally, we spectroscopically identified different species of fluorescent nanodiamonds with overlapping spectra and characterized the heterogeneity of fluorescent nanodiamonds of varying size. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Fiber-optic fluorescence imaging

PubMed Central

Flusberg, Benjamin A; Cocker, Eric D; Piyawattanametha, Wibool; Jung, Juergen C; Cheung, Eunice L M; Schnitzer, Mark J

2010-01-01

Optical fibers guide light between separate locations and enable new types of fluorescence imaging. Fiber-optic fluorescence imaging systems include portable handheld microscopes, flexible endoscopes well suited for imaging within hollow tissue cavities and microendoscopes that allow minimally invasive high-resolution imaging deep within tissue. A challenge in the creation of such devices is the design and integration of miniaturized optical and mechanical components. Until recently, fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on microfabricated device components. PMID:16299479

Combined fluorescence and reflectance spectroscopy for in vivo quantification of cancer biomarkers in low- and high-grade glioma surgery

PubMed Central

Valdés, Pablo A.; Kim, Anthony; Leblond, Frederic; Conde, Olga M.; Harris, Brent T.; Paulsen, Keith D.; Wilson, Brian C.; Roberts, David W.

2011-01-01

Biomarkers are indicators of biological processes and hold promise for the diagnosis and treatment of disease. Gliomas represent a heterogeneous group of brain tumors with marked intra- and inter-tumor variability. The extent of surgical resection is a significant factor influencing post-surgical recurrence and prognosis. Here, we used fluorescence and reflectance spectral signatures for in vivo quantification of multiple biomarkers during glioma surgery, with fluorescence contrast provided by exogenously-induced protoporphyrin IX (PpIX) following administration of 5-aminolevulinic acid. We performed light-transport modeling to quantify multiple biomarkers indicative of tumor biological processes, including the local concentration of PpIX and associated photoproducts, total hemoglobin concentration, oxygen saturation, and optical scattering parameters. We developed a diagnostic algorithm for intra-operative tissue delineation that accounts for the combined tumor-specific predictive capabilities of these quantitative biomarkers. Tumor tissue delineation achieved accuracies of up to 94% (specificity = 94%, sensitivity = 94%) across a range of glioma histologies beyond current state-of-the-art optical approaches, including state-of-the-art fluorescence image guidance. This multiple biomarker strategy opens the door to optical methods for surgical guidance that use quantification of well-established neoplastic processes. Future work would seek to validate the predictive power of this proof-of-concept study in a separate larger cohort of patients. PMID:22112112

Combined fluorescence and reflectance spectroscopy for in vivo quantification of cancer biomarkers in low- and high-grade glioma surgery

NASA Astrophysics Data System (ADS)

Valdés, Pablo A.; Kim, Anthony; Leblond, Frederic; Conde, Olga M.; Harris, Brent T.; Paulsen, Keith D.; Wilson, Brian C.; Roberts, David W.

2011-11-01

Biomarkers are indicators of biological processes and hold promise for the diagnosis and treatment of disease. Gliomas represent a heterogeneous group of brain tumors with marked intra- and inter-tumor variability. The extent of surgical resection is a significant factor influencing post-surgical recurrence and prognosis. Here, we used fluorescence and reflectance spectral signatures for in vivo quantification of multiple biomarkers during glioma surgery, with fluorescence contrast provided by exogenously-induced protoporphyrin IX (PpIX) following administration of 5-aminolevulinic acid. We performed light-transport modeling to quantify multiple biomarkers indicative of tumor biological processes, including the local concentration of PpIX and associated photoproducts, total hemoglobin concentration, oxygen saturation, and optical scattering parameters. We developed a diagnostic algorithm for intra-operative tissue delineation that accounts for the combined tumor-specific predictive capabilities of these quantitative biomarkers. Tumor tissue delineation achieved accuracies of up to 94% (specificity = 94%, sensitivity = 94%) across a range of glioma histologies beyond current state-of-the-art optical approaches, including state-of-the-art fluorescence image guidance. This multiple biomarker strategy opens the door to optical methods for surgical guidance that use quantification of well-established neoplastic processes. Future work would seek to validate the predictive power of this proof-of-concept study in a separate larger cohort of patients.

Vegetation Red-edge Spectral Modeling for Solar-induced Chlorophyll Fluorescence Retrieval at O2-B Band

NASA Astrophysics Data System (ADS)

Huang, C.; Zhang, L.; Qiao, N.; Zhang, X.; Li, Y.

2015-12-01

Remotely sensed solar-induced chlorophyll fluorescence (SIF) has been considered an ideal probe in monitoring global vegetation photosynthesis. However, challenges in accurate estimate of faint SIF (less than 5% of the total reflected radiation in near infrared bands) from the observed apparent reflected radiation greatly limit its wide applications. Currently, the telluric O2-B (~688nm) and O2-A (~761nm) have been proved to be capable of SIF retrieval based on Fraunhofer line depth (FLD) principle. They may still work well even using conventional ground-based commercial spectrometers with typical spectral resolutions of 2~5 nm and high enough signal-to-noise ratio (e.g., the ASD spectrometer). Nevertheless, almost all current FLD based algorithms were mainly developed for O2-A, a few concentrating on the other SIF emission peak in O2-B. One of the critical reasons is that it is very difficult to model the sudden varying reflectance around O2-B band located in the red-edge spectral region (about 680-800 nm). This study investigates a new method by combining the established inverted Gaussian reflectance model (IGM) and FLD principle using diurnal canopy spectra with relative low spectral resolutions of 1 nm (FluorMOD simulations) and 3 nm (measured by ASD spectrometer) respectively. The IGM has been reported to be an objective and good method to characterize the entire vegetation red-edge reflectance. Consequently, the proposed SIF retrieval method (hereinafter called IGMFLD) could exploit all the spectral information along the whole red-edge (680-800 nm) to obtain more reasonable reflectance and fluorescence correction coefficients than traditional FLD methods such as the iFLD. Initial results show that the IGMFLD can better capture the spectrally non-linear characterization of the reflectance in 680-800 nm and thereby yields much more accurate SIFs in O2-B than typical FLD methods, including sFLD, 3FLD and iFLD (see figure 1). Finally, uncertainties and prospect of the IGM-FLD, in contrast with sFLD, 3FLD and iFLD, were discussed here. This study may provide a test-bed for developing more robust methods to retrieve SIF in O2-B from aircraft (e.g. AisaIBIS fluorescence imager) or satellite (FLEX-FLORIS) remote sensing measurements.

Hyperspectral small animal fluorescence imaging: spectral selection imaging

NASA Astrophysics Data System (ADS)

Leavesley, Silas; Jiang, Yanan; Patsekin, Valery; Hall, Heidi; Vizard, Douglas; Robinson, J. Paul

2008-02-01

Molecular imaging is a rapidly growing area of research, fueled by needs in pharmaceutical drug-development for methods for high-throughput screening, pre-clinical and clinical screening for visualizing tumor growth and drug targeting, and a growing number of applications in the molecular biology fields. Small animal fluorescence imaging employs fluorescent probes to target molecular events in vivo, with a large number of molecular targeting probes readily available. The ease at which new targeting compounds can be developed, the short acquisition times, and the low cost (compared to microCT, MRI, or PET) makes fluorescence imaging attractive. However, small animal fluorescence imaging suffers from high optical scattering, absorption, and autofluorescence. Much of these problems can be overcome through multispectral imaging techniques, which collect images at different fluorescence emission wavelengths, followed by analysis, classification, and spectral deconvolution methods to isolate signals from fluorescence emission. We present an alternative to the current method, using hyperspectral excitation scanning (spectral selection imaging), a technique that allows excitation at any wavelength in the visible and near-infrared wavelength range. In many cases, excitation imaging may be more effective at identifying specific fluorescence signals because of the higher complexity of the fluorophore excitation spectrum. Because the excitation is filtered and not the emission, the resolution limit and image shift imposed by acousto-optic tunable filters have no effect on imager performance. We will discuss design of the imager, optimizing the imager for use in small animal fluorescence imaging, and application of spectral analysis and classification methods for identifying specific fluorescence signals.

Multimodal optical imaging database from tumour brain human tissue: endogenous fluorescence from glioma, metastasis and control tissues

NASA Astrophysics Data System (ADS)

Poulon, Fanny; Ibrahim, Ali; Zanello, Marc; Pallud, Johan; Varlet, Pascale; Malouki, Fatima; Abi Lahoud, Georges; Devaux, Bertrand; Abi Haidar, Darine

2017-02-01

Eliminating time-consuming process of conventional biopsy is a practical improvement, as well as increasing the accuracy of tissue diagnoses and patient comfort. We addressed these needs by developing a multimodal nonlinear endomicroscope that allows real-time optical biopsies during surgical procedure. It will provide immediate information for diagnostic use without removal of tissue and will assist the choice of the optimal surgical strategy. This instrument will combine several means of contrast: non-linear fluorescence, second harmonic generation signal, reflectance, fluorescence lifetime and spectral analysis. Multimodality is crucial for reliable and comprehensive analysis of tissue. Parallel to the instrumental development, we currently improve our understanding of the endogeneous fluorescence signal with the different modalities that will be implemented in the stated. This endeavor will allow to create a database on the optical signature of the diseased and control brain tissues. This proceeding will present the preliminary results of this database on three types of tissues: cortex, metastasis and glioblastoma.

Simultaneous acquisition of trajectory and fluorescence lifetime of moving single particles

NASA Astrophysics Data System (ADS)

Wu, Qianqian; Qi, Jing; Lin, Danying; Yan, Wei; Hu, Rui; Peng, Xiao; Qu, Junle

2017-02-01

Fluorescence lifetime imaging (FLIM) has been a powerful tool in life science because it can reveal the interactions of an excited fluorescent molecule and its environment. The combination with two-photon excitation (TPE) and timecorrelated single photon counting (TCSPC) provides it the ability of optical sectioning, high time resolution and detection efficiency. In previous work, we have introduced a two-dimensional acousto-optic deflector (AOD) into TCSPC-based FLIM to achieve fast and flexible FLIM. In this work, we combined the AOD-FLIM system with a single particle tracking (SPT) setup and algorithm and developed an SPT-FLIM system. Using the system, we acquired the trajectory and fluorescence lifetime of a moving particle simultaneously and reconstructed a life-time-marked pseudocolored trajectory, which might reflect dynamic interaction between the moving particle and its local environment along its motion trail. The results indicated the potential of the technique for studying the interaction between specific moving biological macromolecules and the ambient micro-environment in live cells.

Reflectance Hyperspectral Imaging for Investigation of Works of Art: Old Master Paintings and Illuminated Manuscripts.

PubMed

Cucci, Costanza; Delaney, John K; Picollo, Marcello

2016-10-18

Diffuse reflectance hyperspectral imaging, or reflectance imaging spectroscopy, is a sophisticated technique that enables the capture of hundreds of images in contiguous narrow spectral bands (bandwidth < 10 nm), typically in the visible (Vis, 400-750 nm) and the near-infrared (NIR, 750-2500 nm) regions. This sequence of images provides a data set that is called an image-cube or file-cube. Two dimensions of the image-cube are the spatial dimensions of the scene, and the third dimension is the wavelength. In this way, each spatial pixel in the image has an associated reflectance spectrum. This "big data" image-cube allows for the mining of artists' materials and mapping their distribution across the surface of a work of art. Reflectance hyperspectral imaging, introduced in the 1980s by Goetz and co-workers, led to a revolution in the field of remote sensing of the earth and near planets ( Goetz, F. H.; Vane, G.; Solomon, B. N.; Rock, N. Imaging Spectrometry for Earth Remote Sensing . Science , 1985 , 228 , 1147 - 1152 ). In the subsequent decades, thanks to rapid advances in solid-state sensor technology, reflectance hyperspectral imaging, once only available to large government laboratories, was extended to new fields of application, such as monitoring agri-foods, pharmaceutical products, the environment, and cultural heritage. In the 2000s, the potential of this noninvasive technology for the study of artworks became evident and, consequently, the methodology is becoming more widely used in the art conservation science field. Typically hyperspectral reflectance image-cubes contain millions of spectra. Many of these spectra are similar, making the reduction of the data set size an important first step. Thus, image-processing tools based on multivariate techniques, such as principal component analysis (PCA), automated classification methods, or expert knowledge systems, that search for known spectral features are often applied. These algorithms seek to reduce the large number of high-quality spectra to a common subset, which allow identifying and mapping artists' materials and alteration products. Hence, reflectance hyperspectral imaging is finding its place as the starting point to find sites on polychrome surfaces for spot analytical techniques, such as X-ray fluorescence, Raman spectroscopy, and Fourier transform infrared spectroscopy. Reflectance hyperspectral imaging can also provide image products that are a mainstay in the art conservation field, such as color-accurate images, broadband near-infrared images, and false-color products. This Account reports on the research activity carried out by two research groups, one at the "Nello Carrara" Institute of Applied Physics of the Italian National Research Council (IFAC-CNR) in Florence and the other at the National Gallery of Art (NGA) in Washington, D.C. Both groups have conducted parallel research, with frequent interchanges, to develop multispectral and hyperspectral imaging systems to study works of art. In the past decade, they have designed and experimented with some of the earliest spectral imaging prototypes for museum applications. In this Account, a brief presentation of the hyperspectral sensor systems is given with case studies showing how reflectance hyperspectral imaging is answering key questions in cultural heritage.

Field Geology/Processes

NASA Technical Reports Server (NTRS)

Allen, Carlton; Jakes, Petr; Jaumann, Ralf; Marshall, John; Moses, Stewart; Ryder, Graham; Saunders, Stephen; Singer, Robert

1996-01-01

The field geology/process group examined the basic operations of a terrestrial field geologist and the manner in which these operations could be transferred to a planetary lander. Four basic requirements for robotic field geology were determined: geologic content; surface vision; mobility; and manipulation. Geologic content requires a combination of orbital and descent imaging. Surface vision requirements include range, resolution, stereo, and multispectral imaging. The minimum mobility for useful field geology depends on the scale of orbital imagery. Manipulation requirements include exposing unweathered surfaces, screening samples, and bringing samples in contact with analytical instruments. To support these requirements, several advanced capabilities for future development are recommended. Capabilities include near-infrared reflectance spectroscopy, hyper-spectral imaging, multispectral microscopy, artificial intelligence in support of imaging, x ray diffraction, x ray fluorescence, and rock chipping.

Fundus Autofluorescence and Photoreceptor Cell Rosettes in Mouse Models

PubMed Central

Flynn, Erin; Ueda, Keiko; Auran, Emily; Sullivan, Jack M.; Sparrow, Janet R.

2014-01-01

Purpose. This study was conducted to study correlations among fundus autofluorescence (AF), RPE lipofuscin accumulation, and photoreceptor cell degeneration and to investigate the structural basis of fundus AF spots. Methods. Fundus AF images (55° lens; 488-nm excitation) and spectral-domain optical coherence tomography (SD-OCT) scans were acquired in pigmented Rdh8−/−/Abca4−/− mice (ages 1–9 months) with a confocal scanning laser ophthalmoscope (cSLO). For quantitative fundus AF (qAF), gray levels (GLs) were calibrated to an internal fluorescence reference. Retinal bisretinoids were measured by quantitative HPLC. Histometric analysis of outer nuclear layer (ONL) thicknesses was performed, and cryostat sections of retina were examined by fluorescence microscopy. Results. Quantified A2E and qAF intensities increased until age 4 months in the Rdh8−/−/Abca4−/− mice. The A2E levels declined after 4 months of age, but qAF intensity values continued to rise. The decline in A2E levels in the Rdh8−/−/Abca4−/− mice paralleled reduced photoreceptor cell viability as reflected in ONL thinning. Hyperautofluorescent puncta in fundus AF images corresponded to photoreceptor cell rosettes in SD-OCT images and histological sections stained with hematoxylin and eosin. The inner segment/outer segment–containing core of the rosette emitted an autofluorescence detected by fluorescence microscopy. Conclusions. When neural retina is disordered, AF from photoreceptor cells can contribute to noninvasive fundus AF images. Hyperautofluorescent puncta in fundus AF images are attributable, in at least some cases, to photoreceptor cell rosettes. PMID:25015357

Fluorescence Sensing Using DNA Aptamers in Cancer Research and Clinical Diagnostics

PubMed Central

Musumeci, Domenica; Platella, Chiara; Riccardi, Claudia; Moccia, Federica

2017-01-01

Among the various advantages of aptamers over antibodies, remarkable is their ability to tolerate a large number of chemical modifications within their backbone or at the termini without losing significant activity. Indeed, aptamers can be easily equipped with a wide variety of reporter groups or coupled to different carriers, nanoparticles, or other biomolecules, thus producing valuable molecular recognition tools effective for diagnostic and therapeutic purposes. This review reports an updated overview on fluorescent DNA aptamers, designed to recognize significant cancer biomarkers both in soluble or membrane-bound form. In many examples, the aptamer secondary structure switches induced by target recognition are suitably translated in a detectable fluorescent signal using either fluorescently-labelled or label-free aptamers. The fluorescence emission changes, producing an enhancement (“signal-on”) or a quenching (“signal-off”) effect, directly reflect the extent of the binding, thereby allowing for quantitative determination of the target in bioanalytical assays. Furthermore, several aptamers conjugated to fluorescent probes proved to be effective for applications in tumour diagnosis and intraoperative surgery, producing tumour-type specific, non-invasive in vivo imaging tools for cancer pre- and post-treatment assessment. PMID:29261171

Oxygen sensing PLIM together with FLIM of intrinsic cellular fluorophores for metabolic mapping

NASA Astrophysics Data System (ADS)

Kalinina, Sviatlana; Schaefer, Patrick; Breymayer, Jasmin; Bisinger, Dominik; Chakrabortty, Sabyasachi; Rueck, Angelika

2018-02-01

Otical imaging techniques based on time correlated single photon counting (TCSPC) has found wide applications in medicine and biology. Non-invasive and information-rich fluorescence lifetime imaging microscopy (FLIM) is successfully used for monitoring fluorescent intrinsic metabolic coenzymes as NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) and FAD+ (flavin adenine dinucleotide) in living cells and tissues. The ratio between proteinbound and free coenzymes gives an information about the balance between oxidative phosphorylation and glycolysis in the cells. The changes of the ratio reflects major cellular disorders. A correlation exists between metabolic activity, redox ratio and fluorescence lifetime during stem cell differentiation, neurodegenerative diseases, and carcinogenesis. A multichannel FLIM detection system was designed for monitoring the redox state of NAD(P)H and FAD+ and other intrinsic fluorophores as protoporphyrin IX. In addition, the unique upgrade is useful to perform FLIM and PLIM (phosphorescence lifetime imaging microscopy) simultaneously. PLIM is a promising method to investigate oxygen sensing in biomedical samples. In detail, the oxygen-dependent quenching of phosphorescence of some compounds as transition metal complexes enables measuring of oxygen partial pressure (pO2). Using a two-channel FLIM/PLIM system we monitored intrinsic pO2 by PLIM simultaneously with NAD(P)H by FLIM providing complex metabolic and redox imaging of living cells. Physico-chemical properties of oxygen sensitive probes define certain parameters including their localisation. We present results of some ruthenium based complexes including those specifically bound to mitochondria.

Observing the conformation of individual SNARE proteins inside live cells

NASA Astrophysics Data System (ADS)

Weninger, Keith

2010-10-01

Protein conformational dynamics are directly linked to function in many instances. Within living cells, protein dynamics are rarely synchronized so observing ensemble-averaged behaviors can hide details of signaling pathways. Here we present an approach using single molecule fluorescence resonance energy transfer (FRET) to observe the conformation of individual SNARE proteins as they fold to enter the SNARE complex in living cells. Proteins were recombinantly expressed, labeled with small-molecule fluorescent dyes and microinjected for in vivo imaging and tracking using total internal reflection microscopy. Observing single molecules avoids the difficulties of averaging over unsynchronized ensembles. Our approach is easily generalized to a wide variety of proteins in many cellular signaling pathways.

Label-free monitoring of cell death induced by oxidative stress in living human cells using terahertz ATR spectroscopy

PubMed Central

Zou, Yi; Liu, Qiao; Yang, Xia; Huang, Hua-Chuan; Li, Jiang; Du, Liang-Hui; Li, Ze-Ren; Zhao, Jian-Heng; Zhu, Li-Guo

2017-01-01

We demonstrated that attenuated total reflectance terahertz time-domain spectroscopy (ATR THz-TDS) is able to monitor oxidative stress response of living human cells, which is proven in this work that it is an efficient non-invasive, label-free, real-time and in situ monitoring of cell death. Furthermore, the dielectric constant and dielectric loss of cultured living human breast epithelial cells, and along with their evolution under oxidative stress response induced by high concentration of H2O2, were quantitatively determined in the work. Our observation and results were finally confirmed using standard fluorescence-labeled flow cytometry measurements and visible fluorescence imaging. PMID:29359084

High throughput, parallel imaging and biomarker quantification of human spermatozoa by ImageStream flow cytometry.

PubMed

Buckman, Clayton; George, Thaddeus C; Friend, Sherree; Sutovsky, Miriam; Miranda-Vizuete, Antonio; Ozanon, Christophe; Morrissey, Phil; Sutovsky, Peter

2009-12-01

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.

Real-time intraoperative fluorescence imaging system using light-absorption correction.

PubMed

Themelis, George; Yoo, Jung Sun; Soh, Kwang-Sup; Schulz, Ralf; Ntziachristos, Vasilis

2009-01-01

We present a novel fluorescence imaging system developed for real-time interventional imaging applications. The system implements a correction scheme that improves the accuracy of epi-illumination fluorescence images for light intensity variation in tissues. The implementation is based on the use of three cameras operating in parallel, utilizing a common lens, which allows for the concurrent collection of color, fluorescence, and light attenuation images at the excitation wavelength from the same field of view. The correction is based on a ratio approach of fluorescence over light attenuation images. Color images and video is used for surgical guidance and for registration with the corrected fluorescence images. We showcase the performance metrics of this system on phantoms and animals, and discuss the advantages over conventional epi-illumination systems developed for real-time applications and the limits of validity of corrected epi-illumination fluorescence imaging.

In vivo imaging of microscopic structures in the rat retina

PubMed Central

Geng, Ying; Greenberg, Kenneth P.; Wolfe, Robert; Gray, Daniel C.; Hunter, Jennifer J.; Dubra, Alfredo; Flannery, John G.; Williams, David R.; Porter, Jason

2010-01-01

Purpose The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. We have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo. Methods Green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy. Results Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics (AO). AO correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was ∼1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF. Conclusions With perfect aberration compensation, the in vivo resolution in the rat eye could be ∼2× greater than that in the human eye due to its large numerical aperture (∼0.43). While the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using AO to investigate the rodent eye in vivo with high resolution. PMID:19578019

A Hierarchical Convolutional Neural Network for vesicle fusion event classification.

PubMed

Li, Haohan; Mao, Yunxiang; Yin, Zhaozheng; Xu, Yingke

2017-09-01

Quantitative analysis of vesicle exocytosis and classification of different modes of vesicle fusion from the fluorescence microscopy are of primary importance for biomedical researches. In this paper, we propose a novel Hierarchical Convolutional Neural Network (HCNN) method to automatically identify vesicle fusion events in time-lapse Total Internal Reflection Fluorescence Microscopy (TIRFM) image sequences. Firstly, a detection and tracking method is developed to extract image patch sequences containing potential fusion events. Then, a Gaussian Mixture Model (GMM) is applied on each image patch of the patch sequence with outliers rejected for robust Gaussian fitting. By utilizing the high-level time-series intensity change features introduced by GMM and the visual appearance features embedded in some key moments of the fusion process, the proposed HCNN architecture is able to classify each candidate patch sequence into three classes: full fusion event, partial fusion event and non-fusion event. Finally, we validate the performance of our method on 9 challenging datasets that have been annotated by cell biologists, and our method achieves better performances when comparing with three previous methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Shortwave infrared fluorescence imaging with the clinically approved near-infrared dye indocyanine green.

PubMed

Carr, Jessica A; Franke, Daniel; Caram, Justin R; Perkinson, Collin F; Saif, Mari; Askoxylakis, Vasileios; Datta, Meenal; Fukumura, Dai; Jain, Rakesh K; Bawendi, Moungi G; Bruns, Oliver T

2018-04-24

Fluorescence imaging is a method of real-time molecular tracking in vivo that has enabled many clinical technologies. Imaging in the shortwave IR (SWIR; 1,000-2,000 nm) promises higher contrast, sensitivity, and penetration depths compared with conventional visible and near-IR (NIR) fluorescence imaging. However, adoption of SWIR imaging in clinical settings has been limited, partially due to the absence of US Food and Drug Administration (FDA)-approved fluorophores with peak emission in the SWIR. Here, we show that commercially available NIR dyes, including the FDA-approved contrast agent indocyanine green (ICG), exhibit optical properties suitable for in vivo SWIR fluorescence imaging. Even though their emission spectra peak in the NIR, these dyes outperform commercial SWIR fluorophores and can be imaged in the SWIR, even beyond 1,500 nm. We show real-time fluorescence imaging using ICG at clinically relevant doses, including intravital microscopy, noninvasive imaging in blood and lymph vessels, and imaging of hepatobiliary clearance, and show increased contrast compared with NIR fluorescence imaging. Furthermore, we show tumor-targeted SWIR imaging with IRDye 800CW-labeled trastuzumab, an NIR dye being tested in multiple clinical trials. Our findings suggest that high-contrast SWIR fluorescence imaging can be implemented alongside existing imaging modalities by switching the detection of conventional NIR fluorescence systems from silicon-based NIR cameras to emerging indium gallium arsenide-based SWIR cameras. Using ICG in particular opens the possibility of translating SWIR fluorescence imaging to human clinical applications. Indeed, our findings suggest that emerging SWIR-fluorescent in vivo contrast agents should be benchmarked against the SWIR emission of ICG in blood.

Integrated Raman spectroscopy and trimodal wide-field imaging techniques for real-time in vivo tissue Raman measurements at endoscopy.

PubMed

Huang, Zhiwei; Teh, Seng Khoon; Zheng, Wei; Mo, Jianhua; Lin, Kan; Shao, Xiaozhuo; Ho, Khek Yu; Teh, Ming; Yeoh, Khay Guan

2009-03-15

We report an integrated Raman spectroscopy and trimodal (white-light reflectance, autofluorescence, and narrow-band) imaging techniques for real-time in vivo tissue Raman measurements at endoscopy. A special 1.8 mm endoscopic Raman probe with filtering modules is developed, permitting effective elimination of interference of fluorescence background and silica Raman in fibers while maximizing tissue Raman collections. We demonstrate that high-quality in vivo Raman spectra of upper gastrointestinal tract can be acquired within 1 s or subseconds under the guidance of wide-field endoscopic imaging modalities, greatly facilitating the adoption of Raman spectroscopy into clinical research and practice during routine endoscopic inspections.

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

PubMed Central

Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

2011-01-01

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

WE-FG-BRA-04: A Portable Confocal Microscope to Image Live Cell Damage Response Induced by Therapeutic Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

McFadden, C; Flint, D; Grosshans, D

Purpose: To construct a custom and portable fluorescence confocal laser-scanning microscope (FCLSM) that can be placed in the path of therapeutic radiation beams to study real-time radiation-induced damage response in live cells. Methods: We designed and constructed a portable FCLSM with three laser diodes for excitation (405, 488, and 635 nm). An objective lens focuses the excitation light and collects fluorescence from the sample. A pair of galvanometer mirrors scans/collects the laser beam/fluorescence along the focal plane (x/y-directions). A stepper motor stage scans in the axial direction and positions the x/y of the image field. Barrier filters and dichroic mirrorsmore » are used to route the spectral emission bands to the appropriate photodetector. An avalanche photodiode collects near-infrared fluorescence; a photodiode collects back-reflected 635 nm light; and a photomultiplier tube collects green fluorescence in the range of eGFP/eYFP. A 200-µm diameter pinhole was used to implement the confocal geometry for near-infrared and red channels and a 150-µm diameter pinhole for the green channel. Data acquisition and system control were achieved using a high-throughput data acquisition card. In-house software developed in LabVIEW was used to control the hardware, collect data from the photodetectors and reconstruct the confocal images. Results: 6 frames/s can be acquired for a 25 µm{sup 2} (128×128 pixels) field of view, visualizing the entire volume of the cell nucleus (∼10 µm depth) in <10 s. To demonstrate the usefulness of our FCLSM, we imaged gold nanoshells in live cells, radiation-induced damage in fibrosarcoma cells expressing eGFP tagged to a DNA repair protein, and neurons expressing eGFP. The system can also image particle tracks in fluorescent nuclear track detectors. Conclusion: We developed a versatile and portable FCLSM that allows radiobiology studies in live cells exposed to therapeutic radiation. The FCLSM can be placed in any vertical beam line for top-to-bottom exposures. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.« less

Improved facial nerve identification during parotidectomy with fluorescently labeled peptide.

