Quantitative method to assess caries via fluorescence imaging from the perspective of autofluorescence spectral analysis

NASA Astrophysics Data System (ADS)

Chen, Q. G.; Zhu, H. H.; Xu, Y.; Lin, B.; Chen, H.

2015-08-01

A quantitative method to discriminate caries lesions for a fluorescence imaging system is proposed in this paper. The autofluorescence spectral investigation of 39 teeth samples classified by the International Caries Detection and Assessment System levels was performed at 405 nm excitation. The major differences in the different caries lesions focused on the relative spectral intensity range of 565-750 nm. The spectral parameter, defined as the ratio of wavebands at 565-750 nm to the whole spectral range, was calculated. The image component ratio R/(G + B) of color components was statistically computed by considering the spectral parameters (e.g. autofluorescence, optical filter, and spectral sensitivity) in our fluorescence color imaging system. Results showed that the spectral parameter and image component ratio presented a linear relation. Therefore, the image component ratio was graded as <0.66, 0.66-1.06, 1.06-1.62, and >1.62 to quantitatively classify sound, early decay, established decay, and severe decay tissues, respectively. Finally, the fluorescence images of caries were experimentally obtained, and the corresponding image component ratio distribution was compared with the classification result. A method to determine the numerical grades of caries using a fluorescence imaging system was proposed. This method can be applied to similar imaging systems.

Method and apparatus to monitor a beam of ionizing radiation

DOEpatents

Blackburn, Brandon W.; Chichester, David L.; Watson, Scott M.; Johnson, James T.; Kinlaw, Mathew T.

2015-06-02

Methods and apparatus to capture images of fluorescence generated by ionizing radiation and determine a position of a beam of ionizing radiation generating the fluorescence from the captured images. In one embodiment, the fluorescence is the result of ionization and recombination of nitrogen in air.

AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

PubMed

Kong, Jun; Wang, Fusheng; Teodoro, George; Liang, Yanhui; Zhu, Yangyang; Tucker-Burden, Carol; Brat, Daniel J

2015-04-01

A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.

Excitation-resolved multispectral method for imaging pharmacokinetic parameters in dynamic fluorescent molecular tomography

NASA Astrophysics Data System (ADS)

Chen, Maomao; Zhou, Yuan; Su, Han; Zhang, Dong; Luo, Jianwen

2017-04-01

Imaging of the pharmacokinetic parameters in dynamic fluorescence molecular tomography (DFMT) can provide three-dimensional metabolic information for biological studies and drug development. However, owing to the ill-posed nature of the FMT inverse problem, the relatively low quality of the parametric images makes it difficult to investigate the different metabolic processes of the fluorescent targets with small distances. An excitation-resolved multispectral DFMT method is proposed; it is based on the fact that the fluorescent targets with different concentrations show different variations in the excitation spectral domain and can be considered independent signal sources. With an independent component analysis method, the spatial locations of different fluorescent targets can be decomposed, and the fluorescent yields of the targets at different time points can be recovered. Therefore, the metabolic process of each component can be independently investigated. Simulations and phantom experiments are carried out to evaluate the performance of the proposed method. The results demonstrated that the proposed excitation-resolved multispectral method can effectively improve the reconstruction accuracy of the parametric images in DFMT.

Method and apparatus for evaluating structural weakness in polymer matrix composites

DOEpatents

Wachter, E.A.; Fisher, W.G.

1996-01-09

A method and apparatus for evaluating structural weaknesses in polymer matrix composites is described. An object to be studied is illuminated with laser radiation and fluorescence emanating therefrom is collected and filtered. The fluorescence is then imaged and the image is studied to determine fluorescence intensity over the surface of the object being studied and the wavelength of maximum fluorescent intensity. Such images provide a map of the structural integrity of the part being studied and weaknesses, particularly weaknesses created by exposure of the object to heat, are readily visible in the image. 6 figs.

Method and apparatus for evaluating structural weakness in polymer matrix composites

DOEpatents

Wachter, Eric A.; Fisher, Walter G.

1996-01-01

A method and apparatus for evaluating structural weaknesses in polymer matrix composites is described. An object to be studied is illuminated with laser radiation and fluorescence emanating therefrom is collected and filtered. The fluorescence is then imaged and the image is studied to determine fluorescence intensity over the surface of the object being studied and the wavelength of maximum fluorescent intensity. Such images provide a map of the structural integrity of the part being studied and weaknesses, particularly weaknesses created by exposure of the object to heat, are readily visible in the image.

Trace Element Mapping of a Biological Specimen by a Full-Field X-ray Fluorescence Imaging Microscope with a Wolter Mirror

NASA Astrophysics Data System (ADS)

Hoshino, Masato; Yamada, Norimitsu; Ishino, Toyoaki; Namiki, Takashi; Watanabe, Norio; Aoki, Sadao

2007-01-01

A full-field X-ray fluorescence imaging microscope with a Wolter mirror was applied to the element mapping of alfalfa seeds. The X-ray fluorescence microscope was built at the Photon Factory BL3C2 (KEK). X-ray fluorescence images of several growing stages of the alfalfa seeds were obtained. X-ray fluorescence energy spectra were measured with either a solid state detector or a CCD photon counting method. The element distributions of iron and zinc which were included in the seeds were obtained using a photon counting method.

Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates.

PubMed

Cordina, Nicole M; Sayyadi, Nima; Parker, Lindsay M; Everest-Dass, Arun; Brown, Louise J; Packer, Nicolle H

2018-03-14

Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.

Image scanning fluorescence emission difference microscopy based on a detector array.

PubMed

Li, Y; Liu, S; Liu, D; Sun, S; Kuang, C; Ding, Z; Liu, X

2017-06-01

We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

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.

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.

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.

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

Laser-induced fluorescence imaging of bacteria

NASA Astrophysics Data System (ADS)

Hilton, Peter J.

1998-12-01

This paper outlines a method for optically detecting bacteria on various backgrounds, such as meat, by imaging their laser induced auto-fluorescence response. This method can potentially operate in real-time, which is many times faster than current bacterial detection methods, which require culturing of bacterial samples. This paper describes the imaging technique employed whereby a laser spot is scanned across an object while capturing, filtering, and digitizing the returned light. Preliminary results of the bacterial auto-fluorescence are reported and plans for future research are discussed. The results to date are encouraging with six of the eight bacterial strains investigated exhibiting auto-fluorescence when excited at 488 nm. Discrimination of these bacterial strains against red meat is shown and techniques for reducing background fluorescence discussed.

Image Restoration for Fluorescence Planar Imaging with Diffusion Model

PubMed Central

Gong, Yuzhu; Li, Yang

2017-01-01

Fluorescence planar imaging (FPI) is failure to capture high resolution images of deep fluorochromes due to photon diffusion. This paper presents an image restoration method to deal with this kind of blurring. The scheme of this method is conceived based on a reconstruction method in fluorescence molecular tomography (FMT) with diffusion model. A new unknown parameter is defined through introducing the first mean value theorem for definite integrals. System matrix converting this unknown parameter to the blurry image is constructed with the elements of depth conversion matrices related to a chosen plane named focal plane. Results of phantom and mouse experiments show that the proposed method is capable of reducing the blurring of FPI image caused by photon diffusion when the depth of focal plane is chosen within a proper interval around the true depth of fluorochrome. This method will be helpful to the estimation of the size of deep fluorochrome. PMID:29279843

Cell-free measurements of brightness of fluorescently labeled antibodies

PubMed Central

Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

2017-01-01

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures. PMID:28150730

Near-Infrared II Fluorescence for Imaging Hindlimb Vessel Regeneration with Dynamic Tissue Perfusion Measurement

PubMed Central

Hong, Guosong; Lee, Jerry C.; Jha, Arshi; Diao, Shuo; Nakayama, Karina H.; Hou, Luqia; Doyle, Timothy C.; Robinson, Joshua T.; Antaris, Alexander L.; Dai, Hongjie; Cooke, John P.; Huang, Ngan F.

2014-01-01

Background Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000–1400 nm) of photon wavelengths. Methods and Results Owing to the reduced photon scattering of NIR-II fluorescence compared to traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microCT. Furthermore, imaging over 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P < 0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy. Conclusions The penetration depth of millimeters, high spatial resolution and fast acquisition rate of NIR-II imaging makes it a useful imaging tool for murine models of vascular disease. PMID:24657826

Quantitative analysis of phosphoinositide 3-kinase (PI3K) signaling using live-cell total internal reflection fluorescence (TIRF) microscopy.

PubMed

Johnson, Heath E; Haugh, Jason M

2013-12-02

This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors. Copyright © 2013 John Wiley & Sons, Inc.

Analysis of gene expression levels in individual bacterial cells without image segmentation

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

Kwak, In Hae; Son, Minjun; Hagen, Stephen J., E-mail: sjhagen@ufl.edu

2012-05-11

Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on amore » segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.« less

Enhanced speed in fluorescence imaging using beat frequency multiplexing

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Kobayashi, Hirofumi; Wang, Yisen; Hamad, Syed; Ozeki, Yasuyuki; Goda, Keisuke

2016-03-01

Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

NASA Astrophysics Data System (ADS)

Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

2017-03-01

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers.

PubMed

Haring, Martijn T; Liv, Nalan; Zonnevylle, A Christiaan; Narvaez, Angela C; Voortman, Lenard M; Kruit, Pieter; Hoogenboom, Jacob P

2017-03-02

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

PubMed Central

Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

2017-01-01

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample. PMID:28252673

Live visualization of genomic loci with BiFC-TALE

PubMed Central

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-01

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP. PMID:28074901

Live visualization of genomic loci with BiFC-TALE.

PubMed

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-11

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP.

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.

Photonic crystal enhanced fluorescence immunoassay on diatom biosilica.

PubMed

Squire, Kenneth; Kong, Xianming; LeDuff, Paul; Rorrer, Gregory L; Wang, Alan X

2018-05-16

Fluorescence biosensing is one of the most established biosensing methods, particularly fluorescence spectroscopy and microscopy. These are two highly sensitive techniques but require high grade electronics and optics to achieve the desired sensitivity. Efforts have been made to implement these methods using consumer grade electronics and simple optical setups for applications such as point-of-care diagnostics, but the sensitivity inherently suffers. Sensing substrates, capable of enhancing fluorescence are thus needed to achieve high sensitivity for such applications. In this paper, we demonstrate a photonic crystal-enhanced fluorescence immunoassay biosensor using diatom biosilica, which consists of silica frustules with sub-100 nm periodic pores. Utilizing the enhanced local optical field, the Purcell effect and increased surface area from the diatom photonic crystals, we create ultrasensitive immunoassay biosensors that can significantly enhance fluorescence spectroscopy as well as fluorescence imaging. Using standard antibody-antigen-labeled antibody immunoassay protocol, we experimentally achieved 100× and 10× better detection limit with fluorescence spectroscopy and fluorescence imaging respectively. The limit of detection of the mouse IgG goes down to 10 -16 M (14 fg/mL) and 10 -15 M (140 fg/mL) for the two respective detection modalities, virtually sensing a single mouse IgG molecule on each diatom frustule. The effectively enhanced fluorescence imaging in conjunction with the simple hot-spot counting analysis method used in this paper proves the great potential of diatom fluorescence immunoassay for point-of-care biosensing. Scanning electron microscope image of biosilica diatom frustule that enables significant enhancement of fluorescence spectroscopy and fluorescence image. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

PubMed Central

Lidke, Diane S.; Lidke, Keith A.