PubMed

Hussain, Timon; Nguyen, Linda T; Whitney, Michael; Hasselmann, Jonathan; Nguyen, Quyen T

2016-12-01

Additional intraoperative guidance could reduce the risk of iatrogenic injury during parotid gland cancer surgery. We evaluated the intraoperative use of fluorescently labeled nerve binding peptide NP41 to aid facial nerve identification and preservation during parotidectomy in an orthotopic model of murine parotid gland cancer. We also quantified the accuracy of intraoperative nerve detection for surface and buried nerves in the head and neck with NP41 versus white light (WL) alone. Twenty-eight mice underwent parotid gland cancer surgeries with additional fluorescence (FL) guidance versus WL reflectance (WLR) alone. Eight mice were used for additional nerve-imaging experiments. Twenty-eight parotid tumor-bearing mice underwent parotidectomy. Eight mice underwent imaging of both sides of the face after skin removal. Postoperative assessment of facial nerve function measured by automated whisker tracking were compared between FL guidance (n = 13) versus WL alone (n=15). In eight mice, nerve to surrounding tissue contrast was measured under FL versus WLR for all nerve branches detectable in the field of view. Postoperative facial nerve function after parotid gland cancer surgery tended to be better with additional FL guidance. Fluorescent labeling significantly improved nerve to surrounding tissue contrast for both large and smaller buried nerve branches compared to WLR visualization and improved detection sensitivity and specificity. NP41 FL imaging significantly aids the intraoperative identification of nerve braches otherwise nearly invisible to the naked eye. Its application in a murine model of parotid gland cancer surgery tended to improve functional preservation of the facial nerve. NA Laryngoscope, 126:2711-2717, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Improved Facial Nerve Identification During Parotidectomy With Fluorescently Labeled Peptide

PubMed Central

Hussain, Timon; Nguyen, Linda T.; Whitney, Michael; Hasselmann, Jonathan; Nguyen, Quyen T.

2016-01-01

Objectives/Hypothesis Additional intraoperative guidance could reduce the risk of iatrogenic injury during parotid gland cancer surgery. We evaluated the intraoperative use of fluorescently labeled nerve binding peptide NP41 to aid facial nerve identification and preservation during parotidectomy in an orthotopic model of murine parotid gland cancer. We also quantified the accuracy of intraoperative nerve detection for surface and buried nerves in the head and neck with NP41 versus white light (WL) alone. Study Design Twenty-eight mice underwent parotid gland cancer surgeries with additional fluorescence (FL) guidance versus WL reflectance (WLR) alone. Eight mice were used for additional nerve-imaging experiments. Methods Twenty-eight parotid tumor-bearing mice underwent parotidectomy. Eight mice underwent imaging of both sides of the face after skin removal. Postoperative assessment of facial nerve function measured by automated whisker tracking were compared between FL guidance (n = 13) versus WL alone (n = 15). In eight mice, nerve to surrounding tissue contrast was measured under FL versus WLR for all nerve branches detectable in the field of view. Results Postoperative facial nerve function after parotid gland cancer surgery tended to be better with additional FL guidance. Fluorescent labeling significantly improved nerve to surrounding tissue contrast for both large and smaller buried nerve branches compared to WLR visualization and improved detection sensitivity and specificity. Conclusions NP41 FL imaging significantly aids the intraoperative identification of nerve braches otherwise nearly invisible to the naked eye. Its application in a murine model of parotid gland cancer surgery tended to improve functional preservation of the facial nerve. PMID:27171862

Reflective afocal broadband adaptive optics scanning ophthalmoscope

PubMed Central

Dubra, Alfredo; Sulai, Yusufu

2011-01-01

A broadband adaptive optics scanning ophthalmoscope (BAOSO) consisting of four afocal telescopes, formed by pairs of off-axis spherical mirrors in a non-planar arrangement, is presented. The non-planar folding of the telescopes is used to simultaneously reduce pupil and image plane astigmatism. The former improves the adaptive optics performance by reducing the root-mean-square (RMS) of the wavefront and the beam wandering due to optical scanning. The latter provides diffraction limited performance over a 3 diopter (D) vergence range. This vergence range allows for the use of any broadband light source(s) in the 450-850 nm wavelength range to simultaneously image any combination of retinal layers. Imaging modalities that could benefit from such a large vergence range are optical coherence tomography (OCT), multi- and hyper-spectral imaging, single- and multi-photon fluorescence. The benefits of the non-planar telescopes in the BAOSO are illustrated by resolving the human foveal photoreceptor mosaic in reflectance using two different superluminescent diodes with 680 and 796 nm peak wavelengths, reaching the eye with a vergence of 0.76 D relative to each other. PMID:21698035

Reflective afocal broadband adaptive optics scanning ophthalmoscope.

PubMed

Dubra, Alfredo; Sulai, Yusufu

2011-06-01

A broadband adaptive optics scanning ophthalmoscope (BAOSO) consisting of four afocal telescopes, formed by pairs of off-axis spherical mirrors in a non-planar arrangement, is presented. The non-planar folding of the telescopes is used to simultaneously reduce pupil and image plane astigmatism. The former improves the adaptive optics performance by reducing the root-mean-square (RMS) of the wavefront and the beam wandering due to optical scanning. The latter provides diffraction limited performance over a 3 diopter (D) vergence range. This vergence range allows for the use of any broadband light source(s) in the 450-850 nm wavelength range to simultaneously image any combination of retinal layers. Imaging modalities that could benefit from such a large vergence range are optical coherence tomography (OCT), multi- and hyper-spectral imaging, single- and multi-photon fluorescence. The benefits of the non-planar telescopes in the BAOSO are illustrated by resolving the human foveal photoreceptor mosaic in reflectance using two different superluminescent diodes with 680 and 796 nm peak wavelengths, reaching the eye with a vergence of 0.76 D relative to each other.

Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

NASA Astrophysics Data System (ADS)

Darvin, M. E.; Richter, H.; Zhu, Y. J.; Meinke, M. C.; Knorr, F.; Gonchukov, S. A.; Koenig, K.; Lademann, J.

2014-07-01

Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted.

Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Darvin, M E; Richter, H; Zhu, Y J

Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed bymore » using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted. (laser biophotonics)« less

Plane wave analysis of coherent holographic image reconstruction by phase transfer (CHIRPT).

PubMed

Field, Jeffrey J; Winters, David G; Bartels, Randy A

2015-11-01

Fluorescent imaging plays a critical role in a myriad of scientific endeavors, particularly in the biological sciences. Three-dimensional imaging of fluorescent intensity often requires serial data acquisition, that is, voxel-by-voxel collection of fluorescent light emitted throughout the specimen with a nonimaging single-element detector. While nonimaging fluorescence detection offers some measure of scattering robustness, the rate at which dynamic specimens can be imaged is severely limited. Other fluorescent imaging techniques utilize imaging detection to enhance collection rates. A notable example is light-sheet fluorescence microscopy, also known as selective-plane illumination microscopy, which illuminates a large region within the specimen and collects emitted fluorescent light at an angle either perpendicular or oblique to the illumination light sheet. Unfortunately, scattering of the emitted fluorescent light can cause blurring of the collected images in highly turbid biological media. We recently introduced an imaging technique called coherent holographic image reconstruction by phase transfer (CHIRPT) that combines light-sheet-like illumination with nonimaging fluorescent light detection. By combining the speed of light-sheet illumination with the scattering robustness of nonimaging detection, CHIRPT is poised to have a dramatic impact on biological imaging, particularly for in vivo preparations. Here we present the mathematical formalism for CHIRPT imaging under spatially coherent illumination and present experimental data that verifies the theoretical model.

[Development of fluorescent probes for bone imaging in vivo ~Fluorescent probes for intravital imaging of osteoclast activity~.

PubMed

Minoshima, Masafumi; Kikuchi, Kazuya

Fluorescent molecules are widely used as a tool to directly visualize target biomolecules in vivo. Fluorescent probes have the advantage that desired function can be rendered based on rational design. For bone-imaging fluorescent probes in vivo, they should be delivered to bone tissue upon administration. Recently, a fluorescent probe for detecting osteoclast activity was developed. The fluorescent probe has acid-sensitive fluorescence property, specific delivery to bone tissue, and durability against laser irradiation, which enabled real-time intravital imaging of bone-resorbing osteoclasts for a long period of time.

Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

NASA Technical Reports Server (NTRS)

Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

Imaging of NPQ and ROS formation in tobacco leaves: heat inactivation of the water-water cycle prevents down-regulation of PSII.

PubMed

Hideg, Eva; Kós, Péter B; Schreiber, Ulrich

2008-12-01

Non-photochemical chlorophyll fluorescence quenching (NPQ) plays a major role in the protection of the photosynthetic apparatus against damage by excess light, which is closely linked to the production of reactive oxygen species (ROS). The effect of a short heat treatment on NPQ and ROS production was studied with detached tobacco leaves by fluorescence imaging of chlorophyll and of the ROS sensor dye HO-1889NH. NPQ was stimulated >3-fold by 3 min pre-treatment at 44 degrees C, in parallel with suppression of CO(2) uptake, while no ROS formation could be detected. In contrast, after 3 min pre-treatment at 46 degrees C, NPQ was suppressed and ROS formation was indicated by quenching of HO-1889NH fluorescence. After 3 min pre-treatment at 46 degrees C and above, partial inactivation of ascorbate peroxidase and light-driven accumulation of H(2)O(2) was also observed. These data are discussed as evidence for a decisive role of the Mehler ascorbate peroxidase or water-water cycle in the formation of the NPQ that reflects down-regulation of PSII.

Vibrational and UV spectroscopic studies of 2-coumaranone by experimental and density functional theory calculations

NASA Astrophysics Data System (ADS)

Priya, Y. Sushma; Rao, K. Ramachandra; Chalapathi, P. V.; Satyavani, M.; Veeraiah, A.

2017-09-01

The vibrational and electronic properties of 2-coumaranone have been reported in the ground state using experimental techniques (FT-IR, FT-Raman, UV spectra and fluorescence microscopic imaging) and density functional theory (DFT) employing B3LYP correlation with the 6-31G(d, p) basis set. The theoretically reported optimized parameters, vibrational frequencies etc., were compared with the experimental values, which yielded good concurrence between the experimental and calculated values. The assignments of the vibrational spectra were done with the help of normal co-ordinate analysis (NCA) following the Scaled Quantum Mechanical Force Field(SQMFF) methodology. The whole assignments of fundamental modes were based on the potential energy distribution (PED) matrix. The electric dipole moment and the first order hyperpolarizability of the 2-coumaranone have been computed using quantum mechanical calculations. NBO and HOMO, LUMO analyses have been carried out. UV spectrum of 2-coumaranone was recorded in the region 100-300 nm and compared with the theoretical UV spectrum using TD-DFT and SAC-CI methods by which a good agreement is observed. Fluorescence microscopic imaging study reflects that the compound fluoresces in the green-yellow region.

Generation of Micropatterned Substrates Using Micro Photopatterning

PubMed Central

Doyle, Andrew D.

2010-01-01

Micro photopatterning (µPP) has been developed to rapidly test and generate different patterns for extracellular matrix adsorption without being hindered with the process of making physical stamps through nanolithography techniques. It uses two-photon excitation guided through a point-scanning confocal microscope to locally photoablate poly(vinyl) alcohol (PVA) thin films in user-defined computer-controlled patterns. PVA thin films are ideal for surface blocking, being hydrophilic substrates that deter protein adsorption and cell attachment. Because gold substrates are not used during µPP, all live-cell fluorescent imaging techniques including total internal reflection fluorescence microscopy of GFP–linked proteins can be performed with minimal loss of fluorescence signal. Furthermore, because µPP does not require physical stamps for pattern generation, multiple ECMs or other proteins can be localized within microns of each other. This unit details the setup of µPP as well as giving troubleshooting techniques. PMID:20013752

The development of optical microscopy techniques for the advancement of single-particle studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchuk, Kyle

2013-05-15

Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-fieldmore » imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called “non-blinking” quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces.« less

The development of optical microscopy techniques for the advancement of single-particle studies

NASA Astrophysics Data System (ADS)

Marchuk, Kyle

Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called "non-blinking" quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces.

Detection of Sessile Serrated Adenomas in the Proximal Colon Using Wide-Field Fluorescence Endoscopy.

PubMed

Joshi, Bishnu P; Dai, Zhenzhen; Gao, Zhenghong; Lee, Jeong Hoon; Ghimire, Navin; Chen, Jing; Prabhu, Anoop; Wamsteker, Erik J; Kwon, Richard S; Elta, Grace H; Stoffel, Elena M; Pant, Asha; Kaltenbach, Tonya; Soetikno, Roy M; Appelman, Henry D; Kuick, Rork; Turgeon, D Kim; Wang, Thomas D

2017-04-01

Many cancers in the proximal colon develop via from sessile serrated adenomas (SSAs), which have flat, subtle features that are difficult to detect with conventional white-light colonoscopy. Many SSA cells have the V600E mutation in BRAF. We investigated whether this feature could be used with imaging methods to detect SSAs in patients. We used phage display to identify a peptide that binds specifically to SSAs, using subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF and Hs738.St/Int cells as a control. Binding of fluorescently labeled peptide to colorectal cancer cells was evaluated with confocal fluorescence microscopy. Rats received intra-colonic 0.0086 mg/kg, 0.026 mg/kg, or 0.86 mg/kg peptide or vehicle and morbidity, mortality, and injury were monitored twice daily to assess toxicity. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies (3 subjects were given 2.25 μmol/L and 22 patients were given 76.4 μmol/L). We performed blood cell count, chemistry, liver function, and urine analyses approximately 24 hours after peptide administration. In the clinical imaging study, 38 subjects undergoing routine outpatient colonoscopies, at high risk for colorectal cancer, or with a suspected unresected proximal colonic polyp, were first evaluated by white-light endoscopy to identify suspicious regions. The fluorescently labeled peptide (76.4 μmol/L) was administered topically to proximal colon, unbound peptide was washed away, and white-light, reflectance, and fluorescence videos were recorded digitally. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. Endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists (reference standard). We also analyzed the ability of the peptide to identify SSAs vs adenomas, hyperplastic polyps, and normal colonic mucosa in specimens obtained from the tissue bank at the University of Michigan. We identified the peptide sequence KCCFPAQ and measured an apparent dissociation constant of K d  = 72 nM and an apparent association time constant of K = 0.174 min -1 (5.76 minutes). During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. The peptide had no observed toxic effects in animals or patients. In the analysis of ex vivo specimens, peptide bound to SSAs had significantly higher mean fluorescence intensity than to hyperplastic polyps. We have identified a fluorescently labeled peptide that has no observed toxic effects in animals or humans and can be used for wide-field imaging of lesions in the proximal colon. It distinguishes SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. This targeted imaging method might be used in early detection of premalignant serrated lesions during routine colonoscopies. ClinicalTrials.gov ID: NCT02156557. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Detection of Sessile Serrated Adenomas in Proximal Colon Using Wide-field Fluorescence Endoscopy

PubMed Central

Joshi, Bishnu P.; Dai, Zhenzhen; Gao, Zhenghong; Lee, Jeong Hoon; Ghimire, Navin; Chen, Jing; Prabhu, Anoop; Wamsteker, Erik J.; Kwon, Richard S.; Elta, Grace H.; Stoffel, Elena M.; Pant, Asha; Kaltenbach, Tonya; Soetikno, Roy M.; Appelman, Henry D.; Kuick, Rork; Turgeon, D. Kim; Wang, Thomas D.

2018-01-01

Background & Aims Many cancers in the proximal colon develop via from sessile serrated adenomas (SSAs), which have flat, subtle features that are difficult to detect with conventional white-light colonoscopy. Many SSA cells have the V600E mutation in BRAF. We investigated whether this feature could be used with imaging methods to detect SSAs in patients. Methods We used phage display to identify a peptide that binds specifically to SSAs, using subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF and Hs738.St/Int cells as a control. Binding of fluorescently labeled peptide to colorectal cancer cells was evaluated with confocal fluorescence microscopy. Rats received intra-colonic 0.0086 mg/kg, 0.026 mg/kg, or 0.86 mg/kg peptide or vehicle and morbidity, mortality, and injury were monitored twice daily to assess toxicity. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies (3 subjects were given 2.25 µmol/L and 22 patients were given 76.4 µmol/L). We performed blood cell count, chemistry, liver function, and urine analyses approximately 24 hrs after peptide administration. In the clinical imaging study, 38 subjects undergoing routine outpatient colonoscopies, at high risk for colorectal cancer, or with a suspected unresected proximal colonic polyp, were first evaluated by white-light endoscopy, to identify suspicious regions. The fluorescently labeled peptide (76.4 µmol/L) was administered topically to proximal colon, unbound peptide was washed away, and white-light, reflectance, and fluorescence videos were recorded digitally. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. Endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists (reference standard). We also analyzed the ability of the peptide to identify SSAs vs adenomas, hyperplastic polyps, and normal colonic mucosa in specimens obtained from the tissue bank at the University of Michigan. Results We identified the peptide sequence KCCFPAQ, and measured an apparent dissociation constant of kd = 72 nM and an apparent association time constant of k = 0.174 min−1 (5.76 min). During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. The peptide had no observed toxic effects in animals or patients. In the analysis of ex vivo specimens, peptide bound to SSAs had significantly higher mean fluorescence intensity than to hyperplastic polyps. Conclusions We have identified a fluorescently labeled peptide that has no observed toxic effects in animals or humans and can be used for wide-field imaging of lesions in the proximal colon. It distinguishes SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. This targeted imaging method might be used in early detection of pre-malignant serrated lesions during routine colonoscopies. ClinicalTrials.gov no: NCT02156557 PMID:28012848

Interpretative Guidelines and Possible Indications for Indocyanine Green Fluorescence Imaging in Robot-Assisted Sphincter-Saving Operations.

PubMed

Kim, Jin Cheon; Lee, Jong Lyul; Park, Seong Ho

2017-04-01

Since the introduction of indocyanine green angiography more than 25 years ago, few studies have presented interpretative guidelines for indocyanine green fluorescent imaging. We aimed to provide interpretative guidelines for indocyanine green fluorescent imaging through quantitative analysis and to suggest possible indications for indocyanine green fluorescent imaging during robot-assisted sphincter-saving operations. This is a retrospective observational study. This study was conducted at a single center. A cohort of 657 patients with rectal cancer who consecutively underwent curative robot-assisted sphincter-saving operations was enrolled between 2010 and 2016, including 310 patients with indocyanine green imaging (indocyanine green fluorescent imaging+ group) and 347 patients without indocyanine green imaging (indocyanine green fluorescent imaging- group). We tried to quantitatively define the indocyanine green fluorescent imaging findings based on perfusion (mesocolic and colic) time and perfusion intensity (5 grades) to provide probable indications. The anastomotic leakage rate was significantly lower in the indocyanine green fluorescent imaging+ group than in the indocyanine green fluorescent imaging- group (0.6% vs 5.2%) (OR, 0.123; 95% CI, 0.028-0.544; p = 0.006). Anastomotic stricture was closely correlated with anastomotic leakage (p = 0.002) and a short descending mesocolon (p = 0.003). Delayed perfusion (>60 s) and low perfusion intensity (1-2) were more frequently detected in patients with anastomotic stricture and marginal artery defects than in those without these factors (p ≤ 0.001). In addition, perfusion times greater than the mean were more frequently observed in patients aged >58 years, whereas low perfusion intensity was seen more in patients with short descending mesocolon and high ASA classes (≥3). The 300 patients in the indocyanine green fluorescent imaging- group underwent operations 3 years before indocyanine green fluorescent imaging. Quantitative analysis of indocyanine green fluorescent imaging may help prevent anastomotic complications during robot-assisted sphincter-saving operations, and may be of particular value in high-class ASA patients, older patients, and patients with a short descending mesocolon.

Spectroscopy and CCD-photography of extended red emission in reflection nebulae

NASA Technical Reports Server (NTRS)

Witt, A. N.; Schild, R. E.

1986-01-01

Recent spectrographic studies of extended red emission (ERE) seen in the 0.6 to 0.9 micron spectral region in many reflection nebulae have shown fluorescence by amorphous hydrogenated carbon to be the most probable cause of the ERE. Spectrographic observations were performed on the nebulae NGC 2023 and NGC 7023, using the intensified Reticon scanner (IRS) of Kitt Peak National Observatory on the N0-2 0.9 mm telescope. Charge coupled device (CCD) images of NGC2023 and NGC 7023 were obtained with the CfA CCD detector on the 0.6 mm telescope of the Whipple Observatory. Results are discussed.

FLEX: an imaging spectrometer for measurement of vegetation fluorescence

NASA Astrophysics Data System (ADS)

Smorenburg, Kees; Visser, Huib; Court, Andrew; Stoll, Marc Ph.

2017-11-01

Detection of vegetation fluorescence gives information about plant functioning, stress and vitality. During the past decades several ground based laser fluorosensors have been developed to investigate these processes and to demonstrate the value of this technique. FLEX (= FLuorescense EXplorer) is a space mission to measure the fluorescence of vegetation on earth over large areas from space. Such a mission would greatly improve the understanding and enhance the capability to quantify e.g. the role of terrestrial vegetation in global carbon sequestration. Because the fluorescence signal, which is excited by solar irradiation is low with respect to the reflected sunlight the signal from a satellite is proposed to be measured in the solar Fraunhofer lines, where the reflection signal is much reduced. The heart of FLEX is a high resolution imaging spectrometer with 2 channels: channel 1 around the Fraunhofer lines at ‡ = 397 nm, ‡= 423 nm and/or ‡ = 434 nm and channel 2 around the Fraunhofer line at ‡ = 656 nm. The required spectral resolution will depend on the linewidth (0.02-0.3 nm). A first definition of the field of view is 8.4 degrees, leading from an 800 km satellite altitude to a swath of about 120 km. For detection a 1024x1024 pixel frame transfer CCD detector is proposed, with a pixel dimension of 13 x 13 ‡ mm2. The maximum footprint is about 500x500m2. The optical configuration contains a scan mirror for solar calibration, for pointing the FOV in swath direction and for freezing the observed ground scene up to a few seconds to increase the signal to noise performance. At this moment the concept of FLEX is elaborated in a feasibility study. Both the scientific and instrument requirements are updated and the concept is studied in detail. Besides a development plan for FLEX is made. In this paper the idea and the headlines of FLEX are described.

Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions

NASA Astrophysics Data System (ADS)

Gareau, Dan; Bar, Anna; Snaveley, Nicholas; Lee, Ken; Chen, Nathaniel; Swanson, Neil; Simpson, Eric; Jacques, Steve

2012-06-01

For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4× histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.

Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions

PubMed Central

Bar, Anna; Snaveley, Nicholas; Lee, Ken; Chen, Nathaniel; Swanson, Neil; Simpson, Eric; Jacques, Steve

2012-01-01

Abstract. For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4× histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues. PMID:22734774

In vivo assessment of liver fibrosis using diffuse reflectance and fluorescence spectroscopy: a proof of concept.

PubMed

Fabila, Diego; de la Rosa, José Manuel; Stolik, Suren; Moreno, Edgard; Suárez-Álvarez, Karina; López-Navarrete, Giuliana; Guzmán, Carolina; Aguirre-García, Jesús; Acevedo-García, Christian; Kershenobich, David; Escobedo, Galileo

2012-12-01

A novel application of diffuse reflectance and fluorescence spectroscopy in the assessment of liver fibrosis is here reported. To induce different stages of liver fibrosis, a sufficient number of male Wistar rats were differentially exposed to chronic administration with carbon tetrachloride. Then, diffuse reflectance and fluorescence spectra were in vivo measured from the liver surface of each animal by a minimal invasive laparoscopic procedure. The liver fibrosis degree was conventionally determined by means of histological examination using the Mason's Trichrome stain, accompanied by hepatic expression of α-sma, and evaluation of the ALT/AST serum levels. The liver from rats exhibiting higher grades of fibrosis showed a significant increase in diffuse reflectance and fluorescence intensity when compared with control animals. At 365 nm, the diffuse reflectance spectrum exhibited an increase of 4 and 3-fold in mild and advanced fibrotic rats, respectively, when compared to the control group. Similarly, the fluorescence emission at 493 nm was 2-fold higher in fibrotic animals than in controls. By using fluorescence intensity, discrimination algorithms indicated 73% sensitivity and 94% specificity for recognition of hepatic fibrosis, while for diffuse reflectance, these values increased up to 85% and 100%, respectively. Taking into consideration there is a special need for developing new diagnostic approaches focused on detecting different stages of liver fibrosis with minimal invasiveness, these results suggest that diffuse reflectance and fluorescence spectroscopy could be worthy of further exploration in patients with liver disease. Copyright © 2012 Elsevier B.V. All rights reserved.

Near-infrared dental imaging using scanning fiber endoscope

NASA Astrophysics Data System (ADS)

Zhou, Yaxuan; Lee, Robert; Sadr, Alireza; Seibel, Eric J.

2018-02-01

Near-infrared (NIR) wavelength range of 1300-1500nm has the potential to outperform or augment other dental imaging modalities such as fluorescence imaging, owing to its lower scattering coefficient in enamel and trans- parency on stains and non-cariogenic plaque. However, cameras in this wavelength range are bulky and expensive, which lead to difficulties for in-vivo use and commercialization. Thus, we have proposed a new imaging device combining the scanning fiber endoscopy (SFE) and NIR imaging technology. The NIR SFE system has the advantage of miniature size (1.6 mm), flexible shaft, video frame rate (7Hz) and expandable wide field-of-view (60 degrees). Eleven extracted human teeth with or without occlusal caries were scanned by micro-computed X-ray tomography (micro-CT) to obtain 3D micro-CT images, which serve as the standard for comparison. NIR images in reflection mode were then taken on all the occlusal surfaces, using 1310nm super luminescent diode and 1460nm laser diode respectively. Qualitative comparison was performed between near-infrared im- ages and micro-CT images. Enamel demineralization in NIR appeared as areas of increased reflectivity, and distinguished from non-carious staining at the base of occlusal fissures or developmental defects on cusps. This preliminary work presented proof for practicability of combining NIR imaging technology with SFE for reliable and noninvasive dental imaging with miniaturization and low cost.

Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue

PubMed Central

Scolaro, Loretta; Lorenser, Dirk; Madore, Wendy-Julie; Kirk, Rodney W.; Kramer, Anne S.; Yeoh, George C.; Godbout, Nicolas; Sampson, David D.; Boudoux, Caroline; McLaughlin, Robert A.

2015-01-01

Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue. PMID:26137379

Multi-spectral endogenous fluorescence imaging for bacterial differentiation

NASA Astrophysics Data System (ADS)

Chernomyrdin, Nikita V.; Babayants, Margarita V.; Korotkov, Oleg V.; Kudrin, Konstantin G.; Rimskaya, Elena N.; Shikunova, Irina A.; Kurlov, Vladimir N.; Cherkasova, Olga P.; Komandin, Gennady A.; Reshetov, Igor V.; Zaytsev, Kirill I.

2017-07-01

In this paper, the multi-spectral endogenous fluorescence imaging was implemented for bacterial differentiation. The fluorescence imaging was performed using a digital camera equipped with a set of visual bandpass filters. Narrowband 365 nm ultraviolet radiation passed through a beam homogenizer was used to excite the sample fluorescence. In order to increase a signal-to-noise ratio and suppress a non-fluorescence background in images, the intensity of the UV excitation was modulated using a mechanical chopper. The principal components were introduced for differentiating the samples of bacteria based on the multi-spectral endogenous fluorescence images.

Mechanical Damage Detection of Indonesia Local Citrus Based on Fluorescence Imaging

NASA Astrophysics Data System (ADS)

Siregar, T. H.; Ahmad, U.; Sutrisno; Maddu, A.

2018-05-01

Citrus experienced physical damage in peel will produce essential oils that contain polymethoxylated flavone. Polymethoxylated flavone is fluorescence substance; thus can be detected by fluorescence imaging. This study aims to study the fluorescence spectra characteristic and to determine the damage region in citrus peel based on fluorescence image. Pulung citrus from Batu district, East Java, as a famous citrus production area in Indonesia, was used in the experiment. It was observed that the image processing could detect the mechanical damage region. Fluorescence imaging can be used to classify the citrus into two categories, sound and defect citruses.

Shedding quantitative fluorescence light on novel regulatory mechanisms in skeletal biomedicine and biodentistry.

PubMed

Lee, Ji-Won; Iimura, Tadahiro

2017-02-01

Digitalized fluorescence images contain numerical information such as color (wavelength), fluorescence intensity and spatial position. However, quantitative analyses of acquired data and their validation remained to be established. Our research group has applied quantitative fluorescence imaging on tissue sections and uncovered novel findings in skeletal biomedicine and biodentistry. This review paper includes a brief background of quantitative fluorescence imaging and discusses practical applications by introducing our previous research. Finally, the future perspectives of quantitative fluorescence imaging are discussed.

Atomic force-multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells.

PubMed

Trache, Andreea; Meininger, Gerald A

2005-01-01

A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.

Dual PET and Near-Infrared Fluorescence Imaging Probes as Tools for Imaging in Oncology

PubMed Central

An, Fei-Fei; Chan, Mark; Kommidi, Harikrishna; Ting, Richard

2016-01-01

OBJECTIVE The purpose of this article is to summarize advances in PET fluorescence resolution, agent design, and preclinical imaging that make a growing case for clinical PET fluorescence imaging. CONCLUSION Existing SPECT, PET, fluorescence, and MRI contrast imaging techniques are already deeply integrated into the management of cancer, from initial diagnosis to the observation and management of metastases. Combined positron-emitting fluorescent contrast agents can convey new or substantial benefits that improve on these proven clinical contrast agents. PMID:27223168

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

KrF laser-induced OH fluorescence imaging in a supersonic combustion tunnel

NASA Technical Reports Server (NTRS)

Quagliaroli, T. M.; Laufer, G.; Hollo, S. D.; Krauss, R. H.; Whitehurst, R. B., III; Mcdaniel, J. C., Jr.