2015-01-01

Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique. PMID:25860558

ReagentTF: a rapid and versatile optical clearing method for biological imaging(Conference Presentation)

NASA Astrophysics Data System (ADS)

Yu, Tingting; Zhu, Jingtan; Li, Yusha; Qi, Yisong; Xu, Jianyi; Gong, Hui; Luo, Qingming; Zhu, Dan

2017-02-01

The emergence of various optical clearing methods provides a great potential for imaging deep inside tissues by combining with multiple-labelling and microscopic imaging techniques. They were generally developed for specific imaging demand thus presented some non-negligible limitations such as long incubation time, tissue deformation, fluorescence quenching, incompatibility with immunostaining or lipophilic tracers. In this study, we developed a rapid and versatile clearing method, termed ReagentTF, for deep imaging of various fluorescent samples. This method can not only efficiently clear embryos, neonatal whole-brains and adult thick brain sections by simple immersion in aqueous mixtures with minimal volume change, but also can preserve fluorescence of various fluorescent proteins and simultaneously be compatible with immunostaining and lipophilic neuronal dyes. We demonstrate the effectiveness of this method in reconstructing the cell distributions of mouse hippocampus, visualizing the neural projection from CA1 (Cornu Ammonis 1) to HDB (nucleus of the horizontal limb of the diagonal band), and observing the growth of forelimb plexus in whole-mount embryos. These results suggest that ReagentTF is useful for large-volume imaging and will be an option for the deep imaging of biological tissues.

Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice

NASA Astrophysics Data System (ADS)

Dobosz, Michael; Strobel, Steffen; Stubenrauch, Kay-Gunnar; Osl, Franz; Scheuer, Werner

2014-01-01

Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F( fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

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.

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

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

PubMed

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

Boundary segmentation for fluorescence microscopy using steerable filters

NASA Astrophysics Data System (ADS)

Ho, David Joon; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

2017-02-01

Fluorescence microscopy is used to image multiple subcellular structures in living cells which are not readily observed using conventional optical microscopy. Moreover, two-photon microscopy is widely used to image structures deeper in tissue. Recent advancement in fluorescence microscopy has enabled the generation of large data sets of images at different depths, times, and spectral channels. Thus, automatic object segmentation is necessary since manual segmentation would be inefficient and biased. However, automatic segmentation is still a challenging problem as regions of interest may not have well defined boundaries as well as non-uniform pixel intensities. This paper describes a method for segmenting tubular structures in fluorescence microscopy images of rat kidney and liver samples using adaptive histogram equalization, foreground/background segmentation, steerable filters to capture directional tendencies, and connected-component analysis. The results from several data sets demonstrate that our method can segment tubular boundaries successfully. Moreover, our method has better performance when compared to other popular image segmentation methods when using ground truth data obtained via manual segmentation.

Embedding and Chemical Reactivation of Green Fluorescent Protein in the Whole Mouse Brain for Optical Micro-Imaging

PubMed Central

Gang, Yadong; Zhou, Hongfu; Jia, Yao; Liu, Ling; Liu, Xiuli; Rao, Gong; Li, Longhui; Wang, Xiaojun; Lv, Xiaohua; Xiong, Hanqing; Yang, Zhongqin; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2017-01-01

Resin embedding has been widely applied to fixing biological tissues for sectioning and imaging, but has long been regarded as incompatible with green fluorescent protein (GFP) labeled sample because it reduces fluorescence. Recently, it has been reported that resin-embedded GFP-labeled brain tissue can be imaged with high resolution. In this protocol, we describe an optimized protocol for resin embedding and chemical reactivation of fluorescent protein labeled mouse brain, we have used mice as experiment model, but the protocol should be applied to other species. This method involves whole brain embedding and chemical reactivation of the fluorescent signal in resin-embedded tissue. The whole brain embedding process takes a total of 7 days. The duration of chemical reactivation is ~2 min for penetrating 4 μm below the surface in the resin-embedded brain. This protocol provides an efficient way to prepare fluorescent protein labeled sample for high-resolution optical imaging. This kind of sample was demonstrated to be imaged by various optical micro-imaging methods. Fine structures labeled with GFP across a whole brain can be detected. PMID:28352214

Benzofurazan Sulfides for Thiol Imaging and Quantification in Live Cells through Fluorescence Microscopy

PubMed Central

Li, Yinghong; Yang, Yang; Guan, Xiangming

2012-01-01

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a–e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH2, -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 nm and 520 nm as the excitation and emission wavelengths respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an HPLC/UV total thiol assay method. The reagents and method will be of a great value to thiol redox-related research. PMID:22794193

Hyperspectral fluorescence imaging with multi wavelength LED excitation

NASA Astrophysics Data System (ADS)

Luthman, A. Siri; Dumitru, Sebastian; Quirós-Gonzalez, Isabel; Bohndiek, Sarah E.

2016-04-01

Hyperspectral imaging (HSI) can combine morphological and molecular information, yielding potential for real-time and high throughput multiplexed fluorescent contrast agent imaging. Multiplexed readout from targets, such as cell surface receptors overexpressed in cancer cells, could improve both sensitivity and specificity of tumor identification. There remains, however, a need for compact and cost effective implementations of the technology. We have implemented a low-cost wide-field multiplexed fluorescence imaging system, which combines LED excitation at 590, 655 and 740 nm with a compact commercial solid state HSI system operating in the range 600 - 1000 nm. A key challenge for using reflectance-based HSI is the separation of contrast agent fluorescence from the reflectance of the excitation light. Here, we illustrate how it is possible to address this challenge in software, using two offline reflectance removal methods, prior to least-squares spectral unmixing. We made a quantitative comparison of the methods using data acquired from dilutions of contrast agents prepared in well-plates. We then established the capability of our HSI system for non-invasive in vivo fluorescence imaging in small animals using the optimal reflectance removal method. The HSI presented here enables quantitative unmixing of at least four fluorescent contrast agents (Alexa Fluor 610, 647, 700 and 750) simultaneously in living mice. A successful unmixing of the four fluorescent contrast agents was possible both using the pure contrast agents and with mixtures. The system could in principle also be applied to imaging of ex vivo tissue or intraoperative imaging in a clinical setting. These data suggest a promising approach for developing clinical applications of HSI based on multiplexed fluorescence contrast agent imaging.

Scanning fluorescent microthermal imaging apparatus and method

DOEpatents

Barton, Daniel L.; Tangyunyong, Paiboon

1998-01-01

A scanning fluorescent microthermal imaging (FMI) apparatus and method is disclosed, useful for integrated circuit (IC) failure analysis, that uses a scanned and focused beam from a laser to excite a thin fluorescent film disposed over the surface of the IC. By collecting fluorescent radiation from the film, and performing point-by-point data collection with a single-point photodetector, a thermal map of the IC is formed to measure any localized heating associated with defects in the IC.

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

Analysis of gene expression levels in individual bacterial cells without image segmentation.

PubMed

Kwak, In Hae; Son, Minjun; Hagen, Stephen J

2012-05-11

Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly. Copyright © 2012 Elsevier Inc. All rights reserved.

Near-infrared fluorescence image quality test methods for standardized performance evaluation

NASA Astrophysics Data System (ADS)

Kanniyappan, Udayakumar; Wang, Bohan; Yang, Charles; Ghassemi, Pejhman; Wang, Quanzeng; Chen, Yu; Pfefer, Joshua

2017-03-01

Near-infrared fluorescence (NIRF) imaging has gained much attention as a clinical method for enhancing visualization of cancers, perfusion and biological structures in surgical applications where a fluorescent dye is monitored by an imaging system. In order to address the emerging need for standardization of this innovative technology, it is necessary to develop and validate test methods suitable for objective, quantitative assessment of device performance. Towards this goal, we develop target-based test methods and investigate best practices for key NIRF imaging system performance characteristics including spatial resolution, depth of field and sensitivity. Characterization of fluorescence properties was performed by generating excitation-emission matrix properties of indocyanine green and quantum dots in biological solutions and matrix materials. A turbid, fluorophore-doped target was used, along with a resolution target for assessing image sharpness. Multi-well plates filled with either liquid or solid targets were generated to explore best practices for evaluating detection sensitivity. Overall, our results demonstrate the utility of objective, quantitative, target-based testing approaches as well as the need to consider a wide range of factors in establishing standardized approaches for NIRF imaging system performance.

Segmentation-based retrospective shading correction in fluorescence microscopy E. coli images for quantitative analysis

NASA Astrophysics Data System (ADS)

Mai, Fei; Chang, Chunqi; Liu, Wenqing; Xu, Weichao; Hung, Yeung S.

2009-10-01

Due to the inherent imperfections in the imaging process, fluorescence microscopy images often suffer from spurious intensity variations, which is usually referred to as intensity inhomogeneity, intensity non uniformity, shading or bias field. In this paper, a retrospective shading correction method for fluorescence microscopy Escherichia coli (E. Coli) images is proposed based on segmentation result. Segmentation and shading correction are coupled together, so we iteratively correct the shading effects based on segmentation result and refine the segmentation by segmenting the image after shading correction. A fluorescence microscopy E. Coli image can be segmented (based on its intensity value) into two classes: the background and the cells, where the intensity variation within each class is close to zero if there is no shading. Therefore, we make use of this characteristics to correct the shading in each iteration. Shading is mathematically modeled as a multiplicative component and an additive noise component. The additive component is removed by a denoising process, and the multiplicative component is estimated using a fast algorithm to minimize the intra-class intensity variation. We tested our method on synthetic images and real fluorescence E.coli images. It works well not only for visual inspection, but also for numerical evaluation. Our proposed method should be useful for further quantitative analysis especially for protein expression value comparison.

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.

Calibration of fluorescence resonance energy transfer in microscopy

DOEpatents

Youvan, Dougalas C.; Silva, Christopher M.; Bylina, Edward J.; Coleman, William J.; Dilworth, Michael R.; Yang, Mary M.

2003-12-09

Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.

Calibration of fluorescence resonance energy transfer in microscopy

DOEpatents

Youvan, Douglas C.; Silva, Christopher M.; Bylina, Edward J.; Coleman, William J.; Dilworth, Michael R.; Yang, Mary M.

2002-09-24

Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.

Physically-based in silico light sheet microscopy for visualizing fluorescent brain models

PubMed Central

2015-01-01

Background We present a physically-based computational model of the light sheet fluorescence microscope (LSFM). Based on Monte Carlo ray tracing and geometric optics, our method simulates the operational aspects and image formation process of the LSFM. This simulated, in silico LSFM creates synthetic images of digital fluorescent specimens that can resemble those generated by a real LSFM, as opposed to established visualization methods producing visually-plausible images. We also propose an accurate fluorescence rendering model which takes into account the intrinsic characteristics of fluorescent dyes to simulate the light interaction with fluorescent biological specimen. Results We demonstrate first results of our visualization pipeline to a simplified brain tissue model reconstructed from the somatosensory cortex of a young rat. The modeling aspects of the LSFM units are qualitatively analysed, and the results of the fluorescence model were quantitatively validated against the fluorescence brightness equation and characteristic emission spectra of different fluorescent dyes. AMS subject classification Modelling and simulation PMID:26329404

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.

Scanning fluorescent microthermal imaging apparatus and method

DOEpatents

Barton, D.L.; Tangyunyong, P.

1998-01-06

A scanning fluorescent microthermal imaging (FMI) apparatus and method is disclosed, useful for integrated circuit (IC) failure analysis, that uses a scanned and focused beam from a laser to excite a thin fluorescent film disposed over the surface of the IC. By collecting fluorescent radiation from the film, and performing point-by-point data collection with a single-point photodetector, a thermal map of the IC is formed to measure any localized heating associated with defects in the IC. 1 fig.

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

Fluorescent marker-based and marker-free discrimination between healthy and cancerous human tissues using hyper-spectral imaging

NASA Astrophysics Data System (ADS)

Arnold, Thomas; De Biasio, Martin; Leitner, Raimund

2015-06-01

Two problems are addressed in this paper (i) the fluorescent marker-based and the (ii) marker-free discrimination between healthy and cancerous human tissues. For both applications the performance of hyper-spectral methods are quantified. Fluorescent marker-based tissue classification uses a number of fluorescent markers to dye specific parts of a human cell. The challenge is that the emission spectra of the fluorescent dyes overlap considerably. They are, furthermore disturbed by the inherent auto-fluorescence of human tissue. This results in ambiguities and decreased image contrast causing difficulties for the treatment decision. The higher spectral resolution introduced by tunable-filter-based spectral imaging in combination with spectral unmixing techniques results in an improvement of the image contrast and therefore more reliable information for the physician to choose the treatment decision. Marker-free tissue classification is based solely on the subtle spectral features of human tissue without the use of artificial markers. The challenge in this case is that the spectral differences between healthy and cancerous tissues are subtle and embedded in intra- and inter-patient variations of these features. The contributions of this paper are (i) the evaluation of hyper-spectral imaging in combination with spectral unmixing techniques for fluorescence marker-based tissue classification, (ii) the evaluation of spectral imaging for marker-free intra surgery tissue classification. Within this paper, we consider real hyper-spectral fluorescence and endoscopy data sets to emphasize the practical capability of the proposed methods. It is shown that the combination of spectral imaging with multivariate statistical methods can improve the sensitivity and specificity of the detection and the staging of cancerous tissues compared to standard procedures.

Hyperspectral reflectance and fluorescence line-scan imaging system for online detection of fecal contamination on apples

NASA Astrophysics Data System (ADS)

Kim, Moon S.; Cho, Byoung-Kwan; Yang, Chun-Chieh; Chao, Kaunglin; Lefcourt, Alan M.; Chen, Yud-Ren

2006-10-01

We have developed nondestructive opto-electronic imaging techniques for rapid assessment of safety and wholesomeness of foods. A recently developed fast hyperspectral line-scan imaging system integrated with a commercial apple-sorting machine was evaluated for rapid detection of animal feces matter on apples. Apples obtained from a local orchard were artificially contaminated with cow feces. For the online trial, hyperspectral images with 60 spectral channels, reflectance in the visible to near infrared regions and fluorescence emissions with UV-A excitation, were acquired from apples moving at a processing sorting-line speed of three apples per second. Reflectance and fluorescence imaging required a passive light source, and each method used independent continuous wave (CW) light sources. In this paper, integration of the hyperspectral imaging system with the commercial applesorting machine and preliminary results for detection of fecal contamination on apples, mainly based on the fluorescence method, are presented.

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.

Validation of ALFIA: a platform for quantifying near-infrared fluorescent images of lymphatic propulsion in humans

NASA Astrophysics Data System (ADS)

Rasmussen, John C.; Bautista, Merrick; Tan, I.-Chih; Adams, Kristen E.; Aldrich, Melissa; Marshall, Milton V.; Fife, Caroline E.; Maus, Erik A.; Smith, Latisha A.; Zhang, Jingdan; Xiang, Xiaoyan; Zhou, Shaohua Kevin; Sevick-Muraca, Eva M.

2011-02-01

Recently, we demonstrated near-infrared (NIR) fluorescence imaging for quantifying real-time lymphatic propulsion in humans following intradermal injections of microdose amounts of indocyanine green. However computational methods for image analysis are underdeveloped, hindering the translation and clinical adaptation of NIR fluorescent lymphatic imaging. In our initial work we used ImageJ and custom MatLab programs to manually identify lymphatic vessels and individual propulsion events using the temporal transit of the fluorescent dye. In addition, we extracted the apparent velocities of contractile propagation and time periods between propulsion events. Extensive time and effort were required to analyze the 6-8 gigabytes of NIR fluorescent images obtained for each subject. To alleviate this bottleneck, we commenced development of ALFIA, an integrated software platform which will permit automated, near real-time analysis of lymphatic function using NIR fluorescent imaging. However, prior to automation, the base algorithms calculating the apparent velocity and period must be validated to verify that they produce results consistent with the proof-of-concept programs. To do this, both methods were used to analyze NIR fluorescent images of two subjects and the number of propulsive events identified, the average apparent velocities, and the average periods for each subject were compared. Paired Student's t-tests indicate that the differences between their average results are not significant. With the base algorithms validated, further development and automation of ALFIA can be realized, significantly reducing the amount of user interaction required, and potentially enabling the near real-time, clinical evaluation of NIR fluorescent lymphatic imaging.

PH-sensitive fluorescence detection by diffuse fluorescence tomography

NASA Astrophysics Data System (ADS)

Li, Jiao; Gao, Feng; Duan, Linjing; Wang, Xin; Zhang, Limin; Zhao, Huijuan

2012-03-01

The importance of cellular pH has been shown clearly in the study of cell activity, pathological feature, drug metabolism, etc. Monitoring pH changes of living cells and imaging the regions with abnormal pH values in vivo could provide the physiologic and pathologic information for the research of the cell biology, pharmacokinetics, diagnostics and therapeutics of certain diseases such as cancer. Thus, pH-sensitive fluorescence imaging of bulk tissues has been attracting great attention in the regime of near-infrared diffuse fluorescence tomography (DFT), an efficient small-animal imaging tool. In this paper, the feasibility of quantifying pH-sensitive fluorescence targets in turbid medium is investigated using both time-domain and steady-state DFT methods. By use of the specifically designed time-domain and continuous-wave systems and the previously proposed image reconstruction scheme, we validate the method through 2-dimensional imaging experiments on a small-animal-sized phantom with multiply targets of distinct pH values. The results show that the approach can localize the targets with reasonable accuracy and achieve quantitative reconstruction of the pH-sensitive fluorescent yield.

Fluorescence spectroscopy and imaging for noninvasive diagnostics: applications to early cancer detection in the lung

NASA Astrophysics Data System (ADS)

Mycek, Mary-Ann; Urayama, Paul; Zhong, Wei; Sloboda, Roger D.; Dragnev, Konstantin H.; Dmitrovsky, Ethan

2003-10-01

Tissue fluorescence spectroscopy and imaging are being investigated as potential methods for non-invasive detection of pre-neoplastic change in the lung and other organ systems. A substantial contribution to tissue fluorescence is known to arise from endogenous cellular fluorophores. Using steady-state and time-resolved fluorescence spectroscopy and imaging, we characterized the endogenous fluorescence properties of immortalized and carcinogen-transformed human bronchial epithelial cells. Non-invasive sensing of endogenous molecular biomarkers associated with human bronchial pre-neoplasia will be discussed.

Speckle correlation resolution enhancement of wide-field fluorescence imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yilmaz, Hasan

2016-03-01

Structured illumination enables high-resolution fluorescence imaging of nanostructures [1]. We demonstrate a new high-resolution fluorescence imaging method that uses a scattering layer with a high-index substrate as a solid immersion lens [2]. Random scattering of coherent light enables a speckle pattern with a very fine structure that illuminates the fluorescent nanospheres on the back surface of the high-index substrate. The speckle pattern is raster-scanned over the fluorescent nanospheres using a speckle correlation effect known as the optical memory effect. A series of standard-resolution fluorescence images per each speckle pattern displacement are recorded by an electron-multiplying CCD camera using a commercial microscope objective. We have developed a new phase-retrieval algorithm to reconstruct a high-resolution, wide-field image from several standard-resolution wide-field images. We have introduced phase information of Fourier components of standard-resolution images as a new constraint in our algorithm which discards ambiguities therefore ensures convergence to a unique solution. We demonstrate two-dimensional fluorescence images of a collection of nanospheres with a deconvolved Abbe resolution of 116 nm and a field of view of 10 µm × 10 µm. Our method is robust against optical aberrations and stage drifts, therefore excellent for imaging nanostructures under ambient conditions. [1] M. G. L. Gustafsson, J. Microsc. 198, 82-87 (2000). [2] H. Yilmaz, E. G. van Putten, J. Bertolotti, A. Lagendijk, W. L. Vos, and A. P. Mosk, Optica 2, 424-429 (2015).

Accurate quantification of fluorescent targets within turbid media based on a decoupled fluorescence Monte Carlo model.

PubMed

Deng, Yong; Luo, Zhaoyang; Jiang, Xu; Xie, Wenhao; Luo, Qingming

2015-07-01

We propose a method based on a decoupled fluorescence Monte Carlo model for constructing fluorescence Jacobians to enable accurate quantification of fluorescence targets within turbid media. The effectiveness of the proposed method is validated using two cylindrical phantoms enclosing fluorescent targets within homogeneous and heterogeneous background media. The results demonstrate that our method can recover relative concentrations of the fluorescent targets with higher accuracy than the perturbation fluorescence Monte Carlo method. This suggests that our method is suitable for quantitative fluorescence diffuse optical tomography, especially for in vivo imaging of fluorophore targets for diagnosis of different diseases and abnormalities.