1992-01-01

Planar fluorescence images of OH in a continuous-flow, electrical-resistively heated, high enthalpy, hydrogen-air combustion tunnel, induced by a tunable KrF laser, were recorded. These images were compared to previously recorded fluorescence images induced by a doubled-dye laser under similar conditions. Images induced by the doubled-dye laser system demonstrated a severe distortion caused by absorption and fluorescence trapping. By contrast, images of the fluorescence induced by the tunable KrF laser retained the symmetry properties of the flow. Based on signal-to-noise ratio measurements the yield of the fluorescence induced by the doubled-dye laser is larger than the fluorescence yield induced by the KrF laser. The measurements in the present facility of OH fluorescence induced by the KrF laser were limited by the photon-statistical noise. Based 2 on this result, doubled-dye laser systems are recommended for OH imaging in small and OH lean (less than 10 exp 15/cu cm) facilities. KrF lasers should be selected otherwise.

Development of Fluorescence Imaging Lidar for Boat-Based Coral Observation

NASA Astrophysics Data System (ADS)

Sasano, Masahiko; Imasato, Motonobu; Yamano, Hiroya; Oguma, Hiroyuki

2016-06-01

A fluorescence imaging lidar system installed in a boat-towable buoy has been developed for the observation of reef-building corals. Long-range fluorescent images of the sea bed can be recorded in the daytime with this system. The viability of corals is clear in these fluorescent images because of the innate fluorescent proteins. In this study, the specifications and performance of the system are shown.

Specimen illumination apparatus with optical cavity for dark field illumination

DOEpatents

Pinkel, Daniel; Sudar, Damir; Albertson, Donna

1999-01-01

An illumination apparatus with a specimen slide holder, an illumination source, an optical cavity producing multiple reflection of illumination light to a specimen comprising a first and a second reflective surface arranged to achieve multiple reflections of light to a specimen is provided. The apparatus can further include additional reflective surfaces to achieve the optical cavity, a slide for mounting the specimen, a coverslip which is a reflective component of the optical cavity, one or more prisms for directing light within the optical cavity, antifading solutions for improving the viewing properties of the specimen, an array of materials for analysis, fluorescent components, curved reflective surfaces as components of the optical cavity, specimen detection apparatus, optical detection equipment, computers for analysis of optical images, a plane polarizer, fiberoptics, light transmission apertures, microscopic components, lenses for viewing the specimen, and upper and lower mirrors above and below the specimen slide as components of the optical cavity. Methods of using the apparatus are also provided.

Ns-scaled time-gated fluorescence lifetime imaging for forensic document examination

NASA Astrophysics Data System (ADS)

Zhong, Xin; Wang, Xinwei; Zhou, Yan

2018-01-01

A method of ns-scaled time-gated fluorescence lifetime imaging (TFLI) is proposed to distinguish different fluorescent substances in forensic document examination. Compared with Video Spectral Comparator (VSC) which can examine fluorescence intensity images only, TFLI can detect questioned documents like falsification or alteration. TFLI system can enhance weak signal by accumulation method. The two fluorescence intensity images of the interval delay time tg are acquired by ICCD and fitted into fluorescence lifetime image. The lifetimes of fluorescence substances are represented by different colors, which make it easy to detect the fluorescent substances and the sequence of handwritings. It proves that TFLI is a powerful tool for forensic document examination. Furthermore, the advantages of TFLI system are ns-scaled precision preservation and powerful capture capability.

Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: a versatile strategy for multiple molecular imaging modalities.

PubMed

Massa, Sam; Vikani, Niravkumar; Betti, Cecilia; Ballet, Steven; Vanderhaegen, Saskia; Steyaert, Jan; Descamps, Benedicte; Vanhove, Christian; Bunschoten, Anton; van Leeuwen, Fijs W B; Hernot, Sophie; Caveliers, Vicky; Lahoutte, Tony; Muyldermans, Serge; Xavier, Catarina; Devoogdt, Nick

2016-09-01

A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein-ligation capacity of the enzyme Sortase A to label camelid single-domain antibody-fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX-A"-DTPA and NOTA for single-photon emission computed tomography (SPECT) with indium-111 and positron emission tomography (PET) with gallium-68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single-conjugated tracer populations were obtained in high yield (30-50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1-tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A-mediated conjugation as a valuable strategy for the development of site-specifically labeled camelid single-domain antibody-fragments for use in multiple molecular imaging modalities. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

New contrasts for x-ray imaging and synergy with optical imaging

NASA Astrophysics Data System (ADS)

Wang, Ge

2017-02-01

Due to its penetrating power, fine resolution, unique contrast, high-speed, and cost-effectiveness, x-ray imaging is one of the earliest and most popular imaging modalities in biomedical applications. Current x-ray radiographs and CT images are mostly on gray-scale, since they reflect overall energy attenuation. Recent advances in x-ray detection, contrast agent, and image reconstruction technologies have changed our perception and expectation of x-ray imaging capabilities, and generated an increasing interest in imaging biological soft tissues in terms of energy-sensitive material decomposition, phase-contrast, small angle scattering (also referred to as dark-field), x-ray fluorescence and luminescence properties. These are especially relevant to preclinical and mesoscopic studies, and potentially mendable for hybridization with optical molecular tomography. In this article, we review new x-ray imaging techniques as related to optical imaging, suggest some combined x-ray and optical imaging schemes, and discuss our ideas on micro-modulated x-ray luminescence tomography (MXLT) and x-ray modulated opto-genetics (X-Optogenetics).

Compact whole-body fluorescent imaging of nude mice bearing EGFP expressing tumor

NASA Astrophysics Data System (ADS)

Chen, Yanping; Xiong, Tao; Chu, Jun; Yu, Li; Zeng, Shaoqun; Luo, Qingming

2005-01-01

Issue of tumor has been a hotspot of current medicine. It is important for tumor research to detect tumors bearing in animal models easily, fast, repetitively and noninvasivly. Many researchers have paid their increasing interests on the detecting. Some contrast agents, such as green fluorescent protein (GFP) and Discosoma red fluorescent protein (Dsred) were applied to enhance image quality. Three main kinds of imaging scheme were adopted to visualize fluorescent protein expressing tumors in vivo. These schemes based on fluorescence stereo microscope, cooled charge-coupled-device (CCD) or camera as imaging set, and laser or mercury lamp as excitation light source. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes. The authors set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a Kodak digital camera (model DC290), fluorescence filters(B and G2;HB Optical, Shenyang, Liaoning, P.R. China) and a mercury 50-W lamp power supply (U-LH50HG;Olympus Optical, Japan) as excitation light source. The EGFP was excited directly by mercury lamp with D455/70 nm band-pass filter and fluorescence was recorded by digital camera with 520nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For half a year our experiments suggested the imaging scheme was feasible. Whole-body fluorescence optical imaging for fluorescent expressing tumors in nude mouse is an ideal tool for antitumor, antimetastatic, and antiangiogenesis drug screening.

In Vivo Imaging of the Human Retinal Pigment Epithelial Mosaic Using Adaptive Optics Enhanced Indocyanine Green Ophthalmoscopy

PubMed Central

Tam, Johnny; Liu, Jianfei; Dubra, Alfredo; Fariss, Robert

2016-01-01

Purpose The purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye. Methods A customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out. Results The AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection. Conclusions Adaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells. PMID:27564519

In Vivo Imaging of the Human Retinal Pigment Epithelial Mosaic Using Adaptive Optics Enhanced Indocyanine Green Ophthalmoscopy.

PubMed

Tam, Johnny; Liu, Jianfei; Dubra, Alfredo; Fariss, Robert

2016-08-01

The purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye. A customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out. The AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection. Adaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells.

Mathematical modeling of reflectance and intrinsic fluorescence for cancer detection in human pancreatic tissue

NASA Astrophysics Data System (ADS)

Wilson, Robert H.; Chandra, Malavika; Scheiman, James; Simeone, Diane; McKenna, Barbara; Purdy, Julianne; Mycek, Mary-Ann

2009-02-01

Pancreatic adenocarcinoma has a five-year survival rate of only 4%, largely because an effective procedure for early detection has not been developed. In this study, mathematical modeling of reflectance and fluorescence spectra was utilized to quantitatively characterize differences between normal pancreatic tissue, pancreatitis, and pancreatic adenocarcinoma. Initial attempts at separating the spectra of different tissue types involved dividing fluorescence by reflectance, and removing absorption artifacts by applying a "reverse Beer-Lambert factor" when the absorption coefficient was modeled as a linear combination of the extinction coefficients of oxy- and deoxy-hemoglobin. These procedures demonstrated the need for a more complete mathematical model to quantitatively describe fluorescence and reflectance for minimally-invasive fiber-based optical diagnostics in the pancreas.

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

PubMed

Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J; Davis, Wayne M; Jorgensen, Erik M

2012-12-03

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

Fluorescence optical imaging in anticancer drug delivery.

PubMed

Etrych, Tomáš; Lucas, Henrike; Janoušková, Olga; Chytil, Petr; Mueller, Thomas; Mäder, Karsten

2016-03-28

In the past several decades, nanosized drug delivery systems with various targeting functions and controlled drug release capabilities inside targeted tissues or cells have been intensively studied. Understanding their pharmacokinetic properties is crucial for the successful transition of this research into clinical practice. Among others, fluorescence imaging has become one of the most commonly used imaging tools in pre-clinical research. The development of increasing numbers of suitable fluorescent dyes excitable in the visible to near-infrared wavelengths of the spectrum has significantly expanded the applicability of fluorescence imaging. This paper focuses on the potential applications and limitations of non-invasive imaging techniques in the field of drug delivery, especially in anticancer therapy. Fluorescent imaging at both the cellular and systemic levels is discussed in detail. Additionally, we explore the possibility for simultaneous treatment and imaging using theranostics and combinations of different imaging techniques, e.g., fluorescence imaging with computed tomography. Copyright © 2016 Elsevier B.V. All rights reserved.

Real-time quantitative fluorescence imaging using a single snapshot optical properties technique for neurosurgical guidance

NASA Astrophysics Data System (ADS)

Valdes, Pablo A.; Angelo, Joseph; Gioux, Sylvain

2015-03-01

Fluorescence imaging has shown promise as an adjunct to improve the extent of resection in neurosurgery and oncologic surgery. Nevertheless, current fluorescence imaging techniques do not account for the heterogeneous attenuation effects of tissue optical properties. In this work, we present a novel imaging system that performs real time quantitative fluorescence imaging using Single Snapshot Optical Properties (SSOP) imaging. We developed the technique and performed initial phantom studies to validate the quantitative capabilities of the system for intraoperative feasibility. Overall, this work introduces a novel real-time quantitative fluorescence imaging method capable of being used intraoperatively for neurosurgical guidance.

Statistical image segmentation for the detection of skin lesion borders in UV fluorescence excitation

NASA Astrophysics Data System (ADS)

Ortega-Martinez, Antonio; Padilla-Martinez, Juan Pablo; Franco, Walfre

2016-04-01

The skin contains several fluorescent molecules or fluorophores that serve as markers of structure, function and composition. UV fluorescence excitation photography is a simple and effective way to image specific intrinsic fluorophores, such as the one ascribed to tryptophan which emits at a wavelength of 345 nm upon excitation at 295 nm, and is a marker of cellular proliferation. Earlier, we built a clinical UV photography system to image cellular proliferation. In some samples, the naturally low intensity of the fluorescence can make it difficult to separate the fluorescence of cells in higher proliferation states from background fluorescence and other imaging artifacts -- like electronic noise. In this work, we describe a statistical image segmentation method to separate the fluorescence of interest. Statistical image segmentation is based on image averaging, background subtraction and pixel statistics. This method allows to better quantify the intensity and surface distributions of fluorescence, which in turn simplify the detection of borders. Using this method we delineated the borders of highly-proliferative skin conditions and diseases, in particular, allergic contact dermatitis, psoriatic lesions and basal cell carcinoma. Segmented images clearly define lesion borders. UV fluorescence excitation photography along with statistical image segmentation may serve as a quick and simple diagnostic tool for clinicians.

5-ALA induced fluorescent image analysis of actinic keratosis

NASA Astrophysics Data System (ADS)

Cho, Yong-Jin; Bae, Youngwoo; Choi, Eung-Ho; Jung, Byungjo

2010-02-01

In this study, we quantitatively analyzed 5-ALA induced fluorescent images of actinic keratosis using digital fluorescent color and hyperspectral imaging modalities. UV-A was utilized to induce fluorescent images and actinic keratosis (AK) lesions were demarcated from surrounding the normal region with different methods. Eight subjects with AK lesion were participated in this study. In the hyperspectral imaging modality, spectral analysis method was utilized for hyperspectral cube image and AK lesions were demarcated from the normal region. Before image acquisition, we designated biopsy position for histopathology of AK lesion and surrounding normal region. Erythema index (E.I.) values on both regions were calculated from the spectral cube data. Image analysis of subjects resulted in two different groups: the first group with the higher fluorescence signal and E.I. on AK lesion than the normal region; the second group with lower fluorescence signal and without big difference in E.I. between two regions. In fluorescent color image analysis of facial AK, E.I. images were calculated on both normal and AK lesions and compared with the results of hyperspectral imaging modality. The results might indicate that the different intensity of fluorescence and E.I. among the subjects with AK might be interpreted as different phases of morphological and metabolic changes of AK lesions.

Ocular fundus auto-fluorescence observations at different wavelengths in patients with age-related macular degeneration and diabetic retinopathy.

PubMed

Hammer, Martin; Königsdörffer, Ekkehart; Liebermann, Christiane; Framme, Carsten; Schuch, Günter; Schweitzer, Dietrich; Strobel, Jürgen

2008-01-01

Post-translational protein modification by lipid peroxidation products or glycation is a feature of aging as well as pathologic processes in postmitotic cells at the ocular fundus exposed to an oxidative environment. The accumulation of modified proteins such as those found in lipofuscin and advanced glycation end products (AGEs) contribute greatly to the fundus auto-fluorescence. The distinct fluorescence spectra of lipofuscin and AGE enable their differentiation in multispectral fundus fluorescence imaging. A dual-centre consecutive case series of 78 pseudo-phacic patients is reported. Digital colour fundus photographs as well as auto-fluorescence images were taken from 33 patients with age related macular degeneration (AMD), 13 patients with diabetic retinopathy (RD), or from 32 cases without pathologic findings (controls). Fluorescence was excited at 475-515 nm or 476-604 nm and recorded in the emission bands 530-675 nm or 675-715 nm, respectively. Fluorescence images excited at 475-515 nm were taken by a colour CCD-camera (colour-fluorescence imaging) enabling the separate recording of green and red fluorescence. The ratio of green versus red fluorescence was calculated within a representative region of each image. The 530-675 nm auto-fluorescence in AMD patients was dominated by the red emission (green vs. red ratio, g/r = 0.861). In comparison, the fluorescence of the diabetics was green-shifted (g/r = 0.946; controls: g/r = 0.869). Atrophic areas (geographic atrophy, laser scars) showed massive hypo-fluorescence in both emission bands. Hyper-fluorescent drusen and exudates, unobtrusive in the colour fundus images as well as in the fluorescence images with emission >667 nm, showed an impressive green-shift in the colour-fluorescence image. Lipofuscin is the dominant fluorophore at long wavelengths (>675 nm or red channel of the colour fluorescence image). In the green spectral region, we found an additional emission of collagen and elastin (optic disc, sclera) as well as deposits in drusen and exudates. The green shift of the auto-fluorescence in RD may be a hint of increased AGE concentrations.

Fluorescence Spectra of Highlighter Inks

ERIC Educational Resources Information Center

Birriel, Jennifer J.; King, Damon

2018-01-01

Fluorescence spectra excited by laser pointers have been the subject of several papers in "TPT". These papers all describe a fluorescence phenomenon in which the reflected laser light undergoes a change in color: this color change results from the combination of some partially reflected laser light and additional colors generated by…

Information loss and reconstruction in diffuse fluorescence tomography

PubMed Central

Bonfert-Taylor, Petra; Leblond, Frederic; Holt, Robert W.; Tichauer, Kenneth; Pogue, Brian W.; Taylor, Edward C.

2012-01-01

This paper is a theoretical exploration of spatial resolution in diffuse fluorescence tomography. It is demonstrated that, given a fixed imaging geometry, one cannot—relative to standard techniques such as Tikhonov regularization and truncated singular value decomposition—improve the spatial resolution of the optical reconstructions via increasing the node density of the mesh considered for modeling light transport. Using techniques from linear algebra, it is shown that, as one increases the number of nodes beyond the number of measurements, information is lost by the forward model. It is demonstrated that this information cannot be recovered using various common reconstruction techniques. Evidence is provided showing that this phenomenon is related to the smoothing properties of the elliptic forward model that is used in the diffusion approximation to light transport in tissue. This argues for reconstruction techniques that are sensitive to boundaries, such as L1-reconstruction and the use of priors, as well as the natural approach of building a measurement geometry that reflects the desired image resolution. PMID:22472763

Design of a dynamic biofilm imaging cell for white-light interferometric microscopy

DOE PAGES

Larimer, Curtis; Brann, Michelle; Suter, Jonathan D.; ...

2017-05-10

In microbiology research there is a strong need for next generation imaging and sensing instrumentation that will enable minimally invasive and label-free investigation of soft, hydrated structures such as in bacterial biofilms. White light interferometry (WLI) can provide high resolution images of surface topology without the use of fluorescent labels but is not typically used to image biofilms because there is insufficient refractive index contrast to induce reflection from the biofilm’s interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ, especially in a non-destructive manner. In this report, we build onmore » our prior description of static biofilm imaging and describe the design of a dynamic imaging flow cell that enables monitoring the thickness and topology of live biofilms over time using a WLI microscope. The microfluidic system is specifically designed to create a reflective interface on the surface of biofilms while minimizing disruption of fragile structures. The imaging cell was also designed to accommodate limitations imposed by the depth of focus of the microscope’s objective lens. Example images of live biofilm samples are shown in order to illustrate the ability of the flow cell and WLI instrument to 1) support bacterial growth and biofilm development, 2) image biofilm structure that reflects growth in flow conditions, and 3) monitor biofilm development over time non-destructively. In future work, the apparatus described here will enable surface metrology measurements (roughness, surface area, etc.) of biofilms and may be used to observe changes in biofilm structure in response to changes in environmental conditions (e.g., flow velocity, availability of nutrients, and presence of biocides). Furthermore, this development will open new opportunities for the use of WLI in bioimaging.« less

Design of a dynamic biofilm imaging cell for white-light interferometric microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larimer, Curtis; Brann, Michelle; Suter, Jonathan D.

In microbiology research there is a strong need for next generation imaging and sensing instrumentation that will enable minimally invasive and label-free investigation of soft, hydrated structures such as in bacterial biofilms. White light interferometry (WLI) can provide high resolution images of surface topology without the use of fluorescent labels but is not typically used to image biofilms because there is insufficient refractive index contrast to induce reflection from the biofilm’s interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ, especially in a non-destructive manner. In this report, we build onmore » our prior description of static biofilm imaging and describe the design of a dynamic imaging flow cell that enables monitoring the thickness and topology of live biofilms over time using a WLI microscope. The microfluidic system is specifically designed to create a reflective interface on the surface of biofilms while minimizing disruption of fragile structures. The imaging cell was also designed to accommodate limitations imposed by the depth of focus of the microscope’s objective lens. Example images of live biofilm samples are shown in order to illustrate the ability of the flow cell and WLI instrument to 1) support bacterial growth and biofilm development, 2) image biofilm structure that reflects growth in flow conditions, and 3) monitor biofilm development over time non-destructively. In future work, the apparatus described here will enable surface metrology measurements (roughness, surface area, etc.) of biofilms and may be used to observe changes in biofilm structure in response to changes in environmental conditions (e.g., flow velocity, availability of nutrients, and presence of biocides). Furthermore, this development will open new opportunities for the use of WLI in bioimaging.« less

Miniaturized side-viewing imaging probe for fluorescence lifetime imaging (FLIM): validation with fluorescence dyes, tissue structural proteins and tissue specimens

PubMed Central

Elson, D S; Jo, J A

2007-01-01

We report a side viewing fibre-based endoscope that is compatible with intravascular imaging and fluorescence lifetime imaging microscopy (FLIM). The instrument has been validated through testing with fluorescent dyes and collagen and elastin powders using the Laguerre expansion deconvolution technique to calculate the fluorescence lifetimes. The instrument has also been tested on freshly excised unstained animal vascular tissues. PMID:19503759

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Fluorescence lifetime imaging of skin cancer

NASA Astrophysics Data System (ADS)

Patalay, Rakesh; Talbot, Clifford; Munro, Ian; Breunig, Hans Georg; König, Karsten; Alexandrov, Yuri; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

2011-03-01

Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

Instrument pre-development activities for FLEX

NASA Astrophysics Data System (ADS)

Pettinato, L.; Fossati, E.; Coppo, P. M.; Taiti, A.; Labate, D.; Capanni, A.; Taccola, M.; Bézy, J. L.; Francois, M.; Meynart, R.; Erdmann, L.; Triebel, P.

2017-09-01

The FLuorescence Imaging Spectrometer (FLORIS) is the payload of the FLuorescence Explorer Mission (FLEX) of the European Space Agency. The mission objective is to perform quantitative measurements of the solar induced vegetation fluorescence to monitor photosynthetic activity. FLORIS works in a push-broom configuration and it is designed to acquire data in the 500-780 nm spectral range, with a sampling of 0.1 nm in the oxygen bands (759-769 nm and 686- 697 nm) and 0.5-2.0 nm in the red edge, chlorophyll absorption and Photochemical Reflectance Index bands. FLEX will fly in formation with Sentinel-3 to benefit of the measurements made by the Sentinel-3 instruments OLCI and SLSTR, particularly for cloud screening, proper characterization of the atmospheric state and determination of the surface temperature. The instrument concept is based on a common telescope and two modified Offner spectrometers with reflective concave gratings both for the High Resolution (HR) and Low Resolution (LR) spectrometers. In the frame of the instrument pre-development Leonardo Company (I) has built and tested an elegant breadboard of the instrument consisting of the telescope and the HR spectrometer. The development of the LR spectrometer is in charge of OHB System AG (D) and is currently in the manufacturing phase. The main objectives of the activity are: anticipate the development of the instrument and provide early risk retirement of critical components, evaluate the system performances such as imaging quality parameters, straylight, ghost, polarization sensitivity and environmental influences, verify the adequacy of critical tests such as spectral characterization and straylight, define and optimize instrument alignment procedures. Following a brief overview of the FLEX mission, the paper will cover the design and the development of the optics breadboard with emphasis on the results obtained during the tests and the lessons learned for the flight unit.

Refractive Index Imaging of Cells with Variable-Angle Near-Total Internal Reflection (TIR) Microscopy.

PubMed

Bohannon, Kevin P; Holz, Ronald W; Axelrod, Daniel

2017-10-01

The refractive index in the interior of single cells affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence, but often that index is not well known. We here present method to measure and spatially map the absolute index of refraction in a microscopic sample, by imaging a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance-to-substrate can be determined. We apply the technique to liquid calibration samples, silica beads, cultured Chinese hamster ovary cells, and primary culture chromaffin cells. The optical technique suffers from decremented lateral resolution, scattering, and interference artifacts. However, it still provides reasonable results for both refractive index (~1.38) and for distance-to-substrate (~150 nm) for the cells, as well as a lateral resolution to about 1 µm.

Oxygen saturation and perfusion changes during dermatological methylaminolaevulinate photodynamic therapy.

PubMed

Tyrrell, J; Thorn, C; Shore, A; Campbell, S; Curnow, A

2011-12-01

Methylaminolaevulinate (MAL)-photodynamic therapy (PDT) is a successful topical treatment for a number of (pre)cancerous dermatological conditions. In combination, light of the appropriate wavelength, the photosensitizer protoporphyrin IX (PpIX) and tissue oxygen result in the production of singlet oxygen and reactive oxygen species inducing cell death. This study investigates real-time changes in localized tissue blood oxygen saturation and perfusion in conjunction with PpIX fluorescence monitoring for the first time during dermatological MAL-PDT. Oxygen saturation, perfusion and PpIX fluorescence were monitored noninvasively utilizing optical reflectance spectroscopy, laser Doppler perfusion imaging and a fluorescence imaging system, respectively. Patients attending for standard dermatological MAL-PDT were recruited to this ethically approved study and monitored prior to, during and after light irradiation. Significant reductions in mean blood oxygen saturation (P < 0·005) and PpIX fluorescence (P < 0·001) were observed within the first minute of irradiation (4·75 J cm(-2) ) while, in contrast, perfusion was observed to increase significantly (P < 0·01) during treatment. The changes in oxygen saturation and PpIX fluorescence were positively correlated during the initial phase of treatment (r(2) = 0·766). Rapid reductions in the localized blood oxygen saturation have been observed for the first time to occur clinically within the initial minutes of light irradiation and positively correlate with the concurrent PpIX photobleaching. Furthermore, perfusion increases, suggesting that the microvasculature compensates for the PDT-induced oxygen depletion. © 2011 The Authors. BJD © 2011 British Association of Dermatologists 2011.

Ultrasound-mediated Optical Imaging and Focusing in Scattering Media

NASA Astrophysics Data System (ADS)

Suzuki, Yuta

Because of its non-ionizing and molecular sensing nature, light has been an attractive tool in biomedicine. Scanning an optical focus allows not only high-resolution imaging but also manipulation and therapy. However, due to multiple photon scattering events, conventional optical focusing using an ordinary lens is limited to shallow depths of one transport mean free path (lt'), which corresponds to approximately 1 mm in human tissue. To overcome this limitation, ultrasonic modulation (or encoding ) of diffuse light inside scattering media has enabled us to develop both deep-tissue optical imaging and focusing techniques, namely, ultrasound-modulated optical tomography (UOT) and time-reversed ultrasonically encoded (TRUE) optical focusing. While UOT measures the power of the encoded light to obtain an image, TRUE focusing generates a time-reversed (or phase-conjugated) copy of the encoded light, using a phase-conjugate mirror to focus light inside scattering media beyond 1 lt'. However, despite extensive progress in both UOT and TRUE focusing, the low signal-to-noise ratio in encoded-light detection remains a challenge to meeting both the speed and depth requirements for in vivo applications. This dissertation describes technological advancements of both UOT and TRUE focusing, in terms of their signal detection sensitivities, operational depths, and operational speeds. The first part of this dissertation describes sensitivity improvements of encoded-light detection in UOT, achieved by using a large area (˜5 cm x 5 cm) photorefractive polymer. The photorefractive polymer allowed us to improve the detection etendue by more than 10 times that of previous detection schemes. It has enabled us to resolve absorbing objects embedded inside diffused media thicker than 80 lt', using moderate light power and short ultrasound pulses. The second part of this dissertation describes energy enhancement and fluorescent excitation using TRUE focusing in turbid media, using photorefractive materials as the phase-conjugate mirrors. By using a large-area photorefractive polymer as the phase-conjugate mirror, we boosted the focused optical energy by ~40 times over the output of a previously used photorefractive Bi 12SiO20 crystal. Furthermore, using both a photorefractive polymer and a Bi12SiO20 crystal as the phase-conjugate mirrors, we show direct visualization and dynamic control of TRUE focus, and demonstrate fluorescence imaging in a thick turbid medium. The last part of this dissertation describes improvements in the scanning speed of a TRUE focus, using digital phase-conjugate mirrors in both transmission and reflection modes. By employing a multiplex recording of ultrasonically encoded wavefronts in transmission mode, we have accelerated the generation of multiple TRUE foci, using frequency sweeping of both ultrasound and light. With this technique, we obtained a 2-D image of a fluorescent target centered inside a turbid sample having a thickness of 2.4 lt'. Also, by gradually moving the focal position in reflection mode, we show that the TRUE focal intensity is improved, and can be continuously scanned to image fluorescent targets in a shorter time.

Localized surface plasmon enhanced cellular imaging using random metallic structures

NASA Astrophysics Data System (ADS)

Son, Taehwang; Lee, Wonju; Kim, Donghyun

2017-02-01

We have studied fluorescence cellular imaging with randomly distributed localized near-field induced by silver nano-islands. For the fabrication of nano-islands, a 10-nm silver thin film evaporated on a BK7 glass substrate with an adhesion layer of 2-nm thick chromium. Micrometer sized silver square pattern was defined using e-beam lithography and then the film was annealed at 200°C. Raw images were restored using electric field distribution produced on the surface of random nano-islands. Nano-islands were modeled from SEM images. 488-nm p-polarized light source was set to be incident at 60°. Simulation results show that localized electric fields were created among nano-islands and that their average size was found to be 135 nm. The feasibility was tested using conventional total internal reflection fluorescence microscopy while the angle of incidence was adjusted to maximize field enhancement. Mouse microphage cells were cultured on nano-islands, and actin filaments were selectively stained with FITC-conjugated phalloidin. Acquired images were deconvolved based on linear imaging theory, in which molecular distribution was sampled by randomly distributed localized near-field and blurred by point spread function of far-field optics. The optimum fluorophore distribution was probabilistically estimated by repetitively matching a raw image. The deconvolved images are estimated to have a resolution in the range of 100-150 nm largely determined by the size of localized near-fields. We also discuss and compare the results with images acquired with periodic nano-aperture arrays in various optical configurations to excite localized plasmonic fields and to produce super-resolved molecular images.