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

PubMed

Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

2002-10-01

To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

Mapping Diffusion in a Living Cell via the Phasor Approach

PubMed Central

Ranjit, Suman; Lanzano, Luca; Gratton, Enrico

2014-01-01

Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created. PMID:25517145

A fiber-optic-based imaging system for nondestructive assessment of cell-seeded tissue-engineered scaffolds.

PubMed

Hofmann, Matthias C; Whited, Bryce M; Criswell, Tracy; Rylander, Marissa Nichole; Rylander, Christopher G; Soker, Shay; Wang, Ge; Xu, Yong

2012-09-01

A major limitation in tissue engineering is the lack of nondestructive methods that assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot "see" through thick and optically opaque tissue constructs. To address this deficiency, we developed a fiber-optic-based imaging method that is capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque scaffold, contained in a bioreactor. This imaging modality is based on the local excitation of fluorescent cells, the acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells (ECs), stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled cells through a ~500 μm thick scaffold with cell-level resolution and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution on the order of 20-30 μm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8 cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable the nondestructive monitoring of ECs seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future.

Development of ultrasound-assisted fluorescence imaging of indocyanine green.

PubMed

Morikawa, Hiroyasu; Toyota, Shin; Wada, Kenji; Uchida-Kobayashi, Sawako; Kawada, Norifumi; Horinaka, Hiromichi

2017-01-01

Indocyanine green (ICG) accumulation in hepatocellular carcinoma means tumors can be located by fluorescence. However, because of light scattering, it is difficult to detect ICG fluorescence from outside the body. We propose a new fluorescence imaging method that detects changes in the intensity of ICG fluorescence by ultrasound-induced temperature changes. ICG fluorescence intensity decreases as the temperature rises. Therefore, it should theoretically be possible to detect tissue distribution of ICG using ultrasound to heat tissue, moving the point of ultrasound transmission, and monitoring changes in fluorescence intensity. A new probe was adapted for clinical application. It consisted of excitation light from a laser, fluorescence sensing through a light pipe, and heating by ultrasound. We applied the probe to bovine liver to image the accumulation of ICG. ICG emits fluorescence (820 nm) upon light irradiation (783 nm). With a rise in temperature, the fluorescence intensity of ICG decreased by 0.85 %/°C. The distribution of fluorescent ICG was detected using an ultrasonic warming method in a new integrated probe. Modulating fluorescence by changing the temperature using ultrasound can determine where ICG accumulates at a depth, highlighting its potential as a means to locate hepatocellular carcinoma.

Optimization of advanced Wiener estimation methods for Raman reconstruction from narrow-band measurements in the presence of fluorescence background

PubMed Central

Chen, Shuo; Ong, Yi Hong; Lin, Xiaoqian; Liu, Quan

2015-01-01

Raman spectroscopy has shown great potential in biomedical applications. However, intrinsically weak Raman signals cause slow data acquisition especially in Raman imaging. This problem can be overcome by narrow-band Raman imaging followed by spectral reconstruction. Our previous study has shown that Raman spectra free of fluorescence background can be reconstructed from narrow-band Raman measurements using traditional Wiener estimation. However, fluorescence-free Raman spectra are only available from those sophisticated Raman setups capable of fluorescence suppression. The reconstruction of Raman spectra with fluorescence background from narrow-band measurements is much more challenging due to the significant variation in fluorescence background. In this study, two advanced Wiener estimation methods, i.e. modified Wiener estimation and sequential weighted Wiener estimation, were optimized to achieve this goal. Both spontaneous Raman spectra and surface enhanced Raman spectra were evaluated. Compared with traditional Wiener estimation, two advanced methods showed significant improvement in the reconstruction of spontaneous Raman spectra. However, traditional Wiener estimation can work as effectively as the advanced methods for SERS spectra but much faster. The wise selection of these methods would enable accurate Raman reconstruction in a simple Raman setup without the function of fluorescence suppression for fast Raman imaging. PMID:26203387

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.

Near-infrared II fluorescence for imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement.

PubMed

Hong, Guosong; Lee, Jerry C; Jha, Arshi; Diao, Shuo; Nakayama, Karina H; Hou, Luqia; Doyle, Timothy C; Robinson, Joshua T; Antaris, Alexander L; Dai, Hongjie; Cooke, John P; Huang, Ngan F

2014-05-01

Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here, we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000-1400 nm) of photon wavelengths. Because of the reduced photon scattering of NIR-II fluorescence compared with traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microscopic computed tomography. Furthermore, imaging during 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P<0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy. The penetration depth of millimeters, high spatial resolution, and fast acquisition rate of NIR-II imaging make it a useful imaging tool for murine models of vascular disease. © 2014 American Heart Association, Inc.

Compensation of optical heterogeneity-induced artifacts in fluorescence molecular tomography: theory and in vivo validation

NASA Astrophysics Data System (ADS)

Mohajerani, Pouyan; Adibi, Ali; Kempner, Joshua; Yared, Wael

2009-05-01

We present a method for reduction of image artifacts induced by the optical heterogeneities of tissue in fluorescence molecular tomography (FMT) through identification and compensation of image regions that evidence propagation of emission light through thin or low-absorption tunnels in tissue. The light tunneled as such contributes to the emission image as spurious components that might substantially overwhelm the desirable fluorescence emanating from the targeted lesions. The proposed method makes use of the strong spatial correlation between the emission and excitation images to estimate the tunneled components and yield a residual image that mainly consists of the signal due to the desirable fluorescence. This residual image is further refined using a coincidence mask constructed for each excitation-emission image pair. The coincidence mask is essentially a map of the ``hot spots'' that occur in both excitation and emission images, as such areas are often associated with tunneled emission. In vivo studies are performed on a human colon adenocarcinoma xenograft tumor model with subcutaneous tumors and a murine breast adenocarcinoma model with aggressive tumor cell metastasis and growth in the lungs. Results demonstrate significant improvements in the reconstructions achieved by the proposed method.

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

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.

DEVELOPMENT OF EVALUATION OF A QUANTITATIVE VIDEO-FLUORESCENCE IMAGING SYSTEM AND FLUORESCENT TRACER FOR MEASURING TRANSFER OF PESTICIDE RESIDUES FROM SURFACES TO HANDS WITH REPEATED CONTACTS

EPA Science Inventory

A video imaging system and the associated quantification methods have been developed for measurement of the transfers of a fluorescent tracer from surfaces to hands. The highly fluorescent compound riboflavin (Vitamin B2), which is also water soluble and non-toxic, was chosen as...

SIMA: Python software for analysis of dynamic fluorescence imaging data.

PubMed

Kaifosh, Patrick; Zaremba, Jeffrey D; Danielson, Nathan B; Losonczy, Attila

2014-01-01

Fluorescence imaging is a powerful method for monitoring dynamic signals in the nervous system. However, analysis of dynamic fluorescence imaging data remains burdensome, in part due to the shortage of available software tools. To address this need, we have developed SIMA, an open source Python package that facilitates common analysis tasks related to fluorescence imaging. Functionality of this package includes correction of motion artifacts occurring during in vivo imaging with laser-scanning microscopy, segmentation of imaged fields into regions of interest (ROIs), and extraction of signals from the segmented ROIs. We have also developed a graphical user interface (GUI) for manual editing of the automatically segmented ROIs and automated registration of ROIs across multiple imaging datasets. This software has been designed with flexibility in mind to allow for future extension with different analysis methods and potential integration with other packages. Software, documentation, and source code for the SIMA package and ROI Buddy GUI are freely available at http://www.losonczylab.org/sima/.

Optimal optical filters of fluorescence excitation and emission for poultry fecal detection

USDA-ARS?s Scientific Manuscript database

Purpose: An analytic method to design excitation and emission filters of a multispectral fluorescence imaging system is proposed and was demonstrated in an application to poultry fecal inspection. Methods: A mathematical model of a multispectral imaging system is proposed and its system parameters, ...

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.

Methods on observation of fluorescence micro-imaging for microalgae

NASA Astrophysics Data System (ADS)

Ou, Lin; Zhuang, Hui-ru; Chen, Rong; Lei, Jin-pin; Liao, Xiao-hua; Lin, Wen-suo

2007-11-01

Objective: Auto-fluorescence micro-imaging of microalgae are observed by using of laser scanning confocal microscopy (LSCM) and fluorescence microscopy, so as to investigate the effect of auto fluorescence alteration on growth of irradiated microalgae irradiated, meanwhile, the method of microalgae cells stained also to be studied. Methods: Platymonas subcordiformis, Phaeodactylum tricormutum and Isochyrsis zhanjiangensis cells are stained with acridine orange, and observed by fluorescence microscopy; the three types microalgae mentioned above are irradiated by Nd:YAP laser with 10w at 1341nm, irradiating time:12s, 30s, 35s and 55s, than to be cultured 6 days, and the auto fluorescence images and fluorescence spectra of algae cells are obtained by LSCM on lambda scan mode, at excitation 488nm (Ar + laser). Results: It is showed that the shapes and the structural features of microalgae cells stained can be seen clearly, and the cytoplasm and nucleus also can be observed. The chloroplasts in cell is bigger on promoting effects, conversely, it is to be mutilated, deformation and shrink. Contrast to the CK, the peak positions of fluorescence of algae cells irradiated is similar to the whole while the peak light intensity alters. On irradiation of promoting dose, however, the auto fluorescence intensity is enhanced more than control. Conclusions: The method of cell stained can be used to observed genetic material in microalgae. There are obvious effects for laser irradiating to chloroplasts in cells, the bigger chloroplasts the greater fluorescence intensity. Physiological incentive effects of microalgae irradiated can be given expression on fluorescence characteristics and fluorescence intensity alteration of cells.

Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy

PubMed Central

Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

2017-01-01

This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy. PMID:29160812

Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy.

PubMed

Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

2017-11-21

This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology

PubMed Central

Sandell, Lisa L.; Kurosaka, Hiroshi; Trainor, Paul A.

2012-01-01

Here we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional widefield fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images of similar specimens produced by Scanning Electron Microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. PMID:22930523

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

PubMed

Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A

2012-11-01

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. Copyright © 2012 Wiley Periodicals, Inc.

A Rapid and Convenient Method for in Vivo Fluorescent Imaging of Protoscolices of Echinococcus multilocularis.