Reflection imaging of China ink-perfused brain vasculature using confocal laser-scanning microscopy after clarification of brain tissue by the Spalteholz method.

PubMed

Gutierre, R C; Vannucci Campos, D; Mortara, R A; Coppi, A A; Arida, R M

2017-04-01

Confocal laser-scanning microscopy is a useful tool for visualizing neurons and glia in transparent preparations of brain tissue from laboratory animals. Currently, imaging capillaries and venules in transparent brain tissues requires the use of fluorescent proteins. Here, we show that vessels can be imaged by confocal laser-scanning microscopy in transparent cortical, hippocampal and cerebellar preparations after clarification of China ink-injected specimens by the Spalteholz method. This method may be suitable for global, three-dimensional, quantitative analyses of vessels, including stereological estimations of total volume and length and of surface area of vessels, which constitute indirect approaches to investigate angiogenesis. © 2017 Anatomical Society.

The Mechanisms and Biomedical Applications of an NIR BODIPY-Based Switchable Fluorescent Probe

PubMed Central

Cheng, Bingbing; Bandi, Venugopal; Yu, Shuai; D’Souza, Francis; Nguyen, Kytai T.; Hong, Yi; Tang, Liping; Yuan, Baohong

2017-01-01

Highly environment-sensitive fluorophores have been desired for many biomedical applications. Because of the noninvasive operation, high sensitivity, and high specificity to the microenvironment change, they can be used as excellent probes for fluorescence sensing/imaging, cell tracking/imaging, molecular imaging for cancer, and so on (i.e., polarity, viscosity, temperature, or pH measurement). In this work, investigations of the switching mechanism of a recently reported near-infrared environment-sensitive fluorophore, ADP(CA)2, were conducted. Besides, multiple potential biomedical applications of this switchable fluorescent probe have been demonstrated, including wash-free live-cell fluorescence imaging, in vivo tissue fluorescence imaging, temperature sensing, and ultrasound-switchable fluorescence (USF) imaging. The fluorescence of the ADP(CA)2 is extremely sensitive to the microenvironment, especially polarity and viscosity. Our investigations showed that the fluorescence of ADP(CA)2 can be switched on by low polarity, high viscosity, or the presence of protein and surfactants. In wash-free live-cell imaging, the fluorescence of ADP(CA)2 inside cells was found much brighter than the dye-containing medium and was retained for at least two days. In all of the fluorescence imaging applications conducted in this study, high target-to-noise (>5-fold) was achieved. In addition, a high temperature sensitivity (73-fold per Celsius degree) of ADP(CA)2-based temperature probes was found in temperature sensing. PMID:28208666

Fluorescence lifetime imaging ophthalmoscopy.

PubMed

Dysli, Chantal; Wolf, Sebastian; Berezin, Mikhail Y; Sauer, Lydia; Hammer, Martin; Zinkernagel, Martin S

2017-09-01

Imaging techniques based on retinal autofluorescence have found broad applications in ophthalmology because they are extremely sensitive and noninvasive. Conventional fundus autofluorescence imaging measures fluorescence intensity of endogenous retinal fluorophores. It mainly derives its signal from lipofuscin at the level of the retinal pigment epithelium. Fundus autofluorescence, however, can not only be characterized by the spatial distribution of the fluorescence intensity or emission spectrum, but also by a characteristic fluorescence lifetime function. The fluorescence lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo measurement of lifetimes of endogenous retinal fluorophores. Recent reports in this field have contributed to our understanding of the pathophysiology of various macular and retinal diseases. Within this review, the basic concept of fluorescence lifetime imaging is provided. It includes technical background information and correlation with in vitro measurements of individual retinal metabolites. In a second part, clinical applications of fluorescence lifetime imaging and fluorescence lifetime features of selected retinal diseases such as Stargardt disease, age-related macular degeneration, choroideremia, central serous chorioretinopathy, macular holes, diabetic retinopathy, and retinal artery occlusion are discussed. Potential areas of use for fluorescence lifetime imaging ophthalmoscopy will be outlined at the end of this review. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Ultra-sensitive fluorescent imaging-biosensing using biological photonic crystals

NASA Astrophysics Data System (ADS)

Squire, Kenny; Kong, Xianming; Wu, Bo; Rorrer, Gregory; Wang, Alan X.

2018-02-01

Optical biosensing is a growing area of research known for its low limits of detection. Among optical sensing techniques, fluorescence detection is among the most established and prevalent. Fluorescence imaging is an optical biosensing modality that exploits the sensitivity of fluorescence in an easy-to-use process. Fluorescence imaging allows a user to place a sample on a sensor and use an imager, such as a camera, to collect the results. The image can then be processed to determine the presence of the analyte. Fluorescence imaging is appealing because it can be performed with as little as a light source, a camera and a data processor thus being ideal for nontrained personnel without any expensive equipment. Fluorescence imaging sensors generally employ an immunoassay procedure to selectively trap analytes such as antigens or antibodies. When the analyte is present, the sensor fluoresces thus transducing the chemical reaction into an optical signal capable of imaging. Enhancement of this fluorescence leads to an enhancement in the detection capabilities of the sensor. Diatoms are unicellular algae with a biosilica shell called a frustule. The frustule is porous with periodic nanopores making them biological photonic crystals. Additionally, the porous nature of the frustule allows for large surface area capable of multiple analyte binding sites. In this paper, we fabricate a diatom based ultra-sensitive fluorescence imaging biosensor capable of detecting the antibody mouse immunoglobulin down to a concentration of 1 nM. The measured signal has an enhancement of 6× when compared to sensors fabricated without diatoms.

Quantification of tumor fluorescence during intraoperative optical cancer imaging.

PubMed

Judy, Ryan P; Keating, Jane J; DeJesus, Elizabeth M; Jiang, Jack X; Okusanya, Olugbenga T; Nie, Shuming; Holt, David E; Arlauckas, Sean P; Low, Phillip S; Delikatny, E James; Singhal, Sunil

2015-11-13

Intraoperative optical cancer imaging is an emerging technology in which surgeons employ fluorophores to visualize tumors, identify tumor-positive margins and lymph nodes containing metastases. This study compares instrumentation to measure tumor fluorescence. Three imaging systems (Spectropen, Glomax, Flocam) measured and quantified fluorescent signal-to-background ratios (SBR) in vitro, murine xenografts, tissue phantoms and clinically. Evaluation criteria included the detection of small changes in fluorescence, sensitivity of signal detection at increasing depths and practicality of use. In vitro, spectroscopy was superior in detecting incremental differences in fluorescence than luminescence and digital imaging (Ln[SBR] = 6.8 ± 0.6, 2.4 ± 0.3, 2.6 ± 0.1, p = 0.0001). In fluorescent tumor cells, digital imaging measured higher SBRs than luminescence (6.1 ± 0.2 vs. 4.3 ± 0.4, p = 0.001). Spectroscopy was more sensitive than luminometry and digital imaging in identifying murine tumor fluorescence (SBR = 41.7 ± 11.5, 5.1 ± 1.8, 4.1 ± 0.9, p = 0.0001), and more sensitive than digital imaging at detecting fluorescence at increasing depths (SBR = 7.0 ± 3.4 vs. 2.4 ± 0.5, p = 0.03). Lastly, digital imaging was the most practical and least time-consuming. All methods detected incremental differences in fluorescence. Spectroscopy was the most sensitive for small changes in fluorescence. Digital imaging was the most practical considering its wide field of view, background noise filtering capability, and sensitivity to increasing depth.

Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems.

PubMed

Yan, Yuling; Marriott, M Emma; Petchprayoon, Chutima; Marriott, Gerard

2011-02-01

Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.

Coregistered fluorescence-enhanced tumor resection of malignant glioma: relationships between δ-aminolevulinic acid–induced protoporphyrin IX fluorescence, magnetic resonance imaging enhancement, and neuropathological parameters

PubMed Central

Roberts, David W.; Valdés, Pablo A.; Harris, Brent T.; Fontaine, Kathryn M.; Hartov, Alexander; Fan, Xiaoyao; Ji, Songbai; Lollis, S. Scott; Pogue, Brian W.; Leblond, Frederic; Tosteson, Tor D.; Wilson, Brian C.; Paulsen, Keith D.

2010-01-01

Object The aim of this study was to investigate the relationships between intraoperative fluorescence, features on MR imaging, and neuropathological parameters in 11 cases of newly diagnosed glioblastoma multiforme (GBM) treated using protoporphyrin IX (PpIX) fluorescence-guided resection. Methods In 11 patients with a newly diagnosed GBM, δ-aminolevulinic acid (ALA) was administered to enhance endogenous synthesis of the fluorophore PpIX. The patients then underwent fluorescence-guided resection, coregistered with conventional neuronavigational image guidance. Biopsy specimens were collected at different times during surgery and assigned a fluorescence level of 0–3 (0, no fluorescence; 1, low fluorescence; 2, moderate fluorescence; or 3, high fluorescence). Contrast enhancement on MR imaging was quantified using two image metrics: 1) Gd-enhanced signal intensity (GdE) on T1-weighted subtraction MR image volumes, and 2) normalized contrast ratios (nCRs) in T1-weighted, postGd-injection MR image volumes for each biopsy specimen, using the biopsy-specific image-space coordinate transformation provided by the navigation system. Subsequently, each GdE and nCR value was grouped into one of two fluorescence categories, defined by its corresponding biopsy specimen fluorescence assessment as negative fluorescence (fluorescence level 0) or positive fluorescence (fluorescence level 1, 2, or 3). A single neuropathologist analyzed the H & E–stained tissue slides of each biopsy specimen and measured three neuropathological parameters: 1) histopathological score (0–IV); 2) tumor burden score (0–III); and 3) necrotic burden score (0–III). Results Mixed-model analyses with random effects for individuals show a highly statistically significant difference between fluorescing and nonfluorescing tissue in GdE (mean difference 8.33, p = 0.018) and nCRs (mean difference 5.15, p < 0.001). An analysis of association demonstrated a significant relationship between the levels of intraoperative fluorescence and histopathological score (χ2 = 58.8, p < 0.001), between fluorescence levels and tumor burden (χ2 = 42.7, p < 0.001), and between fluorescence levels and necrotic burden (χ2 = 30.9, p < 0.001). The corresponding Spearman rank correlation coefficients were 0.51 (p < 0.001) for fluorescence and histopathological score, and 0.49 (p < 0.001) for fluorescence and tumor burden, suggesting a strongly positive relationship for each of these variables. Conclusions These results demonstrate a significant relationship between contrast enhancement on preoperative MR imaging and observable intraoperative PpIX fluorescence. The finding that preoperative MR image signatures are predictive of intraoperative PpIX fluorescence is of practical importance for identifying candidates for the procedure. Furthermore, this study provides evidence that a strong relationship exists between tumor aggressiveness and the degree of tissue fluorescence that is observable intraoperatively, and that observable fluorescence has an excellent positive predictive value but a low negative predictive value. PMID:20380535

Multi-scale spectrally resolved quantitative fluorescence imaging system: towards neurosurgical guidance in glioma resection

NASA Astrophysics Data System (ADS)

Xie, Yijing; Thom, Maria; Miserocchi, Anna; McEvoy, Andrew W.; Desjardins, Adrien; Ourselin, Sebastien; Vercauteren, Tom

2017-02-01

In glioma resection surgery, the detection of tumour is often guided by using intraoperative fluorescence imaging notably with 5-ALA-PpIX, providing fluorescent contrast between normal brain tissue and the gliomas tissue to achieve improved tumour delineation and prolonged patient survival compared with the conventional white-light guided resection. However, the commercially available fluorescence imaging system relies on surgeon's eyes to visualise and distinguish the fluorescence signals, which unfortunately makes the resection subjective. In this study, we developed a novel multi-scale spectrally-resolved fluorescence imaging system and a computational model for quantification of PpIX concentration. The system consisted of a wide-field spectrally-resolved quantitative imaging device and a fluorescence endomicroscopic imaging system enabling optical biopsy. Ex vivo animal tissue experiments as well as human tumour sample studies demonstrated that the system was capable of specifically detecting the PpIX fluorescent signal and estimate the true concentration of PpIX in brain specimen.

Gold nanoclusters as contrast agents for fluorescent and X-ray dual-modality imaging.

PubMed

Zhang, Aili; Tu, Yu; Qin, Songbing; Li, Yan; Zhou, Juying; Chen, Na; Lu, Qiang; Zhang, Bingbo

2012-04-15

Multimodal imaging technique is an alternative approach to improve sensitivity of early cancer diagnosis. In this study, highly fluorescent and strong X-ray absorption coefficient gold nanoclusters (Au NCs) are synthesized as dual-modality imaging contrast agents (CAs) for fluorescent and X-ray dual-modality imaging. The experimental results show that the as-prepared Au NCs are well constructed with ultrasmall sizes, reliable fluorescent emission, high computed tomography (CT) value and fine biocompatibility. In vivo imaging results indicate that the obtained Au NCs are capable of fluorescent and X-ray enhanced imaging. Copyright © 2012 Elsevier Inc. All rights reserved.

Indocyanine green fluorescence imaging in the surgical management of liver cancers: current facts and future implications.

PubMed

Lim, C; Vibert, E; Azoulay, D; Salloum, C; Ishizawa, T; Yoshioka, R; Mise, Y; Sakamoto, Y; Aoki, T; Sugawara, Y; Hasegawa, K; Kokudo, N

2014-04-01

Imaging detection of liver cancers and identification of the bile ducts during surgery, based on the fluorescence properties of indocyanine green, has recently been developed in liver surgery. The principle of this imaging technique relies on the intravenous administration of indocyanine green before surgery and the illumination of the surface of the liver by an infrared camera that simultaneously induces and collects the fluorescence. Detection by fluorescence is based on the contrast between the (fluorescent) tumoral or peri-tumoral tissues and the healthy (non-fluorescent) liver. Results suggest that indocyanine green fluorescence imaging is capable of identification of new liver cancers and enables the characterization of known hepatic lesions in real time during liver resection. The purpose of this paper is to present the fundamental principles of fluorescence imaging detection, to describe successively the practical and technical aspects of its use and the appearance of hepatic lesions in fluorescence, and to expose the diagnostic and therapeutic perspectives of this innovative imaging technique in liver surgery. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample

NASA Astrophysics Data System (ADS)

Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen

2010-03-01

In conventional fluorescence or confocal microscopy, emitted light is generated not only in the focal plane but also above and below. The situation is different in multiphoton-induced fluorescence and multiphoton-induced higher harmonic generation. Here, restriction of signal generation to a single focal point permits that all emitted photons can contribute to image formation if collected, regardless of their path through the specimen. Often, the intensity of the emitted light is rather low in biological specimens. We present a method to significantly increase the fraction of photons collected by an epi (backward) detector by placing a simple mirror, an aluminum-coated coverslip, directly under the sample. Samples investigated include fluorescent test slides, collagen gels, and thin-layered, intact mouse skeletal muscles. Quantitative analysis revealed an intensity increase of second- and third-harmonic generated signal in skeletal muscle of nine- and sevenfold respectively, and of fluorescent signal in test slides of up to twofold. Our approach thus allows significant signal improvement also for situations were a forward detection is impossible, e.g., due to the anatomy of animals in intravital microscopy.

MRI-guided fluorescence tomography of the breast: a phantom study

NASA Astrophysics Data System (ADS)

Davis, Scott C.; Pogue, Brian W.; Dehghani, Hamid; Paulsen, Keith D.

2009-02-01

Tissue phantoms simulating the human breast were used to demonstrate the imaging capabilities of an MRI-coupled fluorescence molecular tomography (FMT) imaging system. Specifically, phantoms with low tumor-to-normal drug contrast and complex internal structure were imaged with the MR-coupled FMT system. Images of indocyanine green (ICG) fluorescence yield were recovered using a diffusion model-based approach capable of estimating the distribution of fluorescence activity in a tissue volume from tissue-boundary measurements of transmitted light. Tissue structural information, which can be determined from standard T1 and T2 MR images, was used to guide the recovery of fluorescence activity. The study revealed that this spatial guidance is critical for recovering images of fluorescence yield in tissue with low tumor-to-normal drug contrast.

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells.

PubMed

Nygate, Yoav N; Singh, Gyanendra; Barnea, Itay; Shaked, Natan T

2018-06-01

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

Design of a dynamic biofilm imaging cell for white-light interferometric microscopy

NASA Astrophysics Data System (ADS)

Larimer, Curtis; Brann, Michelle; Suter, Jonathan D.; Addleman, R. Shane

2017-11-01

In microbiology research, there is a strong need for next-generation imaging and sensing instrumentation that will enable minimally invasive and label-free investigation of soft, hydrated structures, such as in bacterial biofilms. White-light interferometry (WLI) can provide high-resolution images of surface topology without the use of fluorescent labels but is not typically used to image biofilms because there is insufficient refractive index contrast to induce reflection from the biofilm's interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ, especially in a nondestructive manner. We build on our prior description of static biofilm imaging and describe the design of a dynamic growth flow cell that enables monitoring of the thickness and topology of live biofilms over time using a WLI microscope. The microfluidic system is designed to grow biofilms in dynamic conditions and to create a reflective interface on the surface while minimizing disruption of fragile structures. The imaging cell was also designed to accommodate limitations imposed by the depth of focus of the microscope's objective lens. Example images of live biofilm samples are shown to illustrate the ability of the flow cell and WLI instrument to (1) support bacterial growth and biofilm development, (2) image biofilm structure that reflects growth in flow conditions, and (3) monitor biofilm development over time nondestructively. In future work, the apparatus described here will enable surface metrology measurements (roughness, surface area, etc.) of biofilms and may be used to observe changes in biofilm structure in response to changes in environmental conditions (e.g., flow velocity, availability of nutrients, and presence of biocides). This development will open opportunities for the use of WLI in bioimaging.

Fluorescent image tracking velocimeter

DOEpatents

Shaffer, Franklin D.

1994-01-01

A multiple-exposure fluorescent image tracking velocimeter (FITV) detects and measures the motion (trajectory, direction and velocity) of small particles close to light scattering surfaces. The small particles may follow the motion of a carrier medium such as a liquid, gas or multi-phase mixture, allowing the motion of the carrier medium to be observed, measured and recorded. The main components of the FITV include: (1) fluorescent particles; (2) a pulsed fluorescent excitation laser source; (3) an imaging camera; and (4) an image analyzer. FITV uses fluorescing particles excited by visible laser light to enhance particle image detectability near light scattering surfaces. The excitation laser light is filtered out before reaching the imaging camera allowing the fluoresced wavelengths emitted by the particles to be detected and recorded by the camera. FITV employs multiple exposures of a single camera image by pulsing the excitation laser light for producing a series of images of each particle along its trajectory. The time-lapsed image may be used to determine trajectory and velocity and the exposures may be coded to derive directional information.

Near-Infrared Squaraine Dye Encapsulated Micelles for in Vivo Fluorescence and Photoacoustic Bimodal Imaging.

PubMed

Sreejith, Sivaramapanicker; Joseph, James; Lin, Manjing; Menon, Nishanth Venugopal; Borah, Parijat; Ng, Hao Jun; Loong, Yun Xian; Kang, Yuejun; Yu, Sidney Wing-Kwong; Zhao, Yanli

2015-06-23

Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1(micelle)) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1(micelle) exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1(micelle) in the NIR window of 800-900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1(micelle), as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1(micelle) proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging.

Clinical application of indocyanine green (ICG) fluorescent imaging of hepatoblastoma.

PubMed

Yamamichi, Taku; Oue, Takaharu; Yonekura, Takeo; Owari, Mitsugu; Nakahata, Kengo; Umeda, Satoshi; Nara, Keigo; Ueno, Takehisa; Uehara, Shuichiro; Usui, Noriaki

2015-05-01

Although the usefulness of intraoperative indocyanine green (ICG) fluorescent imaging for the resection of hepatocellular carcinoma has been reported, its usefulness for the resection of hepatoblastoma remains unclear. This study clarifies the feasibility of intraoperative ICG fluorescent imaging for the resection of hepatoblastoma. In three hepatoblastoma patients, a primary tumor, recurrent tumor, and lung metastatic lesions were intraoperatively examined using a near-infrared fluorescence imaging system after the preoperative administration of ICG. ICG fluorescent imaging was useful for the surgical navigation in hepatoblastoma patients. In the first case, the primary hepatoblastoma exhibited intense fluorescence during right hepatectomy, but no fluorescence was detected in the residual liver. In the second case, a recurrent tumor exhibited fluorescence between the residual liver and diaphragm. A complete resection of the residual liver, with a partial resection of the diaphragm, followed by liver transplantation was performed. In the third case with multiple lung metastases, each metastatic lesion showed positive fluorescence, and all were completely resected. These fluorescence-positive lesions were pathologically proven to be viable hepatoblastoma cells. Intraoperative ICG fluorescence imaging for patients with hepatoblastoma was feasible and useful for identifying small viable lesions and confirming that no remnant tumor remained after resection. Copyright © 2015 Elsevier Inc. All rights reserved.

Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter

NASA Astrophysics Data System (ADS)

Wang, Fu; Yang, Kai; Wang, Zhe; Ma, Ying; Gutkind, J. Silvio; Hida, Naoki; Niu, Gang; Tian, Jie

2016-02-01

Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies.Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07308a

Photoreceptor layer map using spectral-domain optical coherence tomography.

PubMed

Lee, Ji Eun; Lim, Dae Won; Bae, Han Yong; Park, Hyun Jin

2009-12-01

To develop a novel method for analysis of the photoreceptor layer map (PLM) generated using spectral-domain optical coherence tomography (OCT). OCT scans were obtained from 20 eyes, 10 with macular holes (MH) and 10 with central serous chorioretinopathy (CSC) using the Macular Cube (512 x 128) protocol of the Cirrus HD-OCT (Carl Zeiss). The scanned data were processed using embedded tools of the advanced visualization. A partial thickness OCT fundus image of the photoreceptor layer was generated by setting the region of interest to a 50-microm thick layer that was parallel and adjacent to the retinal pigment epithelium. The resulting image depicted the photoreceptor layer as a map of the reflectivity in OCT. The PLM was compared with fundus photography, auto-fluorescence, tomography, and retinal thickness map. The signal from the photoreceptor layer of every OCT scan in each case was demonstrated as a single image of PLM in a fundus photograph fashion. In PLM images, detachment of the sensory retina is depicted as a hypo-reflective area, which represents the base of MH and serous detachment in CSC. Relative hypo-reflectivity, which was also noted at closed MH and at recently reattached retina in CSC, was associated with reduced signal from the junction between the inner and outer segments of photoreceptors in OCT images. Using PLM, changes in the area of detachment and reflectivity of the photoreceptor layer could be efficiently monitored. The photoreceptor layer can be analyzed as a map using spectral-domain OCT. In the treatment of both MH and CSC, PLM may provide new pathological information about the photoreceptor layer to expand our understanding of these diseases.

An excitation wavelength-scanning spectral imaging system for preclinical imaging

NASA Astrophysics Data System (ADS)

Leavesley, Silas; Jiang, Yanan; Patsekin, Valery; Rajwa, Bartek; Robinson, J. Paul

2008-02-01

Small-animal fluorescence imaging is a rapidly growing field, driven by applications in cancer detection and pharmaceutical therapies. However, the practical use of this imaging technology is limited by image-quality issues related to autofluorescence background from animal tissues, as well as attenuation of the fluorescence signal due to scatter and absorption. To combat these problems, spectral imaging and analysis techniques are being employed to separate the fluorescence signal from background autofluorescence. To date, these technologies have focused on detecting the fluorescence emission spectrum at a fixed excitation wavelength. We present an alternative to this technique, an imaging spectrometer that detects the fluorescence excitation spectrum at a fixed emission wavelength. The advantages of this approach include increased available information for discrimination of fluorescent dyes, decreased optical radiation dose to the animal, and ability to scan a continuous wavelength range instead of discrete wavelength sampling. This excitation-scanning imager utilizes an acousto-optic tunable filter (AOTF), with supporting optics, to scan the excitation spectrum. Advanced image acquisition and analysis software has also been developed for classification and unmixing of the spectral image sets. Filtering has been implemented in a single-pass configuration with a bandwidth (full width at half maximum) of 16nm at 550nm central diffracted wavelength. We have characterized AOTF filtering over a wide range of incident light angles, much wider than has been previously reported in the literature, and we show how changes in incident light angle can be used to attenuate AOTF side lobes and alter bandwidth. A new parameter, in-band to out-of-band ratio, was defined to assess the quality of the filtered excitation light. Additional parameters were measured to allow objective characterization of the AOTF and the imager as a whole. This is necessary for comparing the excitation-scanning imager to other spectral and fluorescence imaging technologies. The effectiveness of the hyperspectral imager was tested by imaging and analysis of mice with injected fluorescent dyes. Finally, a discussion of the optimization of spectral fluorescence imagers is given, relating the effects of filter quality on fluorescence images collected and the analysis outcome.

Multispectral fluorescence imaging technique for discrimination of cucumber (Cucumis Sativus) seed viability

USDA-ARS?s Scientific Manuscript database

In this study, we developed a nondestructive method for discriminating viable cucumber (Cucumis sativus) seeds based on hyperspectral fluorescence imaging. The fluorescence spectra of cucumber seeds in the 420–700 nm range were extracted from hyperspectral fluorescence images obtained using 365 nm u...

In-line FINCH super resolution digital holographic fluorescence microscopy using a high efficiency transmission liquid crystal GRIN lens.

PubMed

Brooker, Gary; Siegel, Nisan; Rosen, Joseph; Hashimoto, Nobuyuki; Kurihara, Makoto; Tanabe, Ayano

2013-12-15

We report a new optical arrangement that creates high-efficiency, high-quality Fresnel incoherent correlation holography (FINCH) holograms using polarization sensitive transmission liquid crystal gradient index (TLCGRIN) diffractive lenses. In contrast, current universal practice in the field employs a reflective spatial light modulator (SLM) to separate sample and reference beams. Polarization sensitive TLCGRIN lenses enable a straight optical path, have >90% transmission efficiency, are not pixilated, and are free of many limitations of reflective SLM devices. For each sample point, two spherical beams created by a glass lens in combination with a polarization sensitive TLCGRIN lens interfere and create a hologram and resultant super resolution image.

Differentiation of angiogenic burden in human cancer xenografts using a perfusion-type optical contrast agent (SIDAG)

PubMed Central

Wall, Alexander; Persigehl, Thorsten; Hauff, Peter; Licha, Kai; Schirner, Michael; Müller, Silke; von Wallbrunn, Angelika; Matuszewski, Lars; Heindel, Walter; Bremer, Christoph

2008-01-01

Introduction Use of fluorescence imaging in oncology is evolving rapidly, and nontargeted fluorochromes are currently being investigated for clinical application. Here, we investigated whether the degree of tumour angiogenesis can be assessed in vivo by planar and tomographic methods using the perfusion-type cyanine dye SIDAG (1,1'-bis- [4-sulfobutyl]indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium). Method Mice were xenografted with moderately (MCF7, DU4475) or highly vascularized (HT1080, MDA-MB435) tumours and scanned up to 24 hours after intravenous SIDAG injection using fluorescence reflectance imaging. Contrast-to-noise ratio was calculated for all tumours, and fluorochrome accumulation was quantified using fluorescence-mediated tomography. The vascular volume fraction of the xenografts, serving as a surrogate marker for angiogenesis, was measured using magnetic resonance imaging, and blood vessel profile (BVP) density and vascular endothelial growth factor expression were determined. Results SIDAG accumulation correlated well with angiogenic burden, with maximum contrast to noise ratio for MDA-MB435 (P < 0.0001), followed by HT1080, MCF7 and DU4475 tumours. Fluorescence-mediated tomography revealed 4.6-fold higher fluorochrome concentrations in MDA-MB435 than in DU4475 tumours (229 ± 90 nmol/l versus 49 ± 22 nmol/l; P < 0.05). The vascular volume fraction was 4.5-fold (3.58 ± 0.9% versus 0.8 ± 0.53%; P < 0.01), blood vessel profile density 5-fold (399 ± 36 BVPs/mm2 versus 78 ± 16 BVPs/mm2) and vascular endothelial growth factor expression 4-fold higher for MDA-MB435 than for DU4475 tumours. Conclusion Our data suggest that perfusion-type cyanine dyes allow assessment of angiogenesis in vivo using planar or tomographic imaging technology. They may thus facilitate characterization of solid tumours. PMID:18331624

Multispectral laser-induced fluorescence imaging system for large biological samples

NASA Astrophysics Data System (ADS)

Kim, Moon S.; Lefcourt, Alan M.; Chen, Yud-Ren

2003-07-01

A laser-induced fluorescence imaging system developed to capture multispectral fluorescence emission images simultaneously from a relatively large target object is described. With an expanded, 355-nm Nd:YAG laser as the excitation source, the system captures fluorescence emission images in the blue, green, red, and far-red regions of the spectrum centered at 450, 550, 678, and 730 nm, respectively, from a 30-cm-diameter target area in ambient light. Images of apples and of pork meat artificially contaminated with diluted animal feces have demonstrated the versatility of fluorescence imaging techniques for potential applications in food safety inspection. Regions of contamination, including sites that were not readily visible to the human eye, could easily be identified from the images.

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

PubMed Central

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-01-01

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

Biophotonic endoscopy: a review of clinical research techniques for optical imaging and sensing of early gastrointestinal cancer

PubMed Central

Coda, Sergio; Siersema, Peter D.; Stamp, Gordon W. H.; Thillainayagam, Andrew V.

2015-01-01

Detection, characterization, and staging constitute the fundamental elements in the endoscopic diagnosis of gastrointestinal diseases, but histology still remains the diagnostic gold standard. New developments in endoscopic techniques may challenge histopathology in the near future. An ideal endoscopic technique should combine a wide-field, “red flag” screening technique with an optical contrast or microscopy method for characterization and staging, all simultaneously available during the procedure. In theory, biophotonic advances have the potential to unite these elements to allow in vivo “optical biopsy.” These techniques may ultimately offer the potential to increase the rates of detection of high risk lesions and the ability to target biopsies and resections, and so reduce the need for biopsy, costs, and uncertainty for patients. However, their utility and sensitivity in clinical practice must be evaluated against those of conventional histopathology. This review describes some of the most recent applications of biophotonics in endoscopic optical imaging and metrology, along with their fundamental principles and the clinical experience that has been acquired in their deployment as tools for the endoscopist. Particular emphasis has been placed on translational label-free optical techniques, such as fluorescence spectroscopy, fluorescence lifetime imaging microscopy (FLIM), two-photon and multi-photon microscopy, second harmonic generation (SHG) and third harmonic generation (THG) imaging, optical coherence tomography (OCT), diffuse reflectance, Raman spectroscopy, and molecular imaging. PMID:26528489

Guided fluorescence diagnosis of childhood caries: preliminary measures correlate with depth of carious decay

NASA Astrophysics Data System (ADS)

Timoshchuk, Mari-Alina; Zhang, Liang; Dickinson, Brian A.; Ridge, Jeremy S.; Kim, Amy S.; Baltuck, Camille T.; Nelson, Leonard Y.; Berg, Joel H.; Seibel, Eric J.

2014-02-01

The current rise in childhood caries worldwide has increased the demand for portable technologies that can quickly and accurately detect and diagnose early stage carious lesions. These lesions, if identified at an early stage, can be reversed with remineralization treatments, education, and improvements in home care. A multi-modal optical prototype for detecting and diagnosing occlusal caries demineralization in vivo has been developed and pilot tested. The device uses a 405-nm laser as a scanned illumination source to obtain high resolution and high surface contrast reflectance images, which allows the user to quickly image and screen for any signs of demineralized enamel. When a suspicious region is located, the device can be switched to perform dual laser fluorescence spectroscopy using 405-nm and 532-nm laser excitations. These spectra are used to compute an auto-fluorescence (AF) ratio of the suspicious region and the percent difference of AF ratios from a healthy region of the same tooth. The device was tested on 7 children's teeth in vivo with clinically diagnosed carious lesions. Lesion depth was then visually estimated from the video image using the 405-nm scanned light source, and within a month the maximum drill depth was assessed by a clinician. The researcher and clinicians were masked from previous measurements in a blinded study protocol. Preliminary results show that the ratiometric percent difference measurement of the AF spectrum of the tooth correlates with the severity of the demineralization as assessed by the clinician after drilling.

Intraoperative real-time localization of parathyroid gland with near infrared fluorescence imaging

PubMed Central

Kim, Sung Won; Lee, Hyoung Shin

2017-01-01

Surgeons have cited difficulties in identifying the parathyroid glands (PG) during thyroidectomy. To overcome the limitation of naked eye, many studies on near-infrared fluorescence imaging of PGs have been introduced and suggested that fluorescence imaging is useful for both localizing PGs and evaluating their function. This imaging technique has been reported in two ways: (I) imaging using a fluorescent material called indocyanine green (ICG); and (II) autofluorescence using intrinsic fluorophores. These innovative and novel techniques are expected to have a significant impact on performing thyroid or parathyroid surgery. In this article, current papers that describe ICG fluorescence and autofluorescence imaging of PG during thyroid and parathyroid surgery are reviewed. PMID:29142843

Flexible imaging payload for real-time fluorescent biological imaging in parabolic, suborbital and space analog environments

NASA Astrophysics Data System (ADS)

Bamsey, Matthew T.; Paul, Anna-Lisa; Graham, Thomas; Ferl, Robert J.

2014-10-01

Fluorescent imaging offers the ability to monitor biological functions, in this case biological responses to space-related environments. For plants, fluorescent imaging can include general health indicators such as chlorophyll fluorescence as well as specific metabolic indicators such as engineered fluorescent reporters. This paper describes the Flex Imager a fluorescent imaging payload designed for Middeck Locker deployment and now tested on multiple flight and flight-related platforms. The Flex Imager and associated payload elements have been developed with a focus on 'flexibility' allowing for multiple imaging modalities and change-out of individual imaging or control components in the field. The imaging platform is contained within the standard Middeck Locker spaceflight form factor, with components affixed to a baseplate that permits easy rearrangement and fine adjustment of components. The Flex Imager utilizes standard software packages to simplify operation, operator training, and evaluation by flight provider flight test engineers, or by researchers processing the raw data. Images are obtained using a commercial cooled CCD image sensor, with light-emitting diodes for excitation and a suite of filters that allow biological samples to be imaged over wavelength bands of interest. Although baselined for the monitoring of green fluorescent protein and chlorophyll fluorescence from Arabidopsis samples, the Flex Imager payload permits imaging of any biological sample contained within a standard 10 cm by 10 cm square Petri plate. A sample holder was developed to secure sample plates under different flight profiles while permitting sample change-out should crewed operations be possible. In addition to crew-directed imaging, autonomous or telemetric operation of the payload is also a viable operational mode. An infrared camera has also been integrated into the Flex Imager payload to allow concurrent fluorescent and thermal imaging of samples. The Flex Imager has been utilized to assess, in real-time, the response of plants to novel environments including various spaceflight analogs, including several parabolic flight environments as well as hypobaric plant growth chambers. Basic performance results obtained under these operational environments, as well as laboratory-based tests are described. The Flex Imager has also been designed to be compatible with emerging suborbital platforms.

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

PubMed

Higuchi-Sanabria, Ryo; Garcia, Enrique J; Tomoiaga, Delia; Munteanu, Emilia L; Feinstein, Paul; Pon, Liza A

2016-01-01

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

Two-photon excitation laser scanning microscopy of rabbit nasal septal cartilage following Nd:YAG-laser-mediated stress relaxation

NASA Astrophysics Data System (ADS)

Kim, Charlton C.; Wallace, Vincent P.; Coleno, Mariah L.; Dao, Xavier; Tromberg, Bruce J.; Wong, Brian J.

2000-04-01

Laser irradiation of hyaline cartilage result in stable shape changes due to temperature dependent stress relaxation. In this study, we determined the structural changes in chondrocytes within rabbit nasal septal cartilage tissue over a 12-day period using a two-photon laser scanning microscope (TPM) following Nd:YAG laser irradiation. During laser irradiation surface temperature, stress relaxation, and diffuse reflectance, were measured dynamically. Each specimen received one or two sequential laser exposures. The cartilage reached a peak surface temperature of about 61 degrees C during irradiation. Cartilage denatured in 50 percent EtOH was used as a positive control. TPM was performed to detect the fluorescence emission from the chondrocytes. Images of chondrocytes were obtained at depths up to 150 microns, immediately following laser exposure, and also following 12 days in culture. Few differences in the pattern or intensity of fluorescence was observed between controls and irradiated specimens imaged immediately following exposure, regardless of the number of laser pulses. However, following twelve days in tissue culture, the irradiated specimens increase, whereas the native tissue diminishes, in intensity and distribution of fluorescence in the cytoplasm. In contrast, the positive control shows only extracellular matrices and empty lacuna, feature consistent with cell membrane lysis.

Optical properties of mouse brain tissue after optical clearing with FocusClear™

NASA Astrophysics Data System (ADS)

Moy, Austin J.; Capulong, Bernard V.; Saager, Rolf B.; Wiersma, Matthew P.; Lo, Patrick C.; Durkin, Anthony J.; Choi, Bernard

2015-09-01

Fluorescence microscopy is commonly used to investigate disease progression in biological tissues. Biological tissues, however, are strongly scattering in the visible wavelengths, limiting the application of fluorescence microscopy to superficial (<200 μm) regions. Optical clearing, which involves incubation of the tissue in a chemical bath, reduces the optical scattering in tissue, resulting in increased tissue transparency and optical imaging depth. The goal of this study was to determine the time- and wavelength-resolved dynamics of the optical scattering properties of rodent brain after optical clearing with FocusClear™. Light transmittance and reflectance of 1-mm mouse brain sections were measured using an integrating sphere before and after optical clearing and the inverse adding doubling algorithm used to determine tissue optical scattering. The degree of optical clearing was quantified by calculating the optical clearing potential (OCP), and the effects of differing OCP were demonstrated using the optical histology method, which combines tissue optical clearing with optical imaging to visualize the microvasculature. We observed increased tissue transparency with longer optical clearing time and an analogous increase in OCP. Furthermore, OCP did not vary substantially between 400 and 1000 nm for increasing optical clearing durations, suggesting that optical histology can improve ex vivo visualization of several fluorescent probes.

Forensic imaging tools for law enforcement

DOE Office of Scientific and Technical Information (OSTI.GOV)

SMITHPETER,COLIN L.; SANDISON,DAVID R.; VARGO,TIMOTHY D.

2000-01-01

Conventional methods of gathering forensic evidence at crime scenes are encumbered by difficulties that limit local law enforcement efforts to apprehend offenders and bring them to justice. Working with a local law-enforcement agency, Sandia National Laboratories has developed a prototype multispectral imaging system that can speed up the investigative search task and provide additional and more accurate evidence. The system, called the Criminalistics Light-imaging Unit (CLU), has demonstrated the capabilities of locating fluorescing evidence at crime scenes under normal lighting conditions and of imaging other types of evidence, such as untreated fingerprints, by direct white-light reflectance. CLU employs state ofmore » the art technology that provides for viewing and recording of the entire search process on videotape. This report describes the work performed by Sandia to design, build, evaluate, and commercialize CLU.« less

Modeling in vivo fluorescence of small animals using TracePro software

NASA Astrophysics Data System (ADS)

Leavesley, Silas; Rajwa, Bartek; Freniere, Edward R.; Smith, Linda; Hassler, Richard; Robinson, J. Paul

2007-02-01

The theoretical modeling of fluorescence excitation, emission, and propagation within living tissue has been a limiting factor in the development and calibration of in vivo small animal fluorescence imagers. To date, no definitive calibration standard, or phantom, has been developed for use with small animal fluorescence imagers. Our work in the theoretical modeling of fluorescence in small animals using solid modeling software is useful in optimizing the design of small animal imaging systems, and in predicting their response to a theoretical model. In this respect, it is also valuable in the design of a fluorescence phantom for use in in vivo small animal imaging. The use of phantoms is a critical step in the testing and calibration of most diagnostic medical imaging systems. Despite this, a realistic, reproducible, and informative phantom has yet to be produced for use in small animal fluorescence imaging. By modeling the theoretical response of various types of phantoms, it is possible to determine which parameters are necessary for accurately modeling fluorescence within inhomogenous scattering media such as tissue. Here, we present the model that has been developed, the challenges and limitations associated with developing such a model, and the applicability of this model to experimental results obtained in a commercial small animal fluorescence imager.

ALA-induced PpIX spectroscopy for brain tumor image-guided surgery

NASA Astrophysics Data System (ADS)

Valdes, Pablo A.; Leblond, Frederic; Kim, Anthony; Harris, Brent T.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.

2011-03-01

Maximizing the extent of brain tumor resection correlates with improved survival and quality of life outcomes in patients. Optimal surgical resection requires accurate discrimination between normal and abnormal, cancerous tissue. We present our recent experience using quantitative optical spectroscopy in 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence-guided resection. Exogenous administration of ALA leads to preferential accumulation in tumor tissue of the fluorescent compound, PpIX, which can be used for in vivo surgical guidance. Using the state of the art approach with a fluorescence surgical microscope, we have been able to visualize a subset of brain tumors, but the sensitivity and accuracy of fluorescence detection for tumor tissue with this system are low. To take full advantage of the biological selectivity of PpIX accumulation in brain tumors, we used a quantitative optical spectroscopy system for in vivo measurements of PpIX tissue concentrations. We have shown that, using our quantitative approach for determination of biomarker concentrations, ALA-induced PpIX fluorescence-guidance can achieve accuracies of greater than 90% for most tumor histologies. Here we show multivariate analysis of fluorescence and diffuse reflectance signals in brain tumors with comparable diagnostic performance to our previously reported quantitative approach. These results are promising, since they show that technological improvements in current fluorescence-guided surgical technologies and more biologically relevant approaches are required to take full advantage of fluorescent biomarkers, achieve better tumor identification, increase extent of resection, and subsequently, lead to improve survival and quality of life in patients.

Three-dimensional particle tracking in concave structures made by ultraviolet nanoimprint via total internal reflection fluorescence microscopy and refractive-index-matching method

NASA Astrophysics Data System (ADS)

Fujinami, Taku; Kigami, Hiroshi; Unno, Noriyuki; Taniguchi, Jun; Satake, Shin-ichi

2018-06-01

Total internal reflection fluorescence microscopy (TIRFM) is a promising method for measuring fluid flow close to a wall with nanoscale resolution in a process that is termed "multilayer nanoparticle image velocimetry" (MnPIV). TIRFM uses evanescent light that is generated on a substrate (typically a glass slide) by total internal reflection of light. Many researchers have previously studied x- y- z (3D) flows of water close to flat glass slides using MnPIV. On the other hand, a fluid flow close to a structured surface is also important. To measure flows of water near micro-patterns, we previously developed an MnPIV technique that uses a refractive-index-matching method. In previous study, the micropattern is made of a thermoplastic material with a refractive index that closely matches that of water. In this study, ultraviolet nanoimprint lithography was used for fabricating the appropriate micro-patterns because this technique can fabricate a pattern with a high resolution. As a result, we succeeded in performing MnPIV in water with a circular hole array pattern made by ultraviolet nanoimprint using a refractive-index-matching method. We believe that this technique will be helpful in elucidating fluid flows around microstructures.

Three-dimensional particle tracking in concave structures made by ultraviolet nanoimprint via total internal reflection fluorescence microscopy and refractive-index-matching method

NASA Astrophysics Data System (ADS)

Fujinami, Taku; Kigami, Hiroshi; Unno, Noriyuki; Taniguchi, Jun; Satake, Shin-ichi

2018-03-01

Total internal reflection fluorescence microscopy (TIRFM) is a promising method for measuring fluid flow close to a wall with nanoscale resolution in a process that is termed "multilayer nanoparticle image velocimetry" (MnPIV). TIRFM uses evanescent light that is generated on a substrate (typically a glass slide) by total internal reflection of light. Many researchers have previously studied x-y-z (3D) flows of water close to flat glass slides using MnPIV. On the other hand, a fluid flow close to a structured surface is also important. To measure flows of water near micro-patterns, we previously developed an MnPIV technique that uses a refractive-index-matching method. In previous study, the micropattern is made of a thermoplastic material with a refractive index that closely matches that of water. In this study, ultraviolet nanoimprint lithography was used for fabricating the appropriate micro-patterns because this technique can fabricate a pattern with a high resolution. As a result, we succeeded in performing MnPIV in water with a circular hole array pattern made by ultraviolet nanoimprint using a refractive-index-matching method. We believe that this technique will be helpful in elucidating fluid flows around microstructures.

One-Pot Synthesis of Fe3O4@PS@P(AEMH-FITC) Magnetic Fluorescent Nanocomposites for Bimodal Imaging.

PubMed

Wang, Xuandong; Liu, Huiyu; Jun, Ren; Fu, Changhui; Li, Linlin; Li, Tianlong; Tang, Fangqiong; Meng, Xianwei

2016-03-01

Magnetic fluorescent nanocomposites have attracted much attention because of their merging magnetic and fluorescent properties for biomedical application. However, the procedure of synthesis of magnetic fluorescent nanocomposites is always complicated. In addition, the properties of fluorescent component could be easily influenced by magnetic component, retaining both of the magnetic and fluorescent properties into one single nanoparticle considered to be a significant challenge. Herein, we report one-pot method to synthesize multifunctional magnetic fluorescent Fe3O4@PS@P(AEMH-FITC) nanocomposites for bimodal imaging. The asprepared Fe3O4@PS@P(AEMH-FITC) nanocomposites with well-define spherical core/shell structure were stable properties. Moreover, the Fe3O4@PS@P(AEMH-FITC) nanocomposites displayed efficient fluorescent and magnetic properties, respectively. Meanwhile, the magnetic resonance imaging (MRI) and HePG2 cancer cell fluorescent images experiment results suggested that Fe3O4@PS@P(AEMH-FITC) nanocomposites could be used as MRI contrast agents and Fluorescence Imaging (FLI) agents for bioimaging application. Our investigation paves a facile avenue for synthesized magnetic fluorescent nanostructures with well biocompatibility for potential bioimaging application in MRI and FLI.

In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue.

PubMed

Kantelhardt, Sven R; Kalasauskas, Darius; König, Karsten; Kim, Ella; Weinigel, Martin; Uchugonova, Aisada; Giese, Alf

2016-05-01

High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining.

Optical Properties of Plasmonic Nanostructures for Bio-Imaging and Bio-Sensing Applications

NASA Astrophysics Data System (ADS)

Kravets, Vira V.

Kravets, Vira V. (Ph.D., Physics). Optical properties of plasmonic nanostructures for bio-imaging and bio-sensing applications. Dissertation directed by Associate Professor Anatoliy Pinchuk. ABSTRACT. This dissertation explores the physics of free electron excitations in gold nanoparticle chains, silver nanoparticle colloids, and thin gold films. Electron excitations in nanostructures (surface plasmons, SP) are responsible for unique optical properties, which are applied in bio-sensing and bio-imaging applications. For gold nanoparticle chains, the effect of SP on resonance light absorption was studied experimentally and theoretically. Mainly, how the spectral position of the absorption peak depends on inter-particle distances. This dependence is used in “molecular rulers”, providing spatial resolution below the Rayleigh limit. The underlying theory is based on particle interaction via scattered dipole fields. Often in literature only the near-field component of the scattered field is considered. Here, I show that middle and far fields should not be neglected for calculation of extinction by particle chains. In silver nanoparticles, SP excitations produce two independent effects: (a) the intrinsic fluorescence of the particles, and (b) the enhancement of a molecule’s fluorescence by a particle’s surface. The mechanism of (a) is deduced by studying how fluorescence depends on particle size. For (b), I show that fluorescence of a dye molecule on the surface of a nanoparticle is enhanced, when compared to that of the free-standing dye. I demonstrate that the dye’s fluorescent quantum yield is dependent on the particle’s size, making labeled silver nanoparticles attractive candidates as bio-imaging agents. Labeled nanoparticles are applied to cell imaging, and their bio-compatibility with two cell lines is evaluated here. Finally, in gold films under attenuated total internal reflection (ATR) conditions, the SP create a propagating wave (SP-polariton, SPP) when coupled with the incident light. Because of the sensitivity of SPPs to the medium adjacent to the gold film surface, they are widely applied in bio-sensing applications. A toolbox for the description of sputter-deposited gold films is presented here: it employs three experimental techniques (ATR, transmittance and atomic force microscopy) in combination with the effective medium theory for double-layered film model. Our findings have allowed for the avoidance of superficial fitting parameters in our model.

Reflectance and fluorescence spectroscopies in photodynamic therapy

NASA Astrophysics Data System (ADS)

Finlay, Jarod C.

In vivo fluorescence spectroscopy during photodynamic therapy (PDT) has the potential to provide information on the distribution and degradation of sensitizers, the formation of fluorescent photoproducts and changes in tissue autofluorescence induced by photodynamic treatment. Reflectance spectroscopy allows quantification of light absorption and scattering in tissue. We present the results of several related studies of fluorescence and reflectance spectroscopy and their applications to photodynamic dosimetry. First, we develop and test an empirical method for the correction of the distortions imposed on fluorescence spectra by absorption and scattering in turbid media. We characterize the irradiance dependence of the in vivo photobleaching of three sensitizers, protoporphyrin IX (PpIX), Photofrin and mTHPC, in a rat skin model. The photobleaching and photoproduct formation of PpIX exhibit irradiance dependence consistent with singlet oxygen (1O2)-mediated bleaching. The bleaching of mTHPC occurs in two phases, only one of which is consistent with a 1O 2-mediated mechanism. Photofrin's bleaching is independent of irradiance, although its photoproduct formation is not. This can be explained by a mixed-mechanism bleaching model. Second, we develop an algorithm for the determination of tissue optical properties using diffuse reflectance spectra measured at a single source-detector separation and demonstrate the recovery of the hemoglobin oxygen dissociation curve from tissue-simulating phantoms containing human erythrocytes. This method is then used to investigate the heterogeneity of oxygenation response in murine tumors induced by carbogen inhalation. We find that while the response varies among animals and within each tumor, the majority of tumors exhibit an increase in blood oxygenation during carbogen breathing. We present a forward-adjoint model of fluorescence propagation that uses the optical property information acquired from reflectance spectroscopy to obtain the undistorted fluorescence spectrum over a wide range of optical properties. Finally, we investigate the ability of the forward-adjoint theory to extract undistorted fluorescence and optical property information simultaneously from a single measured fluorescence spectrum. This method can recover the hemoglobin oxygen dissociation curve in tissue-simulating phantoms with an accuracy comparable to that of reflectance-based methods while correcting distortions in the fluorescence over a wide range of absorption and scattering coefficients.

In-Field Diffuse Ultraviolet Spectroscopy and Imaging of the Stardust Sample Return Capsule

NASA Technical Reports Server (NTRS)

Pugel, D. Elizabeth; Stackpoole, Mairead; McNamara, Karen; Schwartz, C.; Warren, J.; Kontinos, Dean

2008-01-01

In-field diffuse Ultraviolet (UV) spectroscopy and imaging systems were developed for the purposes of evaluating the surface chemical composition of spacecraft thermal control coatings and materials. The investigation of these systems and the compilation of an associated UV reflectance and luminescence database were conducted using the Stardust Sample Return Capsule (SRC), located at the Johnson Space Center. Spectral responses of the surfaces of the Stardust forebody and aftbody in both reflectance and fluorescence modes were examined post-flight. In this paper, we report on two primary findings of in-field diffuse UV spectroscopy and imaging: (1) deduction of the thermal history of thermal control coatings of the forebody and (2) bond line variations in the aftbody. In the forebody, the thermal history of thermal control coatings may be deduced from the presence of particular semiconducting defect states associated with ZnO, a common emissivity constituent in thermal control coatings. A spatial dependence of this history was mapped for these regions. In the aftbody, luminescing defect states, associated with Si and SiO2 color centers were found along regions of bond variability.

Extending Whole Slide Imaging: Color Darkfield Internal Reflection Illumination (DIRI) for Biological Applications

PubMed Central

Namiki, Kana; Miyawaki, Atsushi; Ishikawa, Takuji

2017-01-01

Whole slide imaging (WSI) is a useful tool for multi-modal imaging, and in our work, we have often combined WSI with darkfield microscopy. However, traditional darkfield microscopy cannot use a single condenser to support high- and low-numerical-aperture objectives, which limits the modality of WSI. To overcome this limitation, we previously developed a darkfield internal reflection illumination (DIRI) microscope using white light-emitting diodes (LEDs). Although the developed DIRI is useful for biological applications, substantial problems remain to be resolved. In this study, we propose a novel illumination technique called color DIRI. The use of three-color LEDs dramatically improves the capability of the system, such that color DIRI (1) enables optimization of the illumination color; (2) can be combined with an oil objective lens; (3) can produce fluorescence excitation illumination; (4) can adjust the wavelength of light to avoid cell damage or reactions; and (5) can be used as a photostimulator. These results clearly illustrate that the proposed color DIRI can significantly extend WSI modalities for biological applications. PMID:28085892

Dendrimer probes for enhanced photostability and localization in fluorescence imaging.

PubMed

Kim, Younghoon; Kim, Sung Hoon; Tanyeri, Melikhan; Katzenellenbogen, John A; Schroeder, Charles M

2013-04-02

Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Frequency division multiplexed multi-color fluorescence microscope system

NASA Astrophysics Data System (ADS)

Le, Vu Nam; Yang, Huai Dong; Zhang, Si Chun; Zhang, Xin Rong; Jin, Guo Fan

2017-10-01

Grayscale camera can only obtain gray scale image of object, while the multicolor imaging technology can obtain the color information to distinguish the sample structures which have the same shapes but in different colors. In fluorescence microscopy, the current method of multicolor imaging are flawed. Problem of these method is affecting the efficiency of fluorescence imaging, reducing the sampling rate of CCD etc. In this paper, we propose a novel multiple color fluorescence microscopy imaging method which based on the Frequency division multiplexing (FDM) technology, by modulating the excitation lights and demodulating the fluorescence signal in frequency domain. This method uses periodic functions with different frequency to modulate amplitude of each excitation lights, and then combine these beams for illumination in a fluorescence microscopy imaging system. The imaging system will detect a multicolor fluorescence image by a grayscale camera. During the data processing, the signal obtained by each pixel of the camera will be processed with discrete Fourier transform, decomposed by color in the frequency domain and then used inverse discrete Fourier transform. After using this process for signals from all of the pixels, monochrome images of each color on the image plane can be obtained and multicolor image is also acquired. Based on this method, this paper has constructed and set up a two-color fluorescence microscope system with two excitation wavelengths of 488 nm and 639 nm. By using this system to observe the linearly movement of two kinds of fluorescent microspheres, after the data processing, we obtain a two-color fluorescence dynamic video which is consistent with the original image. This experiment shows that the dynamic phenomenon of multicolor fluorescent biological samples can be generally observed by this method. Compared with the current methods, this method can obtain the image signals of each color at the same time, and the color video's frame rate is consistent with the frame rate of the camera. The optical system is simpler and does not need extra color separation element. In addition, this method has a good filtering effect on the ambient light or other light signals which are not affected by the modulation process.

A 3D imaging system integrating photoacoustic and fluorescence orthogonal projections for anatomical, functional and molecular assessment of rodent models

NASA Astrophysics Data System (ADS)

Brecht, Hans P.; Ivanov, Vassili; Dumani, Diego S.; Emelianov, Stanislav Y.; Anastasio, Mark A.; Ermilov, Sergey A.

2018-03-01

We have developed a preclinical 3D imaging instrument integrating photoacoustic tomography and fluorescence (PAFT) addressing known deficiencies in sensitivity and spatial resolution of the individual imaging components. PAFT is designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct registration of the two imaging modalities. Orthogonal photoacoustic projections are utilized to reconstruct large (21 cm3 ) volumes showing vascularized anatomical structures and regions of induced optical contrast with spatial resolution exceeding 100 µm. The major advantage of orthogonal fluorescence projections is significant reduction of background noise associated with transmitted or backscattered photons. The fluorescence imaging component of PAFT is used to boost detection sensitivity by providing low-resolution spatial constraint for the fluorescent biomarkers. PAFT performance characteristics were assessed by imaging optical and fluorescent contrast agents in tissue mimicking phantoms and in vivo. The proposed PAFT technology will enable functional and molecular volumetric imaging using fluorescent biomarkers, nanoparticles, and other photosensitive constructs mapped with high fidelity over robust anatomical structures, such as skin, central and peripheral vasculature, and internal organs.

Enhanced fluorescence microscope and its application

NASA Astrophysics Data System (ADS)

Wang, Susheng; Li, Qin; Yu, Xin

1997-12-01

A high gain fluorescence microscope is developed to meet the needs in medical and biological research. By the help of an image intensifier with luminance gain of 4 by 104 the sensitivity of the system can achieve 10-6 1x level and be 104 times higher than ordinary fluorescence microscope. Ultra-weak fluorescence image can be detected by it. The concentration of fluorescent label and emitting light intensity of the system are decreased as much as possible, therefore, the natural environment of the detected call can be kept. The CCD image acquisition set-up controlled by computer obtains the quantitative data of each point according to the gray scale. The relation between luminous intensity and output of CCD is obtained by using a wide range weak photometry. So the system not only shows the image of ultra-weak fluorescence distribution but also gives the intensity of fluorescence of each point. Using this system, we obtained the images of distribution of hypocrellin A (HA) in Hela cell, the images of Hela cell being protected by antioxidant reagent Vit. E, SF and BHT. The images show that the digitized ultra-sensitive fluorescence microscope is a useful tool for medical and biological research.

Deep-tissue reporter-gene imaging with fluorescence and optoacoustic tomography: a performance overview.