PubMed

Yang, Tao; Wang, Sibo; Zhang, Xuyong; Xia, Jie; Guo, Jun; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

2016-04-01

Human and animal alveolar echinococcosis (AE) are important helminth infections endemic in wide areas of the Northern hemisphere. Monitoring Echinococcus multilocularis viability and spread using real-time fluorescent imaging in vivo provides a fast method to evaluate the load of parasite. Here, we generated a kind of fluorescent protoscolices in vivo imaging model and utilized this model to assess the activity against E. multilocularis protoscolices of metformin (Met). Results indicated that JC-1 tagged E. multilocularis can be reliably and confidently used to monitor protoscolices in vitro and in vivo. The availability of this transient in vivo fluorescent imaging of E. multilocularis protoscolices constitutes an important step toward the long term bio-imaging research of the AE-infected mouse models. In addition, this will be of great interest for further research on infection strategies and development of drugs and vaccines against E. multilocularis and other cestodes.

Two-photon fluorescence anisotropy imaging

NASA Astrophysics Data System (ADS)

Li, Wei; Wang, Yi; Shao, Hanrong; He, Yonghong; Ma, Hui

2006-09-01

We have developed a novel method for imaging the fluorescence intensity and anisotropy by two-photon fluorescence microscopy and tested its capability in biological application. This method is applied to model sample including FITC and FITC-CD44 antibody solution and also FITC-CD44 stained cells. The fluorescence anisotropy (FA) of FITC-CD44ab solution is higher than the FITC solution with the same concentration. The fluorescence in cell sample has even higher FA than in solution because the rotation diffusion is restrained in membrane. The method is employed to study the effect of berberine a kind of Chinese medicine, on tumor metastasis. The results indicated that tumor cell membrane fluidity is decreasing with increasing the concentration of berberine in culture medium.

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.

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.

Fluorescence diffuse tomography of small animals with DsRed2 fluorescent protein

NASA Astrophysics Data System (ADS)

Turchin, I. V.; Plehanov, V. I.; Orlova, A. G.; Kamenskiy, V. A.; Kleshnin, M. S.; Shirmanova, M. V.; Shakhova, N. M.; Balalaeva, I. V.; Savitskiy, A. P.

2006-05-01

Fluorescent compounds are used as markers to diagnose oncological diseases, to study molecular processes typical for carcinogenesis, and to investigate metastasis formation and tumor regress under the influence of therapeutics. Different types of tomography, such as continuous wave (CW), frequency-domain (FD), and time-domain (TD) tomography, allow fluorescence imaging of tumors located deep in human or animal tissue. In this work, preliminary results of the frequency domain fluorescent diffuse tomography (FDT) method in application to DsRed2 protein as a fluorescent agent are presented. For the first step of our experiments, we utilized low-frequency amplitude modulation (1 kHz) of second harmonic of Nd: YAG (532 nm). The transilluminative configuration was used in the setup. The results of post mortem experiments with capsules containing DsRed2 inserted inside the esophagus of a 3-day-old hairless rat to simulate tumor are shown. An algorithm of processing fluorescent images based on calculating the zero of maximum curvature has been applied to detect fluorescent inclusion boundaries in the image. This work demonstrates the potential capability of the FDT method for imaging deep fluorescent tumors in human tissue or animal models of human cancer. Improvement of the setup can be accomplished by using high-frequency modulation (using a 110-MHz acoustooptical modulator).

Recent Progress in Fluorescent Imaging Probes

PubMed Central

Pak, Yen Leng; Swamy, K. M. K.; Yoon, Juyoung

2015-01-01

Due to the simplicity and low detection limit, especially the bioimaging ability for cells, fluorescence probes serve as unique detection methods. With the aid of molecular recognition and specific organic reactions, research on fluorescent imaging probes has blossomed during the last decade. Especially, reaction based fluorescent probes have been proven to be highly selective for specific analytes. This review highlights our recent progress on fluorescent imaging probes for biologically important species, such as biothiols, reactive oxygen species, reactive nitrogen species, metal ions including Zn2+, Hg2+, Cu2+ and Au3+, and anions including cyanide and adenosine triphosphate (ATP). PMID:26402684

Recent Progress in Fluorescent Imaging Probes.

PubMed

Pak, Yen Leng; Swamy, K M K; Yoon, Juyoung

2015-09-22

Due to the simplicity and low detection limit, especially the bioimaging ability for cells, fluorescence probes serve as unique detection methods. With the aid of molecular recognition and specific organic reactions, research on fluorescent imaging probes has blossomed during the last decade. Especially, reaction based fluorescent probes have been proven to be highly selective for specific analytes. This review highlights our recent progress on fluorescent imaging probes for biologically important species, such as biothiols, reactive oxygen species, reactive nitrogen species, metal ions including Zn(2+), Hg(2+), Cu(2+) and Au(3+), and anions including cyanide and adenosine triphosphate (ATP).

Visualization of Electrical Field of Electrode Using Voltage-Controlled Fluorescence Release

PubMed Central

Jia, Wenyan; Wu, Jiamin; Gao, Di; Wang, Hao; Sun, Mingui

2016-01-01

In this study we propose an approach to directly visualize electrical current distribution at the electrode-electrolyte interface of a biopotential electrode. High-speed fluorescent microscopic images are acquired when an electric potential is applied across the interface to trigger the release of fluorescent material from the surface of the electrode. These images are analyzed computationally to obtain the distribution of the electric field from the fluorescent intensity of each pixel. Our approach allows direct observation of microscopic electrical current distribution around the electrode. Experiments are conducted to validate the feasibility of the fluorescent imaging method. PMID:27253615

Widely accessible method for superresolution fluorescence imaging of living systems

PubMed Central

Dedecker, Peter; Mo, Gary C. H.; Dertinger, Thomas; Zhang, Jin

2012-01-01

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule–based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging. PMID:22711840

Enabling Histopathological Annotations on Immunofluorescent Images through Virtualization of Hematoxylin and Eosin

PubMed Central

Lahiani, Amal; Klaiman, Eldad; Grimm, Oliver

2018-01-01

Context: Medical diagnosis and clinical decisions rely heavily on the histopathological evaluation of tissue samples, especially in oncology. Historically, classical histopathology has been the gold standard for tissue evaluation and assessment by pathologists. The most widely and commonly used dyes in histopathology are hematoxylin and eosin (H&E) as most malignancies diagnosis is largely based on this protocol. H&E staining has been used for more than a century to identify tissue characteristics and structures morphologies that are needed for tumor diagnosis. In many cases, as tissue is scarce in clinical studies, fluorescence imaging is necessary to allow staining of the same specimen with multiple biomarkers simultaneously. Since fluorescence imaging is a relatively new technology in the pathology landscape, histopathologists are not used to or trained in annotating or interpreting these images. Aims, Settings and Design: To allow pathologists to annotate these images without the need for additional training, we designed an algorithm for the conversion of fluorescence images to brightfield H&E images. Subjects and Methods: In this algorithm, we use fluorescent nuclei staining to reproduce the hematoxylin information and natural tissue autofluorescence to reproduce the eosin information avoiding the necessity to specifically stain the proteins or intracellular structures with an additional fluorescence stain. Statistical Analysis Used: Our method is based on optimizing a transform function from fluorescence to H&E images using least mean square optimization. Results: It results in high quality virtual H&E digital images that can easily and efficiently be analyzed by pathologists. We validated our results with pathologists by making them annotate tumor in real and virtual H&E whole slide images and we obtained promising results. Conclusions: Hence, we provide a solution that enables pathologists to assess tissue and annotate specific structures based on multiplexed fluorescence images. PMID:29531846

Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts.

PubMed

Kolesová, Hana; Čapek, Martin; Radochová, Barbora; Janáček, Jiří; Sedmera, David

2016-08-01

Our goal was to find an optimal tissue clearing protocol for whole-mount imaging of embryonic and adult hearts and whole embryos of transgenic mice that would preserve green fluorescent protein GFP fluorescence and permit comparison of different currently available 3D imaging modalities. We tested various published organic solvent- or water-based clearing protocols intended to preserve GFP fluorescence in central nervous system: tetrahydrofuran dehydration and dibenzylether protocol (DBE), SCALE, CLARITY, and CUBIC and evaluated their ability to render hearts and whole embryos transparent. DBE clearing protocol did not preserve GFP fluorescence; in addition, DBE caused considerable tissue-shrinking artifacts compared to the gold standard BABB protocol. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The SCALE clearing resulted in sufficient tissue transparency up to ED12.5; at later stages the useful depth of imaging was limited by tissue light scattering. The best method for the cardiac specimens proved to be the CUBIC protocol, which preserved GFP fluorescence well, and cleared the specimens sufficiently even at the adult stages. In addition, CUBIC decolorized the blood and myocardium by removing tissue iron. Good 3D renderings of whole fetal hearts and embryos were obtained with optical projection tomography and selective plane illumination microscopy, although at resolutions lower than with a confocal microscope. Comparison of five tissue clearing protocols and three imaging methods for study of GFP mouse embryos and hearts shows that the optimal method depends on stage and level of detail required.

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.

Hyperspectral imaging of endogenous fluorescent metabolic molecules to identify pain states in central nervous system tissue

NASA Astrophysics Data System (ADS)

Staikopoulos, Vasiliki; Gosnell, Martin E.; Anwer, Ayad G.; Mustafa, Sanam; Hutchinson, Mark R.; Goldys, Ewa M.

2016-12-01

Fluorescence-based bio-imaging methods have been extensively used to identify molecular changes occurring in biological samples in various pathological adaptations. Auto-fluorescence generated by endogenous fluorescent molecules within these samples can interfere with signal to background noise making positive antibody based fluorescent staining difficult to resolve. Hyperspectral imaging uses spectral and spatial imaging information for target detection and classification, and can be used to resolve changes in endogenous fluorescent molecules such as flavins, bound and free NADH and retinoids that are involved in cell metabolism. Hyperspectral auto-fluorescence imaging of spinal cord slices was used in this study to detect metabolic differences within pain processing regions of non-pain versus sciatic chronic constriction injury (CCI) animals, an established animal model of peripheral neuropathy. By using an endogenous source of contrast, subtle metabolic variations were detected between tissue samples, making it possible to distinguish between animals from non-injured and injured groups. Tissue maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant tissue regions with compromised mitochondrial function. Taken together, our results demonstrate that hyperspectral imaging provides a new non-invasive method to investigate central changes of peripheral neuropathic injury and other neurodegenerative disease models, and paves the way for novel cellular characterisation in health, disease and during treatment, with proper account of intrinsic cellular heterogeneity.

Laser line illumination scheme allowing the reduction of background signal and the correction of absorption heterogeneities effects for fluorescence reflectance imaging.