PubMed

Deliolanis, Nikolaos C; Ale, Angelique; Morscher, Stefan; Burton, Neal C; Schaefer, Karin; Radrich, Karin; Razansky, Daniel; Ntziachristos, Vasilis

2014-10-01

A primary enabling feature of near-infrared fluorescent proteins (FPs) and fluorescent probes is the ability to visualize deeper in tissues than in the visible. The purpose of this work is to find which is the optimal visualization method that can exploit the advantages of this novel class of FPs in full-scale pre-clinical molecular imaging studies. Nude mice were stereotactically implanted with near-infrared FP expressing glioma cells to from brain tumors. The feasibility and performance metrics of FPs were compared between planar epi-illumination and trans-illumination fluorescence imaging, as well as to hybrid Fluorescence Molecular Tomography (FMT) system combined with X-ray CT and Multispectral Optoacoustic (or Photoacoustic) Tomography (MSOT). It is shown that deep-seated glioma brain tumors are possible to visualize both with fluorescence and optoacoustic imaging. Fluorescence imaging is straightforward and has good sensitivity; however, it lacks resolution. FMT-XCT can provide an improved rough resolution of ∼1 mm in deep tissue, while MSOT achieves 0.1 mm resolution in deep tissue and has comparable sensitivity. We show imaging capacity that can shift the visualization paradigm in biological discovery. The results are relevant not only to reporter gene imaging, but stand as cross-platform comparison for all methods imaging near infrared fluorescent contrast agents.

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.

PubMed

Burnette, Dylan T; Sengupta, Prabuddha; Dai, Yuhai; Lippincott-Schwartz, Jennifer; Kachar, Bechara

2011-12-27

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

Reconstruction algorithms based on l1-norm and l2-norm for two imaging models of fluorescence molecular tomography: a comparative study.

PubMed

Yi, Huangjian; Chen, Duofang; Li, Wei; Zhu, Shouping; Wang, Xiaorui; Liang, Jimin; Tian, Jie

2013-05-01

Fluorescence molecular tomography (FMT) is an important imaging technique of optical imaging. The major challenge of the reconstruction method for FMT is the ill-posed and underdetermined nature of the inverse problem. In past years, various regularization methods have been employed for fluorescence target reconstruction. A comparative study between the reconstruction algorithms based on l1-norm and l2-norm for two imaging models of FMT is presented. The first imaging model is adopted by most researchers, where the fluorescent target is of small size to mimic small tissue with fluorescent substance, as demonstrated by the early detection of a tumor. The second model is the reconstruction of distribution of the fluorescent substance in organs, which is essential to drug pharmacokinetics. Apart from numerical experiments, in vivo experiments were conducted on a dual-modality FMT/micro-computed tomography imaging system. The experimental results indicated that l1-norm regularization is more suitable for reconstructing the small fluorescent target, while l2-norm regularization performs better for the reconstruction of the distribution of fluorescent substance.

Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

PubMed

Lee, Dong-Ryoung; Kim, Young-Duk; Gweon, Dae-Gab; Yoo, Hongki

2013-07-29

We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

Use of a Novel Rover-mounted Fluorescence Imager and Fluorescent Probes to Detect Biological Material in the Atacama Desert in Daylight

NASA Technical Reports Server (NTRS)

Weinstein, S.; Pane, D.; Warren-Rhodes, K.; Cockell, C.; Ernst, L. A.; Minkley, E.; Fisher, G.; Emani, S.; Wettergreen, D. S.; Wagner, M.

2005-01-01

We have developed an imaging system, the Fluorescence Imager (FI), for detecting fluorescence signals from sparse microorganisms and biofilms during autonomous rover exploration. The fluorescence signals arise both from naturally occurring chromophores, such as chlorophyll of cyanobacteria and lichens, and from fluorescent probes applied to soil and rocks. Daylight imaging has been accomplished by a novel use of a high-powered flashlamp synchronized to a CCD camera. The fluorescent probes are cell permanent stains that have extremely low intrinsic fluorescence (quantum yields less than 0.01) and a large fluorescence enhancement (quantum yields greater than 0.4) when bound to the target. Each probe specifically targets either carbohydrates, proteins, nucleic acids or membrane lipids, the four classes of macromolecules found in terrestrial life. The intent of the probes is to interrogate the environment for surface and endolithic life forms.

Entangled-photon coincidence fluorescence imaging

PubMed Central

Scarcelli, Giuliano; Yun, Seok H.

2009-01-01

We describe fluorescence imaging using the second-order correlation of entangled photon pairs. The proposed method is based on the principle that one photon of the pair carries information on where the other photon has been absorbed and has produced fluorescence in a sample. Because fluorescent molecules serve as “detectors” breaking the entanglement, multiply-scattered fluorescence photons within the sample do not cause image blur. We discuss experimental implementations. PMID:18825257

Optical spectroscopy for quantitative sensing in human pancreatic tissues

NASA Astrophysics Data System (ADS)

Wilson, Robert H.; Chandra, Malavika; Lloyd, William; Chen, Leng-Chun; Scheiman, James; Simeone, Diane; McKenna, Barbara; Mycek, Mary-Ann

2011-07-01

Pancreatic adenocarcinoma has a five-year survival rate of only 6%, largely because current diagnostic methods cannot reliably detect the disease in its early stages. Reflectance and fluorescence spectroscopies have the potential to provide quantitative, minimally-invasive means of distinguishing pancreatic adenocarcinoma from normal pancreatic tissue and chronic pancreatitis. The first collection of wavelength-resolved reflectance and fluorescence spectra and time-resolved fluorescence decay curves from human pancreatic tissues was acquired with clinically-compatible instrumentation. Mathematical models of reflectance and fluorescence extracted parameters related to tissue morphology and biochemistry that were statistically significant for distinguishing between pancreatic tissue types. These results suggest that optical spectroscopy has the potential to detect pancreatic disease in a clinical setting.

Three-Dimensional Reflectance Traction Microscopy

PubMed Central

Jones, Christopher A. R.; Groves, Nicholas Scott; Sun, Bo

2016-01-01

Cells in three-dimensional (3D) environments exhibit very different biochemical and biophysical phenotypes compared to the behavior of cells in two-dimensional (2D) environments. As an important biomechanical measurement, 2D traction force microscopy can not be directly extended into 3D cases. In order to quantitatively characterize the contraction field, we have developed 3D reflectance traction microscopy which combines confocal reflection imaging and partial volume correlation postprocessing. We have measured the deformation field of collagen gel under controlled mechanical stress. We have also characterized the deformation field generated by invasive breast cancer cells of different morphologies in 3D collagen matrix. In contrast to employ dispersed tracing particles or fluorescently-tagged matrix proteins, our methods provide a label-free, computationally effective strategy to study the cell mechanics in native 3D extracellular matrix. PMID:27304456

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice.

PubMed

Feeks, James A; Hunter, Jennifer J

2017-05-01

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina.

Ultraviolet signals in birds are special.

PubMed

Hausmann, Franziska; Arnold, Kathryn E; Marshall, N Justin; Owens, Ian P F

2003-01-07

Recent behavioural experiments have shown that birds use ultraviolet (UV)-reflective and fluorescent plumage as cues in mate choice. It remains controversial, however, whether such UV signals play a special role in sexual communication, or whether they are part of general plumage coloration. We use a comparative approach to test for a general association between sexual signalling and either UV-reflective or fluorescent plumage. Among the species surveyed, 72% have UV colours and there is a significant positive association between UV reflectance and courtship displays. Among parrots (Psittaciformes), 68% of surveyed species have fluorescent plumage, and again there is a strong positive association between courtship displays and fluorescence. These associations are not artefacts of the plumage used in courtship displays, being generally more 'colourful' because there is no association between display and colours lacking UV reflectance or fluorescence. Equally, these associations are not phylogenetic artefacts because all results remain unchanged when families or genera, rather than species, are used as independent data points. We also find that, in parrots, fluorescent plumage is usually found adjacent to UV-reflective plumage. Using a simple visual model to examine one parrot, the budgerigar Melopsittacus undulatus, we show that the juxtaposition of UV-reflective and fluorescent plumage leads to a 25-fold increase in chromatic contrast to the budgerigar's visual system. Taken together, these results suggest that signals based on UV contrast are of special importance in the context of active sexual displays. We review briefly six hypotheses on why this may be the case: suitability for short-range signalling; high contrast with backgrounds; invisibility to predators; exploitation of pre-existing sensory biases; advertisement of feather structure; and amplification of behavioural signals.

Ultraviolet signals in birds are special.

PubMed Central

Hausmann, Franziska; Arnold, Kathryn E; Marshall, N Justin; Owens, Ian P F

2003-01-01

Recent behavioural experiments have shown that birds use ultraviolet (UV)-reflective and fluorescent plumage as cues in mate choice. It remains controversial, however, whether such UV signals play a special role in sexual communication, or whether they are part of general plumage coloration. We use a comparative approach to test for a general association between sexual signalling and either UV-reflective or fluorescent plumage. Among the species surveyed, 72% have UV colours and there is a significant positive association between UV reflectance and courtship displays. Among parrots (Psittaciformes), 68% of surveyed species have fluorescent plumage, and again there is a strong positive association between courtship displays and fluorescence. These associations are not artefacts of the plumage used in courtship displays, being generally more 'colourful' because there is no association between display and colours lacking UV reflectance or fluorescence. Equally, these associations are not phylogenetic artefacts because all results remain unchanged when families or genera, rather than species, are used as independent data points. We also find that, in parrots, fluorescent plumage is usually found adjacent to UV-reflective plumage. Using a simple visual model to examine one parrot, the budgerigar Melopsittacus undulatus, we show that the juxtaposition of UV-reflective and fluorescent plumage leads to a 25-fold increase in chromatic contrast to the budgerigar's visual system. Taken together, these results suggest that signals based on UV contrast are of special importance in the context of active sexual displays. We review briefly six hypotheses on why this may be the case: suitability for short-range signalling; high contrast with backgrounds; invisibility to predators; exploitation of pre-existing sensory biases; advertisement of feather structure; and amplification of behavioural signals. PMID:12590772

Detection of Head and Neck Cancer in Surgical Specimens Using Quantitative Hyperspectral Imaging.

PubMed

Lu, Guolan; Little, James V; Wang, Xu; Zhang, Hongzheng; Patel, Mihir R; Griffith, Christopher C; El-Deiry, Mark W; Chen, Amy Y; Fei, Baowei

2017-09-15

Purpose: This study intends to investigate the feasibility of using hyperspectral imaging (HSI) to detect and delineate cancers in fresh, surgical specimens of patients with head and neck cancers. Experimental Design: A clinical study was conducted in order to collect and image fresh, surgical specimens from patients ( N = 36) with head and neck cancers undergoing surgical resection. A set of machine-learning tools were developed to quantify hyperspectral images of the resected tissue in order to detect and delineate cancerous regions which were validated by histopathologic diagnosis. More than two million reflectance spectral signatures were obtained by HSI and analyzed using machine-learning methods. The detection results of HSI were compared with autofluorescence imaging and fluorescence imaging of two vital-dyes of the same specimens. Results: Quantitative HSI differentiated cancerous tissue from normal tissue in ex vivo surgical specimens with a sensitivity and specificity of 91% and 91%, respectively, and which was more accurate than autofluorescence imaging ( P < 0.05) or fluorescence imaging of 2-NBDG ( P < 0.05) and proflavine ( P < 0.05). The proposed quantification tools also generated cancer probability maps with the tumor border demarcated and which could provide real-time guidance for surgeons regarding optimal tumor resection. Conclusions: This study highlights the feasibility of using quantitative HSI as a diagnostic tool to delineate the cancer boundaries in surgical specimens, and which could be translated into the clinic application with the hope of improving clinical outcomes in the future. Clin Cancer Res; 23(18); 5426-36. ©2017 AACR . ©2017 American Association for Cancer Research.

Combined magnetic resonance, fluorescence, and histology imaging strategy in a human breast tumor xenograft model

PubMed Central

Jiang, Lu; Greenwood, Tiffany R.; Amstalden van Hove, Erika R.; Chughtai, Kamila; Raman, Venu; Winnard, Paul T.; Heeren, Ron; Artemov, Dmitri; Glunde, Kristine

2014-01-01

Applications of molecular imaging in cancer and other diseases frequently require combining in vivo imaging modalities, such as magnetic resonance and optical imaging, with ex vivo optical, fluorescence, histology, and immunohistochemical (IHC) imaging, to investigate and relate molecular and biological processes to imaging parameters within the same region of interest. We have developed a multimodal image reconstruction and fusion framework that accurately combines in vivo magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI), ex vivo brightfield and fluorescence microscopic imaging, and ex vivo histology imaging. Ex vivo brightfield microscopic imaging was used as an intermediate modality to facilitate the ultimate link between ex vivo histology and in vivo MRI/MRSI. Tissue sectioning necessary for optical and histology imaging required generation of a three-dimensional (3D) reconstruction module for 2D ex vivo optical and histology imaging data. We developed an external fiducial marker based 3D reconstruction method, which was able to fuse optical brightfield and fluorescence with histology imaging data. Registration of 3D tumor shape was pursued to combine in vivo MRI/MRSI and ex vivo optical brightfield and fluorescence imaging data. This registration strategy was applied to in vivo MRI/MRSI, ex vivo optical brightfield/fluorescence, as well as histology imaging data sets obtained from human breast tumor models. 3D human breast tumor data sets were successfully reconstructed and fused with this platform. PMID:22945331

Fluorescent imaging of cancerous tissues for targeted surgery

PubMed Central

Bu, Lihong; Shen, Baozhong; Cheng, Zhen

2014-01-01

To maximize tumor excision and minimize collateral damage is the primary goal of cancer surgery. Emerging molecular imaging techniques have to “image-guided surgery” developing into “molecular imaging-guided surgery”, which is termed “targeted surgery” in this review. Consequently, the precision of surgery can be advanced from tissue-scale to molecule-scale, enabling “targeted surgery” to be a component of “targeted therapy”. Evidence from numerous experimental and clinical studies has demonstrated significant benefits of fluorescent imaging in targeted surgery with preoperative molecular diagnostic screening. Fluorescent imaging can help to improve intraoperative staging and enable more radical cytoreduction, detect obscure tumor lesions in special organs, highlight tumor margins, better map lymph node metastases, and identify important normal structures intraoperatively. Though limited tissue penetration of fluorescent imaging and tumor heterogeneity are two major hurdles for current targeted surgery, multimodality imaging and multiplex imaging may provide potential solutions to overcome these issues, respectively. Moreover, though many fluorescent imaging techniques and probes have been investigated, targeted surgery remains at a proof-of-principle stage. The impact of fluorescent imaging on cancer surgery will likely be realized through persistent interdisciplinary amalgamation of research in diverse fields. PMID:25064553

[The research of UV-responsive sensitivity enhancement of fluorescent coating films by MgF2 layer].

PubMed

Lu, Zhong-Rong; Ni, Zheng-Ji; Tao, Chun-Xian; Hong, Rui-Jin; Zhang, Da-Wei; Huang, Yuan-Shen

2014-03-01

A low cost and less complicated expansion approach of wavelength responses with a Lumogen phosphor coating was adopted, as they increased the quantum efficiency of CCD and CMOS detectors in ultra-violet by absorbing UV light and then re emitting visible light. In this paper, the sensitivity enhancement of fluorescence coatings was studied by adding an anti-reflection film or barrier film to reduce the loss of the scattering and reflection on the incident interface. The Lumogen and MgF2/Lumogen film were deposited on quartz glasses by physical vacuum deposition. The surface morphology, transmittance spectrum, reflectance spectrum and fluorescence emission spectrum were obtained by atomic force microscope (AFM), spectrophotometer and fluorescence spectrometer, respectively. The results indicated that MgF2 film had obvious positive effect on reducing scattering and reflection loss in 500-700 nm, and enhancing the absorption of Lumogen coating in ultraviolet spectrum. Meanwhile, the fluorescent emission intensity had a substantial increase by smoothing the film surface and thus reducing the light scattering. At the same time, the MgF2 layer could protect Lumogen coating from damaging and contamination, which give a prolong lifetime of the UV-responsive CCD sensors with fluorescent coatings.

Algorithms for differentiating between images of heterogeneous tissue across fluorescence microscopes.

PubMed

Chitalia, Rhea; Mueller, Jenna; Fu, Henry L; Whitley, Melodi Javid; Kirsch, David G; Brown, J Quincy; Willett, Rebecca; Ramanujam, Nimmi

2016-09-01

Fluorescence microscopy can be used to acquire real-time images of tissue morphology and with appropriate algorithms can rapidly quantify features associated with disease. The objective of this study was to assess the ability of various segmentation algorithms to isolate fluorescent positive features (FPFs) in heterogeneous images and identify an approach that can be used across multiple fluorescence microscopes with minimal tuning between systems. Specifically, we show a variety of image segmentation algorithms applied to images of stained tumor and muscle tissue acquired with 3 different fluorescence microscopes. Results indicate that a technique called maximally stable extremal regions followed by thresholding (MSER + Binary) yielded the greatest contrast in FPF density between tumor and muscle images across multiple microscopy systems.

Long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular

NASA Astrophysics Data System (ADS)

Kimura, Hiroaki; Momiyama, Masashi; Tomita, Katsuro; Tsuchiya, Hiroyuki; Hoffman, Robert M.

2010-11-01

We demonstrate the development of a long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular. To observe cytoplasmic and nuclear dynamics of cancer cells in the living mouse, 143B human osteosarcoma cells are labeled with green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm. These dual-color cells are injected by a vascular route in an abdominal skin flap in nude mice. The mice are then imaged with the Olympus MVX10 macroview fluorescence microscope. With the MVX10, the nuclear and cytoplasmic behavior of cancer cells trafficking in blood vessels of live mice is observed. We also image lung metastases in live mice from the macro- to the subcellular level by opening the chest wall and imaging the exposed lung in live mice. Injected splenocytes, expressing cyan fluorescent protein, could also be imaged on the lung of live mice. We demonstrate that the MVX10 microscope offers the possibility of full-range in vivo fluorescence imaging from macro- to subcellular and should enable widespread use of powerful imaging technologies enabled by genetic reporters and other fluorophores.

Fluorescence guided lymph node biopsy in large animals using direct image projection device

NASA Astrophysics Data System (ADS)

Ringhausen, Elizabeth; Wang, Tylon; Pitts, Jonathan; Akers, Walter J.

2016-03-01

The use of fluorescence imaging for aiding oncologic surgery is a fast growing field in biomedical imaging, revolutionizing open and minimally invasive surgery practices. We have designed, constructed, and tested a system for fluorescence image acquisition and direct display on the surgical field for fluorescence guided surgery. The system uses a near-infrared sensitive CMOS camera for image acquisition, a near-infra LED light source for excitation, and DLP digital projector for projection of fluorescence image data onto the operating field in real time. Instrument control was implemented in Matlab for image capture, processing of acquired data and alignment of image parameters with the projected pattern. Accuracy of alignment was evaluated statistically to demonstrate sensitivity to small objects and alignment throughout the imaging field. After verification of accurate alignment, feasibility for clinical application was demonstrated in large animal models of sentinel lymph node biopsy. Indocyanine green was injected subcutaneously in Yorkshire pigs at various locations to model sentinel lymph node biopsy in gynecologic cancers, head and neck cancer, and melanoma. Fluorescence was detected by the camera system during operations and projected onto the imaging field, accurately identifying tissues containing the fluorescent tracer at up to 15 frames per second. Fluorescence information was projected as binary green regions after thresholding and denoising raw intensity data. Promising results with this initial clinical scale prototype provided encouraging results for the feasibility of optical projection of acquired luminescence during open oncologic surgeries.

Non-invasive Photoacoustic and Fluorescence Sentinel Lymph Node Identification using Dye-loaded Perfluorocarbon Nanoparticles

PubMed Central

Akers, Walter J.; Kim, Chulhong; Berezin, Mikhail; Guo, Kevin; Fuhrhop, Ralph; Lanza, Gregory M.; Fischer, Georg M.; Daltrozzo, Ewald; Zumbusch, Andreas; Cai, Xin; Wang, Lihong V.; Achilefu, Samuel

2010-01-01

The contrast mechanisms used for photoacoustic tomography (PAT) and fluorescence imaging differ in subtle but significant ways. Design of contrast agents for each or both modalities requires an understanding of the spectral characteristics as well as intra- and intermolecular interactions that occur during formulation. We found that fluorescence quenching that occurs in the formulation of near infrared (NIR) fluorescent dyes in nanoparticles results in enhanced contrast for PAT. The ability of the new PAT method to utilize strongly absorbing chromophores for signal generation allowed us to convert a highly fluorescent dye into an exceptionally high PA contrast material. Spectroscopic characterization of the developed NIR dye-loaded perfluorocarbon-based nanoparticles for combined fluorescence and PA imaging revealed distinct dye-dependent photophysical behavior. We demonstrate that the enhanced contrast allows detection of regional lymph nodes of rats in vivo with time-domain optical and photoacoustic imaging methods. The results further show that the use of fluorescence lifetime (FLT) imaging, which is less dependent on fluorescence intensity, provides a strategic approach to bridge the disparate contrast reporting mechanisms of fluorescence and PA imaging methods. PMID:21171567

Quantification of tumor fluorescence during intraoperative optical cancer imaging

PubMed Central

Judy, Ryan P.; Keating, Jane J.; DeJesus, Elizabeth M.; Jiang, Jack X.; Okusanya, Olugbenga T.; Nie, Shuming; Holt, David E.; Arlauckas, Sean P.; Low, Phillip S.; Delikatny, E. James; Singhal, Sunil

2015-01-01

Intraoperative optical cancer imaging is an emerging technology in which surgeons employ fluorophores to visualize tumors, identify tumor-positive margins and lymph nodes containing metastases. This study compares instrumentation to measure tumor fluorescence. Three imaging systems (Spectropen, Glomax, Flocam) measured and quantified fluorescent signal-to-background ratios (SBR) in vitro, murine xenografts, tissue phantoms and clinically. Evaluation criteria included the detection of small changes in fluorescence, sensitivity of signal detection at increasing depths and practicality of use. In vitro, spectroscopy was superior in detecting incremental differences in fluorescence than luminescence and digital imaging (Ln[SBR] = 6.8 ± 0.6, 2.4 ± 0.3, 2.6 ± 0.1, p = 0.0001). In fluorescent tumor cells, digital imaging measured higher SBRs than luminescence (6.1 ± 0.2 vs. 4.3 ± 0.4, p = 0.001). Spectroscopy was more sensitive than luminometry and digital imaging in identifying murine tumor fluorescence (SBR = 41.7 ± 11.5, 5.1 ± 1.8, 4.1 ± 0.9, p = 0.0001), and more sensitive than digital imaging at detecting fluorescence at increasing depths (SBR = 7.0 ± 3.4 vs. 2.4 ± 0.5, p = 0.03). Lastly, digital imaging was the most practical and least time-consuming. All methods detected incremental differences in fluorescence. Spectroscopy was the most sensitive for small changes in fluorescence. Digital imaging was the most practical considering its wide field of view, background noise filtering capability, and sensitivity to increasing depth. PMID:26563091

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

PubMed Central

Higuchi-Sanabria, Ryo; Garcia, Enrique J.; Tomoiaga, Delia; Munteanu, Emilia L.; Feinstein, Paul; Pon, Liza A.

2016-01-01

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging. PMID:26727004

Boronic acids for fluorescence imaging of carbohydrates.

PubMed

Sun, Xiaolong; Zhai, Wenlei; Fossey, John S; James, Tony D

2016-02-28

"Fluorescence imaging" is a particularly exciting and rapidly developing area of research; the annual number of publications in the area has increased ten-fold over the last decade. The rapid increase of interest in fluorescence imaging will necessitate the development of an increasing number of molecular receptors and binding agents in order to meet the demand in this rapidly expanding area. Carbohydrate biomarkers are particularly important targets for fluorescence imaging given their pivotal role in numerous important biological events, including the development and progression of many diseases. Therefore, the development of new fluorescent receptors and binding agents for carbohydrates is and will be increasing in demand. This review highlights the development of fluorescence imaging agents based on boronic acids a particularly promising class of receptors given their strong and selective binding with carbohydrates in aqueous media.

Maximizing fluorescence collection efficiency in multiphoton microscopy

PubMed Central

Zinter, Joseph P.; Levene, Michael J.

2011-01-01

Understanding fluorescence propagation through a multiphoton microscope is of critical importance in designing high performance systems capable of deep tissue imaging. Optical models of a scattering tissue sample and the Olympus 20X 0.95NA microscope objective were used to simulate fluorescence propagation as a function of imaging depth for physiologically relevant scattering parameters. The spatio-angular distribution of fluorescence at the objective back aperture derived from these simulations was used to design a simple, maximally efficient post-objective fluorescence collection system. Monte Carlo simulations corroborated by data from experimental tissue phantoms demonstrate collection efficiency improvements of 50% – 90% over conventional, non-optimized fluorescence collection geometries at large imaging depths. Imaging performance was verified by imaging layer V neurons in mouse cortex to a depth of 850 μm. PMID:21934897

Quantitative, spectrally-resolved intraoperative fluorescence imaging

PubMed Central

Valdés, Pablo A.; Leblond, Frederic; Jacobs, Valerie L.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.

2012-01-01

Intraoperative visual fluorescence imaging (vFI) has emerged as a promising aid to surgical guidance, but does not fully exploit the potential of the fluorescent agents that are currently available. Here, we introduce a quantitative fluorescence imaging (qFI) approach that converts spectrally-resolved data into images of absolute fluorophore concentration pixel-by-pixel across the surgical field of view (FOV). The resulting estimates are linear, accurate, and precise relative to true values, and spectral decomposition of multiple fluorophores is also achieved. Experiments with protoporphyrin IX in a glioma rodent model demonstrate in vivo quantitative and spectrally-resolved fluorescence imaging of infiltrating tumor margins for the first time. Moreover, we present images from human surgery which detect residual tumor not evident with state-of-the-art vFI. The wide-field qFI technique has broad implications for intraoperative surgical guidance because it provides near real-time quantitative assessment of multiple fluorescent biomarkers across the operative field. PMID:23152935

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

PubMed

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

Measurement of fluorophore concentrations and fluorescence quantum yield in tissue-simulating phantoms using three diffusion models of steady-state spatially resolved fluorescence.

PubMed

Diamond, Kevin R; Farrell, Thomas J; Patterson, Michael S

2003-12-21

Steady-state diffusion theory models of fluorescence in tissue have been investigated for recovering fluorophore concentrations and fluorescence quantum yield. Spatially resolved fluorescence, excitation and emission reflectance Carlo simulations, and measured using a multi-fibre probe on tissue-simulating phantoms containing either aluminium phthalocyanine tetrasulfonate (AlPcS4), Photofrin meso-tetra-(4-sulfonatophenyl)-porphine dihydrochloride The accuracy of the fluorophore concentration and fluorescence quantum yield recovered by three different models of spatially resolved fluorescence were compared. The models were based on: (a) weighted difference of the excitation and emission reflectance, (b) fluorescence due to a point excitation source or (c) fluorescence due to a pencil beam excitation source. When literature values for the fluorescence quantum yield were used for each of the fluorophores, the fluorophore absorption coefficient (and hence concentration) at the excitation wavelength (mu(a,x,f)) was recovered with a root-mean-square accuracy of 11.4% using the point source model of fluorescence and 8.0% using the more complicated pencil beam excitation model. The accuracy was calculated over a broad range of optical properties and fluorophore concentrations. The weighted difference of reflectance model performed poorly, with a root-mean-square error in concentration of about 50%. Monte Carlo simulations suggest that there are some situations where the weighted difference of reflectance is as accurate as the other two models, although this was not confirmed experimentally. Estimates of the fluorescence quantum yield in multiple scattering media were also made by determining mu(a,x,f) independently from the fitted absorption spectrum and applying the various diffusion theory models. The fluorescence quantum yields for AlPcS4 and TPPS4 were calculated to be 0.59 +/- 0.03 and 0.121 +/- 0.001 respectively using the point source model, and 0.63 +/- 0.03 and 0.129 +/- 0.002 using the pencil beam excitation model. These results are consistent with published values.

An individually coated near-infrared fluorescent protein as a safe and robust nanoprobe for in vivo imaging

NASA Astrophysics Data System (ADS)

Yang, Yu; Xiang, Kun; Yang, Yi-Xin; Wang, Yan-Wen; Zhang, Xin; Cui, Yangdong; Wang, Haifang; Zhu, Qing-Qing; Fan, Liqiang; Liu, Yuanfang; Cao, Aoneng

2013-10-01

A prerequisite for in vivo fluorescence imaging is the safety of fluorescent probes. Among all fluorescent probes, fluorescent proteins (FPs) might be the safest ones, which have been widely used in biological sciences at the gene level. But FPs have not been used in vivo in the purified form yet due to the instability of proteins. Here, we individually coat near-infrared (NIR) FPs (NIRFPs) with a silica nanoshell, resulting in NIRFP@silica, one of the safest and brightest NIR fluorescent nanoprobes with a quantum yield of 0.33 for in vivo imaging. The silica shell not only protects NIRFPs from denaturation and metabolic digestion, but also enhances the quantum yield and photostability of the coated NIRFPs. When injected via the tail vein, NIRFP@silica NPs can distribute all over the mouse body, and then can be efficiently eliminated through urine in 24 h, demonstrating its potential applications as a safe and robust NIR fluorescence probe for whole body imaging.A prerequisite for in vivo fluorescence imaging is the safety of fluorescent probes. Among all fluorescent probes, fluorescent proteins (FPs) might be the safest ones, which have been widely used in biological sciences at the gene level. But FPs have not been used in vivo in the purified form yet due to the instability of proteins. Here, we individually coat near-infrared (NIR) FPs (NIRFPs) with a silica nanoshell, resulting in NIRFP@silica, one of the safest and brightest NIR fluorescent nanoprobes with a quantum yield of 0.33 for in vivo imaging. The silica shell not only protects NIRFPs from denaturation and metabolic digestion, but also enhances the quantum yield and photostability of the coated NIRFPs. When injected via the tail vein, NIRFP@silica NPs can distribute all over the mouse body, and then can be efficiently eliminated through urine in 24 h, demonstrating its potential applications as a safe and robust NIR fluorescence probe for whole body imaging. Electronic supplementary information (ESI) available: A chromatogram of APTS-NIRFP, a TEM image of 40 nm NIRFP@silica, dispersion stability of NIRFP@silica, more whole body fluorescent images, serum biochemical parameters, and optical images of HE stained organ slices. See DOI: 10.1039/c3nr02508j

Laser-scanned fluorescence of nonlased/normal, lased/normal, nonlased/carious, and lased/carious enamel

NASA Astrophysics Data System (ADS)

Zakariasen, Kenneth L.; Barron, Joseph R.; Paton, Barry E.

1992-06-01

Research has shown that low levels of CO2 laser irradiation raise enamel resistance to sub-surface demineralization. Additionally, laser scanned fluorescence analysis of enamel, as well a laser and white light reflection studies, have potential for both clinical diagnosis and comparative research investigations of the caries process. This study was designed to compare laser fluorescence and laser/white light reflection of (1) non-lased/normal with lased/normal enamel and (2) non-lased/normal with non-lased/carious and lased/carious enamel. Specimens were buccal surfaces of extracted third molars, coated with acid resistant varnish except for either two or three 2.25 mm2 windows (two window specimens: non-lased/normal, lased/normal--three window specimens: non-lased/normal, non-lased carious, lased/carious). Teeth exhibiting carious windows were immersed in a demineralizing solution for twelve days. Non-carious windows were covered with wax during immersion. Following immersion, the wax was removed, and fluorescence and laser/white light reflection analyses were performed on all windows utilizing a custom scanning laser fluorescence spectrometer which focuses light from a 25 mWatt He-Cd laser at 442 nm through an objective lens onto a cross-section >= 3 (mu) in diameter. For laser/white light reflection analyses, reflected light intensities were measured. A HeNe laser was used for laser light reflection studies. Following analyses, the teeth are sectioned bucco-lingually into 80 micrometers sections, examined under polarized light microscopy, and the lesions photographed. This permits comparison between fluorescence/reflected light values and the visualized decalcification areas for each section, and thus comparisons between various enamel treatments and normal enamel. The enamel specimens are currently being analyzed.

Clinical application of indocyanine green-fluorescence imaging during hepatectomy

PubMed Central

Ishizawa, Takeaki; Saiura, Akio

2016-01-01

In hepatobiliary surgery, the fluorescence and bile excretion of indocyanine green (ICG) can be used for real-time visualization of biological structure. Fluorescence cholangiography is used to obtain fluorescence images of the bile ducts following intrabiliary injection of 0.025−0.5 mg/mL ICG or intravenous injection of 2.5 mg ICG. Recently, the latter technique has been used in laparoscopic/robotic cholecystectomy. Intraoperative fluorescence imaging can be used to identify subcapsular hepatic tumors. Primary and secondary hepatic malignancy can be identified by intraoperative fluorescence imaging using preoperative intravenous injection of ICG through biliary excretion disorders that exist in cancerous tissues of hepatocellular carcinoma (HCC) and in non-cancerous hepatic parenchyma around adenocarcinoma foci. Intraoperative fluorescence imaging may help detect tumors to be removed, especially during laparoscopic hepatectomy, in which visual inspection and palpation are limited, compared with open surgery. Fluorescence imaging can also be used to identify hepatic segments. Boundaries of hepatic segments can be visualized following injection of 0.25−2.5 mg/mL ICG into the portal veins or by intravenous injection of 2.5 mg ICG following closure of the proximal portal pedicle toward hepatic regions to be removed. These techniques enable identification of hepatic segments before hepatectomy and during parenchymal transection for anatomic resection. Advances in imaging systems will increase the use of fluorescence imaging as an intraoperative navigation tool that can enhance the safety and accuracy of open and laparoscopic/robotic hepatobiliary surgery. PMID:27500144

Characterization of caries progression on dentin after irradiation with Nd:YAG laser by FTIR spectroscopy and fluorescence imaging

NASA Astrophysics Data System (ADS)

Ana, P. A.; Brito, A. M. M.; Zezell, D. M.; Lins, E. C. C. C.

2015-06-01

Considering the use of high intensity lasers for preventing dental caries, this blind in vitro study evaluated the compositional and fluorescence effects promoted by Nd:YAG laser (λ=1064 nm) when applied for prevention of progression of dentin caries, in association or not with topical application of acidulated phosphate fluoride (APF). Sixty bovine root dentin slabs were prepared and demineralized by 32h in order to create early caries lesions. After, the slabs were distributed into six experimental groups: G1- untreated and not submitted to a pH-cycling model; G2- untreated and submitted to a pH-cycling model; G3- acidulated phosphate fluoride application (APF); G4- Nd:YAG irradiation (84.9 J/cm2, 60 mJ/pulse); G5- treated with Nd:YAG+APF; G6- treated with APF+Nd:YAG. After treatments, the samples of groups G2 to G6 were submitted to a 4-day pH-cycling model in order to simulate the progression of early caries lesions. All samples were characterized by the micro-attenuated total reflection technique of Fourier transformed infrared spectroscopy (μATR-FTIR), using a diamond crystal, and by a fluorescence imaging system (FIS), in which it was used an illuminating system at λ= 405±30 nm. Demineralization promoted reduction in carbonate and phosphate contents, exposing the organic matter; as well, it was observed a significant reduction of fluorescence intensity. Nd:YAG laser promoted additional chemical changes, and increased the fluorescence intensity even with the development of caries lesions. It was concluded that the compositional changes promoted by Nd:YAG, when associated to APF, are responsible for the reduction of demineralization progression observed on root dentin.

Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

PubMed

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-10-17

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Optimizing total reflection X-ray fluorescence for direct trace element quantification in proteins I: Influence of sample homogeneity and reflector type

NASA Astrophysics Data System (ADS)

Wellenreuther, G.; Fittschen, U. E. A.; Achard, M. E. S.; Faust, A.; Kreplin, X.; Meyer-Klaucke, W.

2008-12-01

Total reflection X-ray fluorescence (TXRF) is a very promising method for the direct, quick and reliable multi-elemental quantification of trace elements in protein samples. With the introduction of an internal standard consisting of two reference elements, scandium and gallium, a wide range of proteins can be analyzed, regardless of their salt content, buffer composition, additives and amino acid composition. This strategy also enables quantification of matrix effects. Two potential issues associated with drying have been considered in this study: (1) Formation of heterogeneous residues of varying thickness and/or density; and (2) separation of the internal standard and protein during drying (which has to be prevented to allow accurate quantification). These issues were investigated by microbeam X-ray fluorescence (μXRF) with special emphasis on (I) the influence of sample support and (II) the protein / buffer system used. In the first part, a model protein was studied on well established sample supports used in TXRF, PIXE and XRF (Mylar, siliconized quartz, Plexiglas and silicon). In the second part we imaged proteins of different molecular weight, oligomerization state, bound metals and solubility. A partial separation of protein and internal standard was only observed with untreated silicon, suggesting it may not be an adequate support material. Siliconized quartz proved to be the least prone to heterogeneous drying of the sample and yielded the most reliable results.

3D fluorescence anisotropy imaging using selective plane illumination microscopy.

PubMed

Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

2015-08-24

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.

Epi-Fluorescence Microscopy

PubMed Central

Webb, Donna J.; Brown, Claire M.

2012-01-01

Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new “hard” coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes that are the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented. PMID:23026996

Directed molecular evolution to design advanced red fluorescent proteins.

PubMed

Subach, Fedor V; Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-11-29

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

Model for the Interpretation of Hyperspectral Remote-Sensing Reflectance

NASA Technical Reports Server (NTRS)

Lee, Zhongping; Carder, Kendall L.; Hawes, Steve K.; Steward, Robert G.; Peacock, Thomas G.; Davis, Curtiss O.

1994-01-01

Remote-sensing reflectance is easier to interpret for the open ocean than for coastal regions because the optical signals are highly coupled to the phytoplankton (e.g., chlorophyll) concentrations. For estuarine or coastal waters, variable terrigenous colored dissolved organic matter (CDOM), suspended sediments, and bottom reflectance, all factors that do not covary with the pigment concentration, confound data interpretation. In this research, remote-sensing reflectance models are suggested for coastal waters, to which contributions that are due to bottom reflectance, CDOM fluorescence, and water Raman scattering are included. Through the use of two parameters to model the combination of the backscattering coefficient and the Q factor, excellent agreement was achieved between the measured and modeled remote-sensing reflectance for waters from the West Florida Shelf to the Mississippi River plume. These waters cover a range of chlorophyll of 0.2-40 mg/cu m and gelbstoff absorption at 440 nm from 0.02-0.4/m. Data with a spectral resolution of 10 nm or better, which is consistent with that provided by the airborne visible and infrared imaging spectrometer (AVIRIS) and spacecraft spectrometers, were used in the model evaluation.

Setting Standards for Reporting and Quantification in Fluorescence-Guided Surgery.

PubMed

Hoogstins, Charlotte; Burggraaf, Jan Jaap; Koller, Marjory; Handgraaf, Henricus; Boogerd, Leonora; van Dam, Gooitzen; Vahrmeijer, Alexander; Burggraaf, Jacobus

2018-05-29

Intraoperative fluorescence imaging (FI) is a promising technique that could potentially guide oncologic surgeons toward more radical resections and thus improve clinical outcome. Despite the increase in the number of clinical trials, fluorescent agents and imaging systems for intraoperative FI, a standardized approach for imaging system performance assessment and post-acquisition image analysis is currently unavailable. We conducted a systematic, controlled comparison between two commercially available imaging systems using a novel calibration device for FI systems and various fluorescent agents. In addition, we analyzed fluorescence images from previous studies to evaluate signal-to-background ratio (SBR) and determinants of SBR. Using the calibration device, imaging system performance could be quantified and compared, exposing relevant differences in sensitivity. Image analysis demonstrated a profound influence of background noise and the selection of the background on SBR. In this article, we suggest clear approaches for the quantification of imaging system performance assessment and post-acquisition image analysis, attempting to set new standards in the field of FI.

Angular shaping of fluorescence from synthetic opal-based photonic crystal.

PubMed

Boiko, Vitalii; Dovbeshko, Galyna; Dolgov, Leonid; Kiisk, Valter; Sildos, Ilmo; Loot, Ardi; Gorelik, Vladimir

2015-01-01

Spectral, angular, and temporal distributions of fluorescence as well as specular reflection were investigated for silica-based artificial opals. Periodic arrangement of nanosized silica globules in the opal causes a specific dip in the defect-related fluorescence spectra and a peak in the reflectance spectrum. The spectral position of the dip coincides with the photonic stop band. The latter is dependent on the size of silica globules and the angle of observation. The spectral shape and intensity of defect-related fluorescence can be controlled by variation of detection angle. Fluorescence intensity increases up to two times at the edges of the spectral dip. Partial photobleaching of fluorescence was observed. Photonic origin of the observed effects is discussed.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice

PubMed Central

Feeks, James A.; Hunter, Jennifer J.

2017-01-01

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina. PMID:28663886

Use of image analysis to estimate anthocyanin and UV-excited fluorescent phenolic compound levels in strawberry fruit

PubMed Central

Yoshioka, Yosuke; Nakayama, Masayoshi; Noguchi, Yuji; Horie, Hideki

2013-01-01

Strawberry is rich in anthocyanins, which are responsible for the red color, and contains several colorless phenolic compounds. Among the colorless phenolic compounds, some, such as hydroxycinammic acid derivatives, emit blue-green fluorescence when excited with ultraviolet (UV) light. Here, we investigated the effectiveness of image analyses for estimating the levels of anthocyanins and UV-excited fluorescent phenolic compounds in fruit. The fruit skin and cut surface of 12 cultivars were photographed under visible and UV light conditions; colors were evaluated based on the color components of images. The levels of anthocyanins and UV-excited fluorescent compounds in each fruit were also evaluated by spectrophotometric and high performance liquid chromatography (HPLC) analyses, respectively and relationships between these levels and the image data were investigated. Red depth of the fruits differed greatly among the cultivars and anthocyanin content was well estimated based on the color values of the cut surface images. Strong UV-excited fluorescence was observed on the cut surfaces of several cultivars, and the grayscale values of the UV-excited fluorescence images were markedly correlated with the levels of those fluorescent compounds as evaluated by HPLC analysis. These results indicate that image analyses can select promising genotypes rich in anthocyanins and fluorescent phenolic compounds. PMID:23853516

Fluorescent staining for leukocyte chemotaxis. Eosinophil-specific fluorescence with aniline blue.

PubMed

McCrone, E L; Lucey, D R; Weller, P F

1988-11-10

To overcome problems associated with the quantitation of human eosinophil chemotaxis in micropore filters, we have developed a fluorescent method of specifically staining eosinophils in chemotactic filters. A neutral solution of aniline blue yielded bright green fluorescent staining of the cytoplasmic granules of eosinophils. Other leukocytes and contaminating neutrophils potentially present with eosinophils did not fluoresce with aniline blue. The fluorescent staining eosinophils within filters provided bright, non-fading images that facilitated visual microscopic counting and were of sufficiently high contrast, unlike those with conventional eosinophil stains, to allow image analyzer based enumeration of eosinophil chemotactic responses at levels through the filters. Although not cell type-specific, congo red and ethidium bromide also provided high contrast, fluorescent images of all leukocyte types within chemotactic filters. Fluorescent staining with aniline blue constitutes a rapid, stable and eosinophil-specific stain that facilitates the visual or image analyzer-based quantitation of eosinophil chemotaxis.

Self-Assembly of Electron Donor-Acceptor-Based Carbazole Derivatives: Novel Fluorescent Organic Nanoprobes for Both One- and Two-Photon Cellular Imaging.

PubMed

Zhang, Jinfeng; Chen, Wencheng; Kalytchuk, Sergii; Li, King Fai; Chen, Rui; Adachi, Chihaya; Chen, Zhan; Rogach, Andrey L; Zhu, Guangyu; Yu, Peter K N; Zhang, Wenjun; Cheah, Kok Wai; Zhang, Xiaohong; Lee, Chun-Sing

2016-05-11

In this study, we report fluorescent organic nanoprobes with intense blue, green, and orange-red emissions prepared by self-assembling three carbazole derivatives into nanorods/nanoparticles. The three compounds consist of two or four electron-donating carbazole groups linked to a central dicyanobenzene electron acceptor. Steric hindrance from the carbazole groups leads to noncoplanar 3D molecular structures favorable to fluorescence in the solid state, while the donor-acceptor structures endow the molecules with good two-photon excited emission properties. The fluorescent organic nanoprobes exhibit good water dispersibility, low cytotoxicity, superior resistance against photodegradation and photobleaching. Both one- and two-photon fluorescent imaging were shown in the A549 cell line. Two-photon fluorescence imaging with the fluorescent probes was demonstrated to be more effective in visualizing and distinguishing cellular details compared to conventional one-photon fluorescence imaging.

Fluorescence background subtraction technique for hybrid fluorescence molecular tomography/x-ray computed tomography imaging of a mouse model of early stage lung cancer.

PubMed

Ale, Angelique; Ermolayev, Vladimir; Deliolanis, Nikolaos C; Ntziachristos, Vasilis

2013-05-01

The ability to visualize early stage lung cancer is important in the study of biomarkers and targeting agents that could lead to earlier diagnosis. The recent development of hybrid free-space 360-deg fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) imaging yields a superior optical imaging modality for three-dimensional small animal fluorescence imaging over stand-alone optical systems. Imaging accuracy was improved by using XCT information in the fluorescence reconstruction method. Despite this progress, the detection sensitivity of targeted fluorescence agents remains limited by nonspecific background accumulation of the fluorochrome employed, which complicates early detection of murine cancers. Therefore we examine whether x-ray CT information and bulk fluorescence detection can be combined to increase detection sensitivity. Correspondingly, we research the performance of a data-driven fluorescence background estimator employed for subtraction of background fluorescence from acquisition data. Using mice containing known fluorochromes ex vivo, we demonstrate the reduction of background signals from reconstructed images and sensitivity improvements. Finally, by applying the method to in vivo data from K-ras transgenic mice developing lung cancer, we find small tumors at an early stage compared with reconstructions performed using raw data. We conclude with the benefits of employing fluorescence subtraction in hybrid FMT-XCT for early detection studies.

Biological applications of confocal fluorescence polarization microscopy

NASA Astrophysics Data System (ADS)

Bigelow, Chad E.

Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine serum albumin attached to sepharose beads. The action of trypsin and proteinase K on the albumin is monitored to demonstrate validity of the technique. Images of the processing of the albumin in J774 murine macrophages are also presented indicating large intercellular differences in enzyme activity. Future directions for the technique are also presented, including the design of enzyme probes specific for prostate specific antigen based on fluorescently-labeled dendrimers. A technique for enzyme imaging based on extracellular autofluorescence is also proposed.

Spectral characterization of forest damage occurring on Whiteface Mountain, NY - Studies with the Fluorescence Line Imager (FLI) and ground-based spectrometers

NASA Technical Reports Server (NTRS)

Rock, B. N.; Moss, D. M.; Miller, J. R.; Freemantle, J. R.; Boyer, M. G.

1990-01-01

Ground-based spectral characteristics of fir wave damage and an analysis of calibrated FLI data acquired along the same fir wave utilized for the in situ measurements are presented. Derivative curve data were produced from both in situ and FLI reflectance measurements for the red edge spectral region for birch and for various portions of a fir wave. The results suggested that with proper atmospheric correction of airborne imaging spectrometer data sets, the derivative curve approach will provide an accurate means of assessing red edge parameters, and that such data will permit identification of specific types of forest damage on the basis of spectral fine features.

Visualization of subcapsular hepatic malignancy by indocyanine-green fluorescence imaging during laparoscopic hepatectomy.

PubMed

Kudo, Hiroki; Ishizawa, Takeaki; Tani, Keigo; Harada, Nobuhiro; Ichida, Akihiko; Shimizu, Atsushi; Kaneko, Junichi; Aoki, Taku; Sakamoto, Yoshihiro; Sugawara, Yasuhiko; Hasegawa, Kiyoshi; Kokudo, Norihiro

2014-08-01

Although laparoscopic hepatectomy has increasingly been used to treat cancers in the liver, the accuracy of intraoperative diagnosis may be inferior to that of open surgery because the ability to visualize and palpate the liver surface during laparoscopy is relatively limited. Fluorescence imaging has the potential to provide a simple compensatory diagnostic tool for identification of cancers in the liver during laparoscopic hepatectomy. In 17 patients who were to undergo laparoscopic hepatectomy, 0.5 mg/kg body weight of indocyanine green (ICG) was administered intravenously within the 2 weeks prior to surgery. Intraoperatively, a laparoscopic fluorescence imaging system obtained fluorescence images of its surfaces during mobilization of the liver. In all, 16 hepatocellular carcinomas (HCCs) and 16 liver metastases (LMs) were resected. Of these, laparoscopic ICG fluorescence imaging identified 12 HCCs (75%) and 11 LMs (69%) on the liver surfaces distributed over Couinaud's segments 1-8, including the 17 tumors that had not been identified by visual inspections of normal color images. The 23 tumors that were identified by fluorescence imaging were located closer to the liver surfaces than another nine tumors that were not identified by fluorescence imaging (median [range] depth 1 [0-5] vs. 11 [8-30] mm; p < 0.001). Like palpation during open hepatectomy, laparoscopic ICG fluorescence imaging enables real-time identification of subcapsular liver cancers, thus facilitating estimation of the required extent of hepatic mobilization and determination of the location of an appropriate hepatic transection line.

Fluorescent probes for nucleic Acid visualization in fixed and live cells.

PubMed

Boutorine, Alexandre S; Novopashina, Darya S; Krasheninina, Olga A; Nozeret, Karine; Venyaminova, Alya G

2013-12-11

This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i) sequence-specific peptides and proteins; (ii) triplex-forming oligonucleotides and (iii) polyamide oligo(N-methylpyrrole/N-methylimidazole) minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy

PubMed Central

Hunter, Jennifer J.; Masella, Benjamin; Dubra, Alfredo; Sharma, Robin; Yin, Lu; Merigan, William H.; Palczewska, Grazyna; Palczewski, Krzysztof; Williams, David R.

2011-01-01

In vivo two-photon imaging through the pupil of the primate eye has the potential to become a useful tool for functional imaging of the retina. Two-photon excited fluorescence images of the macaque cone mosaic were obtained using a fluorescence adaptive optics scanning laser ophthalmoscope, overcoming the challenges of a low numerical aperture, imperfect optics of the eye, high required light levels, and eye motion. Although the specific fluorophores are as yet unknown, strong in vivo intrinsic fluorescence allowed images of the cone mosaic. Imaging intact ex vivo retina revealed that the strongest two-photon excited fluorescence signal comes from the cone inner segments. The fluorescence response increased following light stimulation, which could provide a functional measure of the effects of light on photoreceptors. PMID:21326644

Comparison of Measurements and FluorMOD Simulations for Solar Induced Chlorophyll Fluorescence and Reflectance of a Corn Crop under Nitrogen Treatments [SIF and Reflectance for Corn

NASA Technical Reports Server (NTRS)

Middleton, Elizabeth M.; Corp, Lawrence A.; Campbell, Petya K. E.

2007-01-01

The FLuorescence Explorer (FLEX) satellite concept is one of six semifinalist mission proposals selected in 2006 for pre-Phase studies by the European Space Agency (ESA). The FLEX concept proposes to measure passive solar induced chlorophyll fluorescence (SIF) of terrestrial ecosystems. A new spectral vegetation Fluorescence Model (FluorMOD) was developed to include the effects of steady state SIF on canopy reflectance. We used our laboratory and field measurements previously acquired from foliage and canopies of corn (Zea mays L.) under controlled nitrogen (N) fertilization to parameterize and evaluate FluorMOD. Our data included biophysical properties, fluorescence (F) and reflectance spectra for leaves; reflectance spectra of canopies and soil; solar irradiance; plot-level leaf area index; and canopy SIF emissions determined using the Fraunhofer Line Depth principal for the atmospheric telluric oxygen absorption features at 688 nm (O2-beta) and 760 nm (O2-alpha). FluorMOD simulations implemented in the default "look-up-table" mode did not reproduce the observed magnitudes of leaf F, canopy SIF, or canopy reflectance. However, simulations for all of these parameters agreed with observations when the default FluorMOD information was replaced with measurements, although N treatment responses were underestimated. Recommendations were provided to enhance FluorMOD's potential utility in support of SIF field experiments and studies of agriculture and ecosystems.

Sculpting the internal architecture of fluorescent silica particles via a template-free approach.

PubMed

Rosu, Cornelia; Gorman, Andrew J; Cueto, Rafael; Dooley, Kerry M; Russo, Paul S

2016-04-01

Particles with an open, porous structure can be used to deliver payloads. It is often of interest to detect such particles in tissue or materials, which is facilitated by addition of dye. A straightforward approach leading to fluorescent, porous silica particles is described. The particles are etched with 3mM aqueous sodium hydroxide, taking advantage of the etching rate difference between normal silica and an interior band of silica that contains covalently attached dye. No additional steps, such as dye labeling or thermal annealing, are required. Etching modeled the internal structure of the fluorescent silica particles by creating meso/macropores and voids, as reflected by nitrogen absorption measurements. In order to investigate whether a polymer shell influences etching, certain composite particles are top-coated with poly(l-lysine) representing neutral or positive charged surfaces under typical pH conditions in living systems. The polypeptide-coated fluorescent silica cores exhibit the same porous morphology as uncoated homologs. The polypeptide topcoat does little to alter the permeation by the etching agent. Preservation of size during etching, confirmed by dynamic light scattering, transmission electron microscopy and small-angle X-ray scattering, simplifies the use of these template-free porous fluorescent particles as platforms for drug encapsulation, drug carriers and in vivo imaging. Copyright © 2016 Elsevier Inc. All rights reserved.

Spatio-temporal imaging of EGF-induced activation of protein kinase A by FRET in living cells

NASA Astrophysics Data System (ADS)

Wang, Jin Jun; Chen, Xiao-Chuan; Xing, Da

2004-07-01

Intracellular molecular interaction is important for the study of cell physiology, yet current relevant methods require fixation or microinjection and lack temporal or spatial resolution. We introduced a new method -- fluorescence resonance energy transfer (FRET) to detect molecular interaction in living cells. On the basis of FRET principle, A-kinase activity reporter (AKAR) protein was designed to consist of the fusions of cyan fluorescent protein (CFP), a phosphoamino acid binding domain, a consensus substrate for protein kinase-A (PKA), and yellow fluorescent protein (YFP). In this study, the designed pAKAR plasmid was used to transfect a human lung cancer cell line (ASTC-a-1). When the AKAR-transfected cells were treated by forskolin (Fsk), we were able to observe the efficient transfer of energy from excited CFP to YFP within the AKAR molecule by fluorescence microcopy, whereas no FRET was detected in the transfected cells without the treatment of Fsk. When the cells were treated by Epidermal growth factor (EGF), the change of FRET was observed at different subcellular locations, reflecting PKA activation inside the cells upon EGF stimulation. The successful design of a fluorescence reporter of PKA activation and its application demonstrated the superiority of this technology in the research of intracellular protein-protein interaction.

FLIM data analysis of NADH and Tryptophan autofluorescence in prostate cancer cells

NASA Astrophysics Data System (ADS)

O'Melia, Meghan J.; Wallrabe, Horst; Svindrych, Zdenek; Rehman, Shagufta; Periasamy, Ammasi

2016-03-01

Fluorescence lifetime imaging microscopy (FLIM) is one of the most sensitive techniques to measure metabolic activity in living cells, tissues and whole animals. We used two- and three-photon fluorescence excitation together with time-correlated single photon counting (TCSPC) to acquire FLIM signals from normal and prostate cancer cell lines. FLIM requires complex data fitting and analysis; we explored different ways to analyze the data to match diverse cellular morphologies. After non-linear least square fitting of the multi-photon TCSPC images by the SPCImage software (Becker & Hickl), all image data are exported and further processed in ImageJ. Photon images provide morphological, NAD(P)H signal-based autofluorescent features, for which regions of interest (ROIs) are created. Applying these ROIs to all image data parameters with a custom ImageJ macro, generates a discrete, ROI specific database. A custom Excel (Microsoft) macro further analyzes the data with charts and statistics. Applying this highly automated assay we compared normal and cancer prostate cell lines with respect to their glycolytic activity by analyzing the NAD(P)H-bound fraction (a2%), NADPH/NADH ratio and efficiency of energy transfer (E%) for Tryptophan (Trp). Our results show that this assay is able to differentiate the effects of glucose stimulation and Doxorubicin in these prostate cell lines by tracking the changes in a2% of NAD(P)H, NADPH/NADH ratio and the changes in Trp E%. The ability to isolate a large, ROI-based data set, reflecting the heterogeneous cellular environment and highlighting even subtle changes -- rather than whole cell averages - makes this assay particularly valuable.

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

PubMed

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

Towards pH-sensitive imaging of small animals with photon-counting difference diffuse fluorescence tomography

NASA Astrophysics Data System (ADS)

Li, Jiao; Wang, Xin; Yi, Xi; Zhang, Limin; Zhou, Zhongxing; Zhao, Huijuan; Gao, Feng

2012-09-01

The importance of cellular pH has been shown clearly in the study of cell activity, pathological feature, and drug metabolism. Monitoring pH changes of living cells and imaging the regions with abnormal pH-values, in vivo, could provide invaluable physiological and pathological information for the research of the cell biology, pharmacokinetics, diagnostics, and therapeutics of certain diseases such as cancer. Naturally, pH-sensitive fluorescence imaging of bulk tissues has been attracting great attentions from the realm of near infrared diffuse fluorescence tomography (DFT). Herein, the feasibility of quantifying pH-induced fluorescence changes in turbid medium is investigated using a continuous-wave difference-DFT technique that is based on the specifically designed computed tomography-analogous photon counting system and the Born normalized difference image reconstruction scheme. We have validated the methodology using two-dimensional imaging experiments on a small-animal-sized phantom, embedding an inclusion with varying pH-values. The results show that the proposed approach can accurately localize the target with a quantitative resolution to pH-sensitive variation of the fluorescent yield, and might provide a promising alternative method of pH-sensitive fluorescence imaging in addition to the fluorescence-lifetime imaging.

FluoroSim: A Visual Problem-Solving Environment for Fluorescence Microscopy

PubMed Central

Quammen, Cory W.; Richardson, Alvin C.; Haase, Julian; Harrison, Benjamin D.; Taylor, Russell M.; Bloom, Kerry S.