PubMed

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

2015-10-01

Intraoperative fluorescence imaging in reflectance geometry is an attractive imaging modality as it allows to noninvasively monitor the fluorescence targeted tumors located below the tissue surface. Some drawbacks of this technique are the background fluorescence decreasing the contrast and absorption heterogeneities leading to misinterpretations concerning fluorescence concentrations. We propose a correction technique based on a laser line scanning illumination scheme. We scan the medium with the laser line and acquire, at each position of the line, both fluorescence and excitation images. We then use the finding that there is a relationship between the excitation intensity profile and the background fluorescence one to predict the amount of signal to subtract from the fluorescence images to get a better contrast. As the light absorption information is contained both in fluorescence and excitation images, this method also permits us to correct the effects of absorption heterogeneities. This technique has been validated on simulations and experimentally. Fluorescent inclusions are observed in several configurations at depths ranging from 1 mm to 1 cm. Results obtained with this technique are compared with those obtained with a classical wide-field detection scheme for contrast enhancement and with the fluorescence by an excitation ratio approach for absorption correction.

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

Fluorescence endoscopy using fiber speckle illumination

NASA Astrophysics Data System (ADS)

Nakano, Shuhei; Katagiri, Takashi; Matsuura, Yuji

2018-02-01

An endoscopic fluorescence imaging system based on fiber speckle illumination is proposed. In this system, a multimode fiber for transmission of excitation laser light and collection of fluorescence is inserted into a conventional flexible endoscope. Since the excitation laser light has random speckle structure, one can detect fluorescence signal corresponding to the irradiation pattern if the sample contains fluorophores. The irradiation pattern can be captured by the endoscope camera when the excitation wavelength is within the sensitivity range of the camera. By performing multiple measurements while changing the irradiation pattern, a fluorescence image is reconstructed by solving a norm minimization problem. The principle of our method was experimentally demonstrated. A 2048 pixels image of quantum dots coated on a frosted glass was successfully reconstructed by 32 measurements. We also confirmed that our method can be applied on biological tissues.

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.

Optimized optical clearing method for imaging central nervous system

NASA Astrophysics Data System (ADS)

Yu, Tingting; Qi, Yisong; Gong, Hui; Luo, Qingming; Zhu, Dan

2015-03-01

The development of various optical clearing methods provides a great potential for imaging entire central nervous system by combining with multiple-labelling and microscopic imaging techniques. These methods had made certain clearing contributions with respective weaknesses, including tissue deformation, fluorescence quenching, execution complexity and antibody penetration limitation that makes immunostaining of tissue blocks difficult. The passive clarity technique (PACT) bypasses those problems and clears the samples with simple implementation, excellent transparency with fine fluorescence retention, but the passive tissue clearing method needs too long time. In this study, we not only accelerate the clearing speed of brain blocks but also preserve GFP fluorescence well by screening an optimal clearing temperature. The selection of proper temperature will make PACT more applicable, which evidently broaden the application range of this method.

Multiresolution multiscale active mask segmentation of fluorescence microscope images

NASA Astrophysics Data System (ADS)

Srinivasa, Gowri; Fickus, Matthew; Kovačević, Jelena

2009-08-01

We propose an active mask segmentation framework that combines the advantages of statistical modeling, smoothing, speed and flexibility offered by the traditional methods of region-growing, multiscale, multiresolution and active contours respectively. At the crux of this framework is a paradigm shift from evolving contours in the continuous domain to evolving multiple masks in the discrete domain. Thus, the active mask framework is particularly suited to segment digital images. We demonstrate the use of the framework in practice through the segmentation of punctate patterns in fluorescence microscope images. Experiments reveal that statistical modeling helps the multiple masks converge from a random initial configuration to a meaningful one. This obviates the need for an involved initialization procedure germane to most of the traditional methods used to segment fluorescence microscope images. While we provide the mathematical details of the functions used to segment fluorescence microscope images, this is only an instantiation of the active mask framework. We suggest some other instantiations of the framework to segment different types of images.

Adaptive optical fluorescence microscopy.

PubMed

Ji, Na

2017-03-31

The past quarter century has witnessed rapid developments of fluorescence microscopy techniques that enable structural and functional imaging of biological specimens at unprecedented depth and resolution. The performance of these methods in multicellular organisms, however, is degraded by sample-induced optical aberrations. Here I review recent work on incorporating adaptive optics, a technology originally applied in astronomical telescopes to combat atmospheric aberrations, to improve image quality of fluorescence microscopy for biological imaging.

Indocyanine green fluorescence imaging in hepatobiliary surgery.

PubMed

Majlesara, Ali; Golriz, Mohammad; Hafezi, Mohammadreza; Saffari, Arash; Stenau, Esther; Maier-Hein, Lena; Müller-Stich, Beat P; Mehrabi, Arianeb

2017-03-01

Indocyanine green (ICG) is a fluorescent dye that has been widely used for fluorescence imaging during hepatobiliary surgery. ICG is injected intravenously, selectively taken up by the liver, and then secreted into the bile. The catabolism and fluorescence properties of ICG permit a wide range of visualization methods in hepatobiliary surgery. We have characterized the applications of ICG during hepatobiliary surgery into: 1) liver mapping, 2) cholangiography, 3) tumor visualization, and 4) partial liver graft evaluation. In this literature review, we summarize the current understanding of ICG use during hepatobiliary surgery. Intra-operative ICG fluorescence imaging is a safe, simple, and feasible method that improves the visualization of hepatobiliary anatomy and liver tumors. Intravenous administration of ICG is not toxic and avoids the drawbacks of conventional imaging. In addition, it reduces post-operative complications without any known side effects. ICG fluorescence imaging provides a safe and reliable contrast for extra-hepatic cholangiography when detecting intra-hepatic bile leakage following liver resection. In addition, liver tumors can be visualized and well-differentiated hepatocellular carcinoma tumors can be accurately identified. Moreover, vascular reconstruction and outflow can be evaluated following partial liver transplantation. However, since tissue penetration is limited to 5-10mm, deeper tissue cannot be visualized using this method. Many instances of false positive or negative results have been reported, therefore further characterization is required. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous fluorescence and quantitative phase microscopy with single-pixel detectors

NASA Astrophysics Data System (ADS)

Liu, Yang; Suo, Jinli; Zhang, Yuanlong; Dai, Qionghai

2018-02-01

Multimodal microscopy offers high flexibilities for biomedical observation and diagnosis. Conventional multimodal approaches either use multiple cameras or a single camera spatially multiplexing different modes. The former needs expertise demanding alignment and the latter suffers from limited spatial resolution. Here, we report an alignment-free full-resolution simultaneous fluorescence and quantitative phase imaging approach using single-pixel detectors. By combining reference-free interferometry with single-pixel detection, we encode the phase and fluorescence of the sample in two detection arms at the same time. Then we employ structured illumination and the correlated measurements between the sample and the illuminations for reconstruction. The recovered fluorescence and phase images are inherently aligned thanks to single-pixel detection. To validate the proposed method, we built a proof-of-concept setup for first imaging the phase of etched glass with the depth of a few hundred nanometers and then imaging the fluorescence and phase of the quantum dot drop. This method holds great potential for multispectral fluorescence microscopy with additional single-pixel detectors or a spectrometer. Besides, this cost-efficient multimodal system might find broad applications in biomedical science and neuroscience.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

PubMed

Zhao, Ming; Li, Yu; Peng, Leilei

2014-05-05

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

Simple and rapid determination of homozygous transgenic mice via in vivo fluorescence imaging.

PubMed

Lin, Xiaolin; Jia, Junshuang; Qin, Yujuan; Lin, Xia; Li, Wei; Xiao, Gaofang; Li, Yanqing; Xie, Raoying; Huang, Hailu; Zhong, Lin; Wu, Qinghong; Wang, Wanshan; Huang, Wenhua; Yao, Kaitai; Xiao, Dong; Sun, Yan

2015-11-17

Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study.

Simple and rapid determination of homozygous transgenic mice via in vivo fluorescence imaging

PubMed Central

Li, Wei; Xiao, Gaofang; Li, Yanqing; Xie, Raoying; Huang, Hailu; Zhong, Lin; Wu, Qinghong; Wang, Wanshan; Huang, Wenhua; Yao, Kaitai; Xiao, Dong; Sun, Yan

2015-01-01

Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study. PMID:26472024

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.

A method for optical imaging and monitoring of the excretion of fluorescent nanocomposites from the body using artificial neural networks.

PubMed

Sarmanova, Olga E; Burikov, Sergey A; Dolenko, Sergey A; Isaev, Igor V; Laptinskiy, Kirill A; Prabhakar, Neeraj; Karaman, Didem Şen; Rosenholm, Jessica M; Shenderova, Olga A; Dolenko, Tatiana A

2018-04-12

In this study, a new approach to the implementation of optical imaging of fluorescent nanoparticles in a biological medium using artificial neural networks is proposed. The studies were carried out using new synthesized nanocomposites - nanometer graphene oxides, covered by the poly(ethylene imine)-poly(ethylene glycol) copolymer and by the folic acid. We present an example of a successful solution of the problem of monitoring the removal of nanocomposites based on nGO and their components with urine using fluorescent spectroscopy and artificial neural networks. However, the proposed method is applicable for optical imaging of any fluorescent nanoparticles used as theranostic agents in biological tissue. Copyright © 2018. Published by Elsevier Inc.

GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method

PubMed Central

Kim, Byungyeon; Park, Byungjun; Lee, Seungrag; Won, Youngjae

2016-01-01

We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection. PMID:28018724

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.

GPU-Accelerated Hybrid Algorithm for 3D Localization of Fluorescent Emitters in Dense Clusters

NASA Astrophysics Data System (ADS)

Jung, Yoon; Barsic, Anthony; Piestun, Rafael; Fakhri, Nikta

In stochastic switching-based super-resolution imaging, a random subset of fluorescent emitters are imaged and localized for each frame to construct a single high resolution image. However, the condition of non-overlapping point spread functions (PSFs) imposes constraints on experimental parameters. Recent development in post processing methods such as dictionary-based sparse support recovery using compressive sensing has shown up to an order of magnitude higher recall rate than single emitter fitting methods. However, the computational complexity of this approach scales poorly with the grid size and requires long runtime. Here, we introduce a fast and accurate compressive sensing algorithm for localizing fluorescent emitters in high density in 3D, namely sparse support recovery using Orthogonal Matching Pursuit (OMP) and L1-Homotopy algorithm for reconstructing STORM images (SOLAR STORM). SOLAR STORM combines OMP with L1-Homotopy to reduce computational complexity, which is further accelerated by parallel implementation using GPUs. This method can be used in a variety of experimental conditions for both in vitro and live cell fluorescence imaging.