2010-01-01

Fluorescence microscopy provides a powerful method for localization of structures in biological specimens. However, aspects of the image formation process such as noise and blur from the microscope's point-spread function combine to produce an unintuitive image transformation on the true structure of the fluorescing molecules in the specimen, hindering qualitative and quantitative analysis of even simple structures in unprocessed images. We introduce FluoroSim, an interactive fluorescence microscope simulator that can be used to train scientists who use fluorescence microscopy to understand the artifacts that arise from the image formation process, to determine the appropriateness of fluorescence microscopy as an imaging modality in an experiment, and to test and refine hypotheses of model specimens by comparing the output of the simulator to experimental data. FluoroSim renders synthetic fluorescence images from arbitrary geometric models represented as triangle meshes. We describe three rendering algorithms on graphics processing units for computing the convolution of the specimen model with a microscope's point-spread function and report on their performance. We also discuss several cases where the microscope simulator has been used to solve real problems in biology. PMID:20431698

Handheld Fluorescence Microscopy based Flow Analyzer.

PubMed

Saxena, Manish; Jayakumar, Nitin; Gorthi, Sai Siva

2016-03-01

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

Applications of two-photon fluorescence microscopy in deep-tissue imaging

NASA Astrophysics Data System (ADS)

Dong, Chen-Yuan; Yu, Betty; Hsu, Lily L.; Kaplan, Peter D.; Blankschstein, D.; Langer, Robert; So, Peter T. C.

2000-07-01

Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

Medical imaging systems

DOEpatents

Frangioni, John V [Wayland, MA

2012-07-24

A medical imaging system provides simultaneous rendering of visible light and fluorescent images. The system may employ dyes in a small-molecule form that remains in a subject's blood stream for several minutes, allowing real-time imaging of the subject's circulatory system superimposed upon a conventional, visible light image of the subject. The system may also employ dyes or other fluorescent substances associated with antibodies, antibody fragments, or ligands that accumulate within a region of diagnostic significance. In one embodiment, the system provides an excitation light source to excite the fluorescent substance and a visible light source for general illumination within the same optical guide that is used to capture images. In another embodiment, the system is configured for use in open surgical procedures by providing an operating area that is closed to ambient light. More broadly, the systems described herein may be used in imaging applications where a visible light image may be usefully supplemented by an image formed from fluorescent emissions from a fluorescent substance that marks areas of functional interest.

Image-Guided Surgery using Invisible Near-Infrared Light: Fundamentals of Clinical Translation

PubMed Central

Gioux, Sylvain; Choi, Hak Soo; Frangioni, John V.

2011-01-01

The field of biomedical optics has matured rapidly over the last decade and is poised to make a significant impact on patient care. In particular, wide-field (typically > 5 cm), planar, near-infrared (NIR) fluorescence imaging has the potential to revolutionize human surgery by providing real-time image guidance to surgeons for tissue that needs to be resected, such as tumors, and tissue that needs to be avoided, such as blood vessels and nerves. However, to become a clinical reality, optimized imaging systems and NIR fluorescent contrast agents will be needed. In this review, we introduce the principles of NIR fluorescence imaging, analyze existing NIR fluorescence imaging systems, and discuss the key parameters that guide contrast agent development. We also introduce the complexities surrounding clinical translation using our experience with the Fluorescence-Assisted Resection and Exploration (FLARE™) imaging system as an example. Finally, we introduce state-of-the-art optical imaging techniques that might someday improve image-guided surgery even further. PMID:20868625

Indocyanine-green-loaded microballoons for biliary imaging in cholecystectomy

NASA Astrophysics Data System (ADS)

Mitra, Kinshuk; Melvin, James; Chang, Shufang; Park, Kyoungjin; Yilmaz, Alper; Melvin, Scott; Xu, Ronald X.

2012-11-01

We encapsulate indocyanine green (ICG) in poly[(D,L-lactide-co-glycolide)-co-PEG] diblock (PLGA-PEG) microballoons for real-time fluorescence and hyperspectral imaging of biliary anatomy. ICG-loaded microballoons show superior fluorescence characteristics and slower degradation in comparison with pure ICG. The use of ICG-loaded microballoons in biliary imaging is demonstrated in both biliary-simulating phantoms and an ex vivo tissue model. The biliary-simulating phantoms are prepared by embedding ICG-loaded microballoons in agar gel and imaged by a fluorescence imaging module in a Da Vinci surgical robot. The ex vivo model consists of liver, gallbladder, common bile duct, and part of the duodenum freshly dissected from a domestic swine. After ICG-loaded microballoons are injected into the gallbladder, the biliary structure is imaged by both hyperspectral and fluorescence imaging modalities. Advanced spectral analysis and image processing algorithms are developed to classify the tissue types and identify the biliary anatomy. While fluorescence imaging provides dynamic information of movement and flow in the surgical region of interest, data from hyperspectral imaging allow for rapid identification of the bile duct and safe exclusion of any contaminant fluorescence from tissue not part of the biliary anatomy. Our experiments demonstrate the technical feasibility of using ICG-loaded microballoons for biliary imaging in cholecystectomy.

Label-free reflectance hyperspectral imaging for tumor margin assessment: a pilot study on surgical specimens of cancer patients

NASA Astrophysics Data System (ADS)

Fei, Baowei; Lu, Guolan; Wang, Xu; Zhang, Hongzheng; Little, James V.; Patel, Mihir R.; Griffith, Christopher C.; El-Diery, Mark W.; Chen, Amy Y.

2017-08-01

A label-free, hyperspectral imaging (HSI) approach has been proposed for tumor margin assessment. HSI data, i.e., hypercube (x,y,λ), consist of a series of high-resolution images of the same field of view that are acquired at different wavelengths. Every pixel on an HSI image has an optical spectrum. In this pilot clinical study, a pipeline of a machine-learning-based quantification method for HSI data was implemented and evaluated in patient specimens. Spectral features from HSI data were used for the classification of cancer and normal tissue. Surgical tissue specimens were collected from 16 human patients who underwent head and neck (H&N) cancer surgery. HSI, autofluorescence images, and fluorescence images with 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) and proflavine were acquired from each specimen. Digitized histologic slides were examined by an H&N pathologist. The HSI and classification method were able to distinguish between cancer and normal tissue from the oral cavity with an average accuracy of 90%±8%, sensitivity of 89%±9%, and specificity of 91%±6%. For tissue specimens from the thyroid, the method achieved an average accuracy of 94%±6%, sensitivity of 94%±6%, and specificity of 95%±6%. HSI outperformed autofluorescence imaging or fluorescence imaging with vital dye (2-NBDG or proflavine). This study demonstrated the feasibility of label-free, HSI for tumor margin assessment in surgical tissue specimens of H&N cancer patients. Further development of the HSI technology is warranted for its application in image-guided surgery.

Hyperspectral imaging fluorescence excitation scanning for colon cancer detection

NASA Astrophysics Data System (ADS)

Leavesley, Silas J.; Walters, Mikayla; Lopez, Carmen; Baker, Thomas; Favreau, Peter F.; Rich, Thomas C.; Rider, Paul F.; Boudreaux, Carole W.

2016-10-01

Optical spectroscopy and hyperspectral imaging have shown the potential to discriminate between cancerous and noncancerous tissue with high sensitivity and specificity. However, to date, these techniques have not been effectively translated to real-time endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents new technology that may be well suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The goal of this study was to evaluate the initial feasibility of using fluorescence excitation-scanning hyperspectral imaging for measuring changes in fluorescence excitation spectrum concurrent with colonic adenocarcinoma using a small pre-pilot-scale sample size. Ex vivo analysis was performed using resected pairs of colorectal adenocarcinoma and normal mucosa. Adenocarcinoma was confirmed by histologic evaluation of hematoxylin and eosin (H&E) permanent sections. Specimens were imaged using a custom hyperspectral imaging fluorescence excitation-scanning microscope system. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation range of 390 to 450 nm that could be the basis for wavelength-dependent detection of colorectal cancers. Hence, excitation-scanning hyperspectral imaging may offer an alternative approach for discriminating adenocarcinoma from surrounding normal colonic mucosa, but further studies will be required to evaluate the accuracy of this approach using a larger patient cohort.

Facilitating in vivo tumor localization by principal component analysis based on dynamic fluorescence molecular imaging

NASA Astrophysics Data System (ADS)

Gao, Yang; Chen, Maomao; Wu, Junyu; Zhou, Yuan; Cai, Chuangjian; Wang, Daliang; Luo, Jianwen

2017-09-01

Fluorescence molecular imaging has been used to target tumors in mice with xenograft tumors. However, tumor imaging is largely distorted by the aggregation of fluorescent probes in the liver. A principal component analysis (PCA)-based strategy was applied on the in vivo dynamic fluorescence imaging results of three mice with xenograft tumors to facilitate tumor imaging, with the help of a tumor-specific fluorescent probe. Tumor-relevant features were extracted from the original images by PCA and represented by the principal component (PC) maps. The second principal component (PC2) map represented the tumor-related features, and the first principal component (PC1) map retained the original pharmacokinetic profiles, especially of the liver. The distribution patterns of the PC2 map of the tumor-bearing mice were in good agreement with the actual tumor location. The tumor-to-liver ratio and contrast-to-noise ratio were significantly higher on the PC2 map than on the original images, thus distinguishing the tumor from its nearby fluorescence noise of liver. The results suggest that the PC2 map could serve as a bioimaging marker to facilitate in vivo tumor localization, and dynamic fluorescence molecular imaging with PCA could be a valuable tool for future studies of in vivo tumor metabolism and progression.

In vivo tumor-targeted dual-modal fluorescence/CT imaging using a nanoprobe co-loaded with an aggregation-induced emission dye and gold nanoparticles.

PubMed

Zhang, Jimei; Li, Chan; Zhang, Xu; Huo, Shuaidong; Jin, Shubin; An, Fei-Fei; Wang, Xiaodan; Xue, Xiangdong; Okeke, C I; Duan, Guiyun; Guo, Fengguang; Zhang, Xiaohong; Hao, Jifu; Wang, Paul C; Zhang, Jinchao; Liang, Xing-Jie

2015-02-01

As an intensely studied computed tomography (CT) contrast agent, gold nanoparticle has been suggested to be combined with fluorescence imaging modality to offset the low sensitivity of CT. However, the strong quenching of gold nanoparticle on fluorescent dyes requires complicated design and shielding to overcome. Herein, we report a unique nanoprobe (M-NPAPF-Au) co-loading an aggregation-induced emission (AIE) red dye and gold nanoparticles into DSPE-PEG(2000) micelles for dual-modal fluorescence/CT imaging. The nanoprobe was prepared based on a facile method of "one-pot ultrasonic emulsification". Surprisingly, in the micelles system, fluorescence dye (NPAPF) efficiently overcame the strong fluorescence quenching of shielding-free gold nanoparticles and retained the crucial AIE feature. In vivo studies demonstrated the nanoprobe had superior tumor-targeting ability, excellent fluorescence and CT imaging effects. The totality of present studies clearly indicates the significant potential application of M-NPAPF-Au as a dual-modal non-invasive fluorescence/X-ray CT nanoprobe for in vivo tumor-targeted imaging and diagnosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Improved sensitivity to fluorescence for cancer detection in wide-field image-guided neurosurgery

PubMed Central

Jermyn, Michael; Gosselin, Yoann; Valdes, Pablo A.; Sibai, Mira; Kolste, Kolbein; Mercier, Jeanne; Angulo, Leticia; Roberts, David W.; Paulsen, Keith D.; Petrecca, Kevin; Daigle, Olivier; Wilson, Brian C.; Leblond, Frederic

2015-01-01

In glioma surgery, Protoporphyrin IX (PpIX) fluorescence may identify residual tumor that could be resected while minimizing damage to normal brain. We demonstrate that improved sensitivity for wide-field spectroscopic fluorescence imaging is achieved with minimal disruption to the neurosurgical workflow using an electron-multiplying charge-coupled device (EMCCD) relative to a state-of-the-art CMOS system. In phantom experiments the EMCCD system can detect at least two orders-of-magnitude lower PpIX. Ex vivo tissue imaging on a rat glioma model demonstrates improved fluorescence contrast compared with neurosurgical fluorescence microscope technology, and the fluorescence detection is confirmed with measurements from a clinically-validated spectroscopic probe. Greater PpIX sensitivity in wide-field fluorescence imaging may improve the residual tumor detection during surgery with consequent impact on survival. PMID:26713218

Fluorescence imaging as a diagnostic of M-band x-ray drive condition in hohlraum with fluorescent Si targets

NASA Astrophysics Data System (ADS)

Li, Qi; Hu, Zhimin; Yao, Li; Huang, Chengwu; Yuan, Zheng; Zhao, Yang; Xiong, Gang; Qing, Bo; Lv, Min; Zhu, Tuo; Deng, Bo; Li, Jin; Wei, Minxi; Zhan, Xiayu; Li, Jun; Yang, Yimeng; Su, Chunxiao; Yang, Guohong; Zhang, Jiyan; Li, Sanwei; Yang, Jiamin; Ding, Yongkun

2017-01-01

Fluorescence imaging of surrogate Si-doped CH targets has been used to provide a measurement for drive condition of high-energy x-ray (i.e. M-band x-ray) drive symmetry upon the capsule in hohlraum on Shenguang-II laser facility. A series of experiments dedicated to the study of photo-pumping and fluorescence effect in Si-plasma are presented. To investigate the feasibility of fluorescence imaging in Si-plasma, an silicon plasma in Si-foil target is pre-formed at ground state by the soft x-ray from a half-hohlraum, which is then photo-pumped by the K-shell lines from a spatially distinct laser-produced Si-plasma. The resonant Si photon pump is used to improve the fluorescence signal and cause visible image in the Si-foil. Preliminary fluorescence imaging of Si-ball target is performed in both Si-doped and pure Au hohlraum. The usual capsule at the center of the hohlraum is replaced with a solid Si-doped CH-ball (Si-ball). Since the fluorescence is proportional to the photon pump upon the Si-plasma, high-energy x-ray drive symmetry is equal to the fluorescence distribution of the Si-ball.

Mitigating fluorescence spectral overlap in wide-field endoscopic imaging

PubMed Central

Hou, Vivian; Nelson, Leonard Y.; Seibel, Eric J.

2013-01-01

Abstract. The number of molecular species suitable for multispectral fluorescence imaging is limited due to the overlap of the emission spectra of indicator fluorophores, e.g., dyes and nanoparticles. To remove fluorophore emission cross-talk in wide-field multispectral fluorescence molecular imaging, we evaluate three different solutions: (1) image stitching, (2) concurrent imaging with cross-talk ratio subtraction algorithm, and (3) frame-sequential imaging. A phantom with fluorophore emission cross-talk is fabricated, and a 1.2-mm ultrathin scanning fiber endoscope (SFE) is used to test and compare these approaches. Results show that fluorophore emission cross-talk could be successfully avoided or significantly reduced. Near term, the concurrent imaging method of wide-field multispectral fluorescence SFE is viable for early stage cancer detection and localization in vivo. Furthermore, a means to enhance exogenous fluorescence target-to-background ratio by the reduction of tissue autofluorescence background is demonstrated. PMID:23966226

Improving limited-projection-angle fluorescence molecular tomography using a co-registered x-ray computed tomography scan.

PubMed

Radrich, Karin; Ale, Angelique; Ermolayev, Vladimir; Ntziachristos, Vasilis

2012-12-01

We examine the improvement in imaging performance, such as axial resolution and signal localization, when employing limited-projection-angle fluorescence molecular tomography (FMT) together with x-ray computed tomography (XCT) measurements versus stand-alone FMT. For this purpose, we employed living mice, bearing a spontaneous lung tumor model, and imaged them with FMT and XCT under identical geometrical conditions using fluorescent probes for cancer targeting. The XCT data was employed, herein, as structural prior information to guide the FMT reconstruction. Gold standard images were provided by fluorescence images of mouse cryoslices, providing the ground truth in fluorescence bio-distribution. Upon comparison of FMT images versus images reconstructed using hybrid FMT and XCT data, we demonstrate marked improvements in image accuracy. This work relates to currently disseminated FMT systems, using limited projection scans, and can be employed to enhance their performance.

Three-dimensional super-resolved live cell imaging through polarized multi-angle TIRF.

PubMed

Zheng, Cheng; Zhao, Guangyuan; Liu, Wenjie; Chen, Youhua; Zhang, Zhimin; Jin, Luhong; Xu, Yingke; Kuang, Cuifang; Liu, Xu

2018-04-01

Measuring three-dimensional nanoscale cellular structures is challenging, especially when the structure is dynamic. Owing to the informative total internal reflection fluorescence (TIRF) imaging under varied illumination angles, multi-angle (MA) TIRF has been examined to offer a nanoscale axial and a subsecond temporal resolution. However, conventional MA-TIRF still performs badly in lateral resolution and fails to characterize the depth image in densely distributed regions. Here, we emphasize the lateral super-resolution in the MA-TIRF, exampled by simply introducing polarization modulation into the illumination procedure. Equipped with a sparsity and accelerated proximal algorithm, we examine a more precise 3D sample structure compared with previous methods, enabling live cell imaging with a temporal resolution of 2 s and recovering high-resolution mitochondria fission and fusion processes. We also shared the recovery program, which is the first open-source recovery code for MA-TIRF, to the best of our knowledge.

Hyperspectral imaging applied to medical diagnoses and food safety

NASA Astrophysics Data System (ADS)

Carrasco, Oscar; Gomez, Richard B.; Chainani, Arun; Roper, William E.

2003-08-01

This paper analyzes the feasibility and performance of HSI systems for medical diagnosis as well as for food safety. Illness prevention and early disease detection are key elements for maintaining good health. Health care practitioners worldwide rely on innovative electronic devices to accurately identify disease. Hyperspectral imaging (HSI) is an emerging technique that may provide a less invasive procedure than conventional diagnostic imaging. By analyzing reflected and fluorescent light applied to the human body, a HSI system serves as a diagnostic tool as well as a method for evaluating the effectiveness of applied therapies. The safe supply and production of food is also of paramount importance to public health illness prevention. Although this paper will focus on imaging and spectroscopy in food inspection procedures -- the detection of contaminated food sources -- to ensure food quality, HSI also shows promise in detecting pesticide levels in food production (agriculture.)

Anti-translational research: from the bedside back to the bench for reflectance confocal microscopy

NASA Astrophysics Data System (ADS)

Gareau, Daniel

2014-03-01

The reflectance confocal microscope has made translational progress in dermatology. 0.5 micrometer lateral resolution, 0.75mm field-of-view and excellent temporal resolution at ~15 frames/second serve the VivaScope well in the clinic, but it may be overlooked in basic research. This work reviews high spatiotemporal confocal microscopy and presents images acquired of various samples: zebra fish embryo where melanocytes with excellent contrast overly the spinal column, chicken embryo, where myocardium is seen moving at 15 frames/ second, calcium spikes in dendrites (fluorescence mode) just beyond the temporal resolution, and human skin where blood cells race through the artereovenous microvasculature. For an introduction to confocal microscopy, see: http://dangareau.net.s69818.gridserver.com/science/confocal-microscopy

Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

NASA Astrophysics Data System (ADS)

Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

2016-02-01

Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

Multiparametric Analysis of the Tumor Microenvironment: Hypoxia Markers and Beyond.

PubMed

Mayer, Arnulf; Vaupel, Peter

2017-01-01

We have established a novel in situ protein analysis pipeline, which is built upon highly sensitive, multichannel immunofluorescent staining of paraffin sections of human and xenografted tumor tissue. Specimens are digitized using slide scanners equipped with suitable light sources and fluorescence filter combinations. Resulting digital images are subsequently subjected to quantitative image analysis using a primarily object-based approach, which comprises segmentation of single cells or higher-order structures (e.g., blood vessels), cell shape approximation, measurement of signal intensities in individual fluorescent channels and correlation of these data with positional information for each object. Our approach could be particularly useful for the study of the hypoxic tumor microenvironment as it can be utilized to systematically explore the influence of spatial factors on cell phenotypes, e.g., the distance of a given cell type from the nearest blood vessel on the cellular expression of hypoxia-associated biomarkers and other proteins reflecting their specific state of activation or function. In this report, we outline the basic methodology and provide an outlook on possible use cases.

Intravital Fluorescence Excitation in Whole-Animal Optical Imaging.

PubMed

Nooshabadi, Fatemeh; Yang, Hee-Jeong; Bixler, Joel N; Kong, Ying; Cirillo, Jeffrey D; Maitland, Kristen C

2016-01-01

Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU.

Intravital Fluorescence Excitation in Whole-Animal Optical Imaging

PubMed Central

Bixler, Joel N.; Kong, Ying; Cirillo, Jeffrey D.; Maitland, Kristen C.

2016-01-01

Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU. PMID:26901051

Lucky Imaging: Improved Localization Accuracy for Single Molecule Imaging

PubMed Central

Cronin, Bríd; de Wet, Ben; Wallace, Mark I.

2009-01-01

We apply the astronomical data-analysis technique, Lucky imaging, to improve resolution in single molecule fluorescence microscopy. We show that by selectively discarding data points from individual single-molecule trajectories, imaging resolution can be improved by a factor of 1.6 for individual fluorophores and up to 5.6 for more complex images. The method is illustrated using images of fluorescent dye molecules and quantum dots, and the in vivo imaging of fluorescently labeled linker for activation of T cells. PMID:19348772

In Vivo Deep Tissue Fluorescence and Magnetic Imaging Employing Hybrid Nanostructures.

PubMed

Ortgies, Dirk H; de la Cueva, Leonor; Del Rosal, Blanca; Sanz-Rodríguez, Francisco; Fernández, Nuria; Iglesias-de la Cruz, M Carmen; Salas, Gorka; Cabrera, David; Teran, Francisco J; Jaque, Daniel; Martín Rodríguez, Emma

2016-01-20

Breakthroughs in nanotechnology have made it possible to integrate different nanoparticles in one single hybrid nanostructure (HNS), constituting multifunctional nanosized sensors, carriers, and probes with great potential in the life sciences. In addition, such nanostructures could also offer therapeutic capabilities to achieve a wider variety of multifunctionalities. In this work, the encapsulation of both magnetic and infrared emitting nanoparticles into a polymeric matrix leads to a magnetic-fluorescent HNS with multimodal magnetic-fluorescent imaging abilities. The magnetic-fluorescent HNS are capable of simultaneous magnetic resonance imaging and deep tissue infrared fluorescence imaging, overcoming the tissue penetration limits of classical visible-light based optical imaging as reported here in living mice. Additionally, their applicability for magnetic heating in potential hyperthermia treatments is assessed.

Fluorescent screens and image processing for the APS linac test stand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berg, W.; Ko, K.

A fluorescent screen was used to monitor relative beam position and spot size of a 56-MeV electron beam in the linac test stand. A chromium doped alumina ceramic screen inserted into the beam was monitored by a video camera. The resulting image was captured using a frame grabber and stored into memory. Reconstruction and analysis of the stored image was performed using PV-WAVE. This paper will discuss the hardware and software implementation of the fluorescent screen and imaging system. Proposed improvements for the APS linac fluorescent screens and image processing will also be discussed.

Multiphoton microscopy for the in-situ investigation of cellular processes and integrity in cryopreservation.

PubMed

Doerr, Daniel; Stark, Martin; Ehrhart, Friederike; Zimmermann, Heiko; Stracke, Frank

2009-08-01

In this study we demonstrate a new noninvasive imaging method to monitor freezing processes in biological samples and to investigate life in the frozen state. It combines a laser scanning microscope with a computer-controlled cryostage. Nearinfrared (NIR) femtosecond laser pulses evoke the fluorescence of endogenous fluorophores and fluorescent labels due to multiphoton absorption.The inherent optical nonlinearity of multiphoton absorption allows 3D fluorescence imaging for optical tomography of frozen biological material in-situ. As an example for functional imaging we use fluorescence lifetime imaging (FLIM) to create images with chemical and physical contrast.

Fluorescence image excited by a scanning UV-LED light

NASA Astrophysics Data System (ADS)

Tsai, Hsin-Yi; Chen, Yi-Ju; Huang, Kuo-Cheng

2013-03-01

An optical scanning system using UV-LED light to induced fluorescence technology can enhance a fluorescence image significantly in a short period. It has several advantages such as lower power consumption, no scattering effect in skins, and multilayer images can be obtained to analyze skin disease. From the experiment results, the light intensity increases with increase spot size and decrease scanning speed, but the image resolution is oppositely. Moreover, the system could be widely used in clinical diagnosis and photodynamic therapy for skin disease because even the irradiated time of fluorescence substance is short but it will provide accurately positioning of fluorescence object.

Method and apparatus for imaging and documenting fingerprints

DOEpatents

Fernandez, Salvador M.

2002-01-01

The invention relates to a method and apparatus for imaging and documenting fingerprints. A fluorescent dye brought in intimate proximity with the lipid residues of a latent fingerprint is caused to fluoresce on exposure to light energy. The resulting fluorescing image may be recorded photographically.

Lipidots: competitive organic alternative to quantum dots for in vivo fluorescence imaging

NASA Astrophysics Data System (ADS)

Gravier, Julien; Navarro, Fabrice P.; Delmas, Thomas; Mittler, Frédérique; Couffin, Anne-Claude; Vinet, Françoise; Texier, Isabelle

2011-09-01

The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker®705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC50 > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.

Combination of Fluorescence-Guided Surgery With Photodynamic Therapy for the Treatment of Cancer

PubMed Central

He, Jun; Yang, Leping; Yi, Wenjun; Fan, Wentao; Wen, Yu; Miao, Xiongying; Xiong, Li

2017-01-01

Specific visualization of body parts is needed during surgery. Fluorescence-guided surgery (FGS) uses a fluorescence contrast agent for in vivo tumor imaging to detect and identify both malignant and normal tissues. There are several advantages and clinical benefits of FGS over other conventional medical imaging modalities, such as its safety, effectiveness, and suitability for real-time imaging in the operating room. Recent advancements in contrast agents and intraoperative fluorescence imaging devices have led to a greater potential for intraoperative fluorescence imaging in clinical applications. Photodynamic therapy (PDT) is an alternative modality to treat tumors, which uses a light-sensitive drug (photosensitizers) and special light to destroy the targeted tissues. In this review, we discuss the fluorescent contrast agents, some newly developed imaging devices, and the successful clinical application of FGS. Additionally, we present the combined strategy of FGS with PDT to further improve the therapeutic effect for patients with cancer. Taken together, this review provides a unique perspective and summarization of FGS. PMID:28849712

Before In Vivo Imaging: Evaluation of Fluorescent Probes Using Fluorescence Microscopy, Multiplate Reader, and Cytotoxicity Assays.

PubMed

Zhang, Shaojuan

2016-01-01

Fluorescent probes are widely utilized for noninvasive fluorescence imaging. Continuing efforts have been made in developing novel fluorescent probes with improved fluorescence quantum yield, enhanced target-specificity, and lower cytotoxicity. Before such probes are administrated into a living system, it is essential to evaluate the subcellular uptake, targeting specificity, and cytotoxicity in vitro. In this chapter, we briefly outline common methods used to evaluate fluorescent probes using fluorescence microscopy, multiplate reader, and cytotoxicity assay.

Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.

PubMed

Hirata, Tomoya; Terai, Takuya; Yamamura, Hisao; Shimonishi, Manabu; Komatsu, Toru; Hanaoka, Kenjiro; Ueno, Tasuku; Imaizumi, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-03-01

K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.

Design of small confocal endo-microscopic probe working under multiwavelength environment

NASA Astrophysics Data System (ADS)

Kim, Young-Duk; Ahn, MyoungKi; Gweon, Dae-Gab

2010-02-01

Recently, optical imaging system is widely used in medical purpose. By using optical imaging system specific diseases can be easily diagnosed at early stage because optical imaging system has high resolution performance and various imaging method. These methods are used to get high resolution image of human body and can be used to verify whether the cell is infected by virus. Confocal microscope is one of the famous imaging systems which is used for in-vivo imaging. Because most of diseases are accompanied with cellular level changes, doctors can diagnosis at early stage by observing the cellular image of human organ. Current research is focused in the development of endo-microscope that has great advantage in accessibility to human body. In this research, I designed small probe that is connected to confocal microscope through optical fiber bundle and work as endo-microscope. And this small probe is mainly designed to correct chromatic aberration to use various laser sources for both fluorescence type and reflection type confocal images. By using two kinds of laser sources at the same time we demonstrated multi-modality confocal endo-microscope.

Preparation and characterization of alginate based-fluorescent magnetic nanoparticles for fluorescence/magnetic resonance multimodal imaging applications

NASA Astrophysics Data System (ADS)

Kwon, Yong-Su; Choi, Kee-Bong; Lim, Hyungjun; Lee, Sunghwi; Lee, Jae-Jong

2018-06-01

Simple and versatile methodologies have been reported that customize the surface of superparamagnetic iron oxide (SPIO) nanoparticles and impart additional fluorescence capabilities to these contrast agents. Herein, we present the rational design, synthesis, characterization, and biological applications of a new magnetic-based fluorescent probe. The dual modality imaging protocol was developed by labeling fluorophore with alginate natural polymers that have excellent biocompatibility and biodegradability, and using gelification method to form nanocomposites containing SPIO. The formation of alginate-based fluorescent magnetic (AFM) nanoparticles was observed in spherical and elliptical forms with a diameter of less than 500 nm by a transmission electron microscope (TEM). The fluorescent wavelength band in the range of 560 nm was also confirmed in the UV–visible spectrophotometer. In this study, we demonstrate that the multi-tasking design of AFM nanoparticles provides an ideal platform for building balanced dual-image probes of magnetic resonance imaging and optical imaging.