Hyper-spectral imaging in scanning-confocal-fluorescence microscopy using a novel broadband diffractive optic

NASA Astrophysics Data System (ADS)

Wang, Peng; Ebeling, Carl G.; Gerton, Jordan; Menon, Rajesh

In this paper, we demonstrate hyper-spectral imaging of fluorescent microspheres in a scanning-confocal-fluorescence microscope by spatially dispersing the spectra using a novel broadband diffractive optic, and applying a nonlinear optimization technique to extract the full-incident spectra. This broadband diffractive optic has a designed optical efficiency of over 90% across the entire visible spectrum. We used this technique to create two-color images of two fluorophores and also extracted their emission spectra with good fidelity. This method can be extended to image both spatially and spectrally overlapping fluorescent samples. Full control in the number of emission spectra and the feasibility of enhanced imaging speed are demonstrated as well.

Fluorescence imaging host pathogen interactions: fifteen years benefit of hindsight….

PubMed

Aulner, Nathalie; Danckaert, Anne; Fernandes, Julien; Nicola, Marie-Anne; Roux, Pascal; Salles, Audrey; Tinevez, Jean-Yves; Shorte, Spencer L

2018-03-19

We consider in review current state-of-the-art fluorescence microscopy for investigating the host-pathogen interface. Our perspective is honed from years with literally thousands of microbiologists using the variety of imaging technologies available within our dedicated BSL2/BSL3 optical imaging research service facilities at the Institut Pasteur Paris founded from scratch in 2001. During fifteen years learning from the success and failures of introducing different fluorescence imaging technologies, methods, and technical development strategies we provide here a synopsis review of our experience to date and a synthesis of how we see the future in perspective for fluorescence imaging at the host-pathogen interface. Copyright © 2018. Published by Elsevier Ltd.

Extraction of the number of peroxisomes in yeast cells by automated image analysis.

PubMed

Niemistö, Antti; Selinummi, Jyrki; Saleem, Ramsey; Shmulevich, Ilya; Aitchison, John; Yli-Harja, Olli

2006-01-01

An automated image analysis method for extracting the number of peroxisomes in yeast cells is presented. Two images of the cell population are required for the method: a bright field microscope image from which the yeast cells are detected and the respective fluorescent image from which the number of peroxisomes in each cell is found. The segmentation of the cells is based on clustering the local mean-variance space. The watershed transformation is thereafter employed to separate cells that are clustered together. The peroxisomes are detected by thresholding the fluorescent image. The method is tested with several images of a budding yeast Saccharomyces cerevisiae population, and the results are compared with manually obtained results.

High-speed bioimaging with frequency-division-multiplexed fluorescence confocal microscopy

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Harmon, Jeffrey; Ozeki, Yasuyuki; Goda, Keisuke

2017-04-01

We present methods of fluorescence confocal microscopy that enable unprecedentedly high frame rate of > 10,000 fps. The methods are based on a frequency-division multiplexing technique, which was originally developed in the field of communication engineering. Specifically, we achieved a broad bandwidth ( 400 MHz) of detection signals using a dual- AOD method and overcame limitations in frame rate, due to a scanning device, by using a multi-line focusing method, resulting in a significant increase in frame rate. The methods have potential biomedical applications such as observation of sub-millisecond dynamics in biological tissues, in-vivo three-dimensional imaging, and fluorescence imaging flow cytometry.

Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

PubMed Central

Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

2016-01-01

We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

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.

Ultrafast Method for the Analysis of Fluorescence Lifetime Imaging Microscopy Data Based on the Laguerre Expansion Technique

PubMed Central

Jo, Javier A.; Fang, Qiyin; Marcu, Laura

2007-01-01

We report a new deconvolution method for fluorescence lifetime imaging microscopy (FLIM) based on the Laguerre expansion technique. The performance of this method was tested on synthetic and real FLIM images. The following interesting properties of this technique were demonstrated. 1) The fluorescence intensity decay can be estimated simultaneously for all pixels, without a priori assumption of the decay functional form. 2) The computation speed is extremely fast, performing at least two orders of magnitude faster than current algorithms. 3) The estimated maps of Laguerre expansion coefficients provide a new domain for representing FLIM information. 4) The number of images required for the analysis is relatively small, allowing reduction of the acquisition time. These findings indicate that the developed Laguerre expansion technique for FLIM analysis represents a robust and extremely fast deconvolution method that enables practical applications of FLIM in medicine, biology, biochemistry, and chemistry. PMID:19444338

Double-excitation fluorescence spectral imaging: eliminating tissue auto-fluorescence from in vivo PPIX measurements

NASA Astrophysics Data System (ADS)

Torosean, Sason; Flynn, Brendan; Samkoe, Kimberley S.; Davis, Scott C.; Gunn, Jason; Axelsson, Johan; Pogue, Brian W.

2012-02-01

An ultrasound coupled handheld-probe-based optical fluorescence molecular tomography (FMT) system has been in development for the purpose of quantifying the production of Protoporphyrin IX (PPIX) in aminolevulinic acid treated (ALA), Basal Cell Carcinoma (BCC) in vivo. The design couples fiber-based spectral sampling of PPIX fluorescence emission with a high frequency ultrasound imaging system, allowing regionally localized fluorescence intensities to be quantified [1]. The optical data are obtained by sequential excitation of the tissue with a 633nm laser, at four source locations and five parallel detections at each of the five interspersed detection locations. This method of acquisition permits fluorescence detection for both superficial and deep locations in ultrasound field. The optical boundary data, tissue layers segmented from ultrasound image and diffusion theory are used to estimate the fluorescence in tissue layers. To improve the recovery of the fluorescence signal of PPIX, eliminating tissue autofluorescence is of great importance. Here the approach was to utilize measurements which straddled the steep Qband excitation peak of PPIX, via the integration of an additional laser source, exciting at 637 nm; a wavelength with a 2 fold lower PPIX excitation value than 633nm.The auto-fluorescence spectrum acquired from the 637 nm laser is then used to spectrally decouple the fluorescence data and produce an accurate fluorescence emission signal, because the two wavelengths have very similar auto-fluorescence but substantially different PPIX excitation levels. The accuracy of this method, using a single source detector pair setup, is verified through animal tumor model experiments, and the result is compared to different methods of fluorescence signal recovery.

Example-Based Super-Resolution Fluorescence Microscopy.

PubMed

Jia, Shu; Han, Boran; Kutz, J Nathan

2018-04-23

Capturing biological dynamics with high spatiotemporal resolution demands the advancement in imaging technologies. Super-resolution fluorescence microscopy offers spatial resolution surpassing the diffraction limit to resolve near-molecular-level details. While various strategies have been reported to improve the temporal resolution of super-resolution imaging, all super-resolution techniques are still fundamentally limited by the trade-off associated with the longer image acquisition time that is needed to achieve higher spatial information. Here, we demonstrated an example-based, computational method that aims to obtain super-resolution images using conventional imaging without increasing the imaging time. With a low-resolution image input, the method provides an estimate of its super-resolution image based on an example database that contains super- and low-resolution image pairs of biological structures of interest. The computational imaging of cellular microtubules agrees approximately with the experimental super-resolution STORM results. This new approach may offer potential improvements in temporal resolution for experimental super-resolution fluorescence microscopy and provide a new path for large-data aided biomedical 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

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

Simultaneous AFM and fluorescence imaging: A method for aligning an AFM-tip with an excitation beam using a 2D galvanometer

NASA Astrophysics Data System (ADS)

Moores, A. N.; Cadby, A. J.

2018-02-01

Correlative fluorescence and atomic force microscopy (AFM) imaging is a highly attractive technique for use in biological imaging, enabling force and mechanical measurements of particular structures whose locations are known due to the specificity of fluorescence imaging. The ability to perform these two measurements simultaneously (rather than consecutively with post-processing correlation) is highly valuable because it would allow the mechanical properties of a structure to be tracked over time as changes in the sample occur. We present an instrument which allows simultaneous AFM and fluorescence imaging by aligning an incident fluorescence excitation beam with an AFM-tip. Alignment was performed by calibrating a 2D galvanometer present in the excitation beam path and using it to reposition the incident beam. Two programs were developed (one manual and one automated) which correlate sample features between the AFM and fluorescence images, calculating the distance required to translate the incident beam towards the AFM-tip. Using this method, we were able to obtain beam-tip alignment (and therefore field-of-view alignment) from an offset of >15 μm to within one micron in two iterations of the program. With the program running alongside data acquisition for real-time feedback between AFM and optical images, this offset was maintained over a time period of several hours. Not only does this eliminate the need to image large areas with both techniques to ensure that fields-of-view overlap, but it also raises the possibility of using this instrument for tip-enhanced fluorescence applications, a technique in which super-resolution images have previously been achieved.

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.

New method of acne disease fluorescent diagnostics in natural and fluorescent light and treatment control

NASA Astrophysics Data System (ADS)

Karimova, L. N.; Berezin, A. N.; Shevchik, S. A.; Kharnas, S. S.; Kusmin, S. G.; Loschenov, V. B.

2005-08-01

In the given research the new method of fluorescent diagnostics (FD) and photodynamic therapy (PDT) control of acne disease is submitted. Method is based on simultaneous diagnostics in natural and fluorescent light. PDT was based on using 5-ALA (5- aminolevulinic acid) preparation and 600-730 nanometers radiation. If the examined site of a skin possessed a high endogenous porphyrin fluorescence level, PDT was carried out without 5-ALA. For FD and treatment control a dot spectroscopy and the fluorescent imaging of the affected skin were used.

Multimodality molecular imaging and extracellular vesicle release based genetic profiling with porphyrin nanodroplets (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zemp, Roger J.; Paproski, Robert J.

2017-03-01

For emerging tissue-engineering applications, transplants, and cell-based therapies it is important to assess cell viability and function in vivo in deep tissues. Bioluminescence and fluorescence methods are poorly suited to deep monitoring applications with high resolution and require genetically-engineered reporters which are not always feasible. We report on a method for imaging cell viability using deep, high-resolution photoacoustic imaging. We use an exogenous dye, Resazurin, itself weakly fluorescent until it is reduced from blue to a pink color with bright red fluorescence. Upon cell death fluorescence is lost and an absorption shift is observed. The irreversible reaction of resazurin to resorufin is proportional to aerobic respiration. We detect colorimetric absorption shifts using multispectral photoacoustic imaging and quantify the fraction of viable cells. SKOV-3 cells with and without ±80oC heat treatment were imaged after Resazurin treatment. High 575nm:620nm ratiometric absorption and photoacoustic signals in viable cells were observed with a much lower ratio in low-viability populations.

Diagnosis of basal cell carcinoma by two photon excited fluorescence combined with lifetime imaging

NASA Astrophysics Data System (ADS)

Fan, Shunping; Peng, Xiao; Liu, Lixin; Liu, Shaoxiong; Lu, Yuan; Qu, Junle

2014-02-01

Basal cell carcinoma (BCC) is the most common type of human skin cancer. The traditional diagnostic procedure of BCC is histological examination with haematoxylin and eosin staining of the tissue biopsy. In order to reduce complexity of the diagnosis procedure, a number of noninvasive optical methods have been applied in skin examination, for example, multiphoton tomography (MPT) and fluorescence lifetime imaging microscopy (FLIM). In this study, we explored two-photon optical tomography of human skin specimens using two-photon excited autofluorescence imaging and FLIM. There are a number of naturally endogenous fluorophores in skin sample, such as keratin, melanin, collagen, elastin, flavin and porphyrin. Confocal microscopy was used to obtain structures of the sample. Properties of epidermic and cancer cells were characterized by fluorescence emission spectra, as well as fluorescence lifetime imaging. Our results show that two-photon autofluorescence lifetime imaging can provide accurate optical biopsies with subcellular resolution and is potentially a quantitative optical diagnostic method in skin cancer diagnosis.

Label-free imaging and spectroscopy for early detection of cervical cancer.

PubMed

Jing, Yueyue; Wang, Yulan; Wang, Xinyi; Song, Chuan; Ma, Jiong; Xie, Yonghui; Fei, Yiyan; Zhang, Qinghua; Mi, Lan

2018-05-01

The label-free imaging and spectroscopy method was studied on cervical unstained tissue sections obtained from 36 patients. The native fluorescence spectra of tissues are analyzed by the optical redox ratio (ORR), which is defined as fluorescence intensity ratio between NADH and FAD, and indicates the metabolism change with the cancer development. The ORRs of normal tissues are consistently higher than those of precancer or cancerous tissues. A criterion line of ORR at 5.0 can be used to discriminate cervical precancer/cancer from normal tissues. The sensitivity and specificity of the native fluorescence spectroscopy method for cervical cancer diagnosis are determined as 100% and 91%. Moreover, the native fluorescence spectroscopy study is much more sensitive on the healthy region of cervical precancer/cancer patients compared with the traditional clinical staining method. The results suggest label-free imaging and spectroscopy is a fast, highly sensitive and specific method on the detection of cervical cancer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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.

Electron-Beam Diagnostic Methods for Hypersonic Flow Diagnostics

NASA Technical Reports Server (NTRS)

1994-01-01

The purpose of this work was the evaluation of the use of electron-bean fluorescence for flow measurements during hypersonic flight. Both analytical and numerical models were developed in this investigation to evaluate quantitatively flow field imaging concepts based upon the electron beam fluorescence technique for use in flight research and wind tunnel applications. Specific models were developed for: (1) fluorescence excitation/emission for nitrogen, (2) rotational fluorescence spectrum for nitrogen, (3) single and multiple scattering of electrons in a variable density medium, (4) spatial and spectral distribution of fluorescence, (5) measurement of rotational temperature and density, (6) optical filter design for fluorescence imaging, and (7) temperature accuracy and signal acquisition time requirements. Application of these models to a typical hypersonic wind tunnel flow is presented. In particular, the capability of simulating the fluorescence resulting from electron impact ionization in a variable density nitrogen or air flow provides the capability to evaluate the design of imaging instruments for flow field mapping. The result of this analysis is a recommendation that quantitative measurements of hypersonic flow fields using electron-bean fluorescence is a tractable method with electron beam energies of 100 keV. With lower electron energies, electron scattering increases with significant beam divergence which makes quantitative imaging difficult. The potential application of the analytical and numerical models developed in this work is in the design of a flow field imaging instrument for use in hypersonic wind tunnels or onboard a flight research vehicle.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging

PubMed Central

Zhao, Ming; Li, Yu; Peng, Leilei

2014-01-01

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community. PMID:24921725

Simultaneous dual-color fluorescence microscope: a characterization study.

PubMed

Li, Zheng; Chen, Xiaodong; Ren, Liqiang; Song, Jie; Li, Yuhua; Zheng, Bin; Liu, Hong

2013-01-01

High spatial resolution and geometric accuracy is crucial for chromosomal analysis of clinical cytogenetic applications. High resolution and rapid simultaneous acquisition of multiple fluorescent wavelengths can be achieved by utilizing concurrent imaging with multiple detectors. However, such class of microscopic systems functions differently from traditional fluorescence microscopes. To develop a practical characterization framework to assess and optimize the performance of a high resolution and dual-color fluorescence microscope designed for clinical chromosomal analysis. A dual-band microscopic imaging system utilizes a dichroic mirror, two sets of specially selected optical filters, and two detectors to simultaneously acquire two fluorescent wavelengths. The system's geometric distortion, linearity, the modulation transfer function, and the dual detectors' alignment were characterized. Experiment results show that the geometric distortion at lens periphery is less than 1%. Both fluorescent channels show linear signal responses, but there exists discrepancy between the two due to the detectors' non-uniform response ratio to different wavelengths. In terms of the spatial resolution, the two contrast transfer function curves trend agreeably with the spatial frequency. The alignment measurement allows quantitatively assessing the cameras' alignment. A result image of adjusted alignment is demonstrated to show the reduced discrepancy by using the alignment measurement method. In this paper, we present a system characterization study and its methods for a specially designed imaging system for clinical cytogenetic applications. The presented characterization methods are not only unique to this dual-color imaging system but also applicable to evaluation and optimization of other similar multi-color microscopic image systems for improving their clinical utilities for future cytogenetic applications.

Detection of melanomas by digital imaging of spectrally resolved UV light-induced autofluorescence of human skin

NASA Astrophysics Data System (ADS)

Chwirot, B. W.; Chwirot, S.; Jedrzejczyk, W.; Redzinski, J.; Raczynska, A. M.; Telega, K.

2001-07-01

We studied spectral and spatial distributions of the intensity of the ultraviolet light-excited fluorescence of human skin. Our studied performed in situ in 162 patients with malignant and non-malignant skin lesions resulted in a new method of detecting melanomas in situ using digital imaging of the spectrally resolved fluorescence. With our diagnostic algorithm we could successfully detect 88.5% of the cases of melanoma in the group of patients subject to examinations with the fluorescence method. A patent application for the method has been submitted to the Patent Office in Warsaw.

Statistical Deconvolution for Superresolution Fluorescence Microscopy

PubMed Central

Mukamel, Eran A.; Babcock, Hazen; Zhuang, Xiaowei

2012-01-01

Superresolution microscopy techniques based on the sequential activation of fluorophores can achieve image resolution of ∼10 nm but require a sparse distribution of simultaneously activated fluorophores in the field of view. Image analysis procedures for this approach typically discard data from crowded molecules with overlapping images, wasting valuable image information that is only partly degraded by overlap. A data analysis method that exploits all available fluorescence data, regardless of overlap, could increase the number of molecules processed per frame and thereby accelerate superresolution imaging speed, enabling the study of fast, dynamic biological processes. Here, we present a computational method, referred to as deconvolution-STORM (deconSTORM), which uses iterative image deconvolution in place of single- or multiemitter localization to estimate the sample. DeconSTORM approximates the maximum likelihood sample estimate under a realistic statistical model of fluorescence microscopy movies comprising numerous frames. The model incorporates Poisson-distributed photon-detection noise, the sparse spatial distribution of activated fluorophores, and temporal correlations between consecutive movie frames arising from intermittent fluorophore activation. We first quantitatively validated this approach with simulated fluorescence data and showed that deconSTORM accurately estimates superresolution images even at high densities of activated fluorophores where analysis by single- or multiemitter localization methods fails. We then applied the method to experimental data of cellular structures and demonstrated that deconSTORM enables an approximately fivefold or greater increase in imaging speed by allowing a higher density of activated fluorophores/frame. PMID:22677393

UV fluorescence excitation imaging of healing of wounds in skin: Evaluation of wound closure in organ culture model.

PubMed

Wang, Ying; Gutierrez-Herrera, Enoch; Ortega-Martinez, Antonio; Anderson, Richard Rox; Franco, Walfre

2016-09-01

Molecules native to tissue that fluoresce upon light excitation can serve as reporters of cellular activity and protein structure. In skin, the fluorescence ascribed to tryptophan is a marker of cellular proliferation, whereas the fluorescence ascribed to cross-links of collagen is a structural marker. In this work, we introduce and demonstrate a simple but robust optical method to image the functional process of epithelialization and the exposed dermal collagen in wound healing of human skin in an organ culture model. Non-closing non-grafted, partial closing non-grafted, and grafted wounds were created in ex vivo human skin and kept in culture. A wide-field UV fluorescence excitation imaging system was used to visualize epithelialization of the exposed dermis and quantitate wound area, closure, and gap. Histology (H&E staining) was also used to evaluate epithelialization. The endogenous fluorescence excitation of cross-links of collagen at 335 nm clearly shows the dermis missing epithelium, while the endogenous fluorescence excitation of tryptophan at 295 nm shows keratinocytes in higher proliferating state. The size of the non-closing wound was 11.4 ± 1.8 mm and remained constant during the observation period, while the partial-close wound reached 65.5 ± 4.9% closure by day 16. Evaluations of wound gaps using fluorescence excitation images and histology images are in agreement. We have established a fluorescence imaging method for studying epithelialization processes, evaluating keratinocyte proliferation, and quantitating closure during wound healing of skin in an organ culture model: the dermal fluorescence of pepsin-digestible collagen cross-links can be used to quantitate wound size, closure extents, and gaps; and, the epidermal fluorescence ascribed to tryptophan can be used to monitor and quantitate functional states of epithelialization. UV fluorescence excitation imaging has the potential to become a valuable tool for research, diagnostic and educational purposes on evaluating the healing of wounds. Lasers Surg. Med. 48:678-685, 2016. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

A Model-Based Approach for Microvasculature Structure Distortion Correction in Two-Photon Fluorescence Microscopy Images

PubMed Central

Dao, Lam; Glancy, Brian; Lucotte, Bertrand; Chang, Lin-Ching; Balaban, Robert S; Hsu, Li-Yueh

2015-01-01

SUMMARY This paper investigates a post-processing approach to correct spatial distortion in two-photon fluorescence microscopy images for vascular network reconstruction. It is aimed at in vivo imaging of large field-of-view, deep-tissue studies of vascular structures. Based on simple geometric modeling of the object-of-interest, a distortion function is directly estimated from the image volume by deconvolution analysis. Such distortion function is then applied to sub volumes of the image stack to adaptively adjust for spatially varying distortion and reduce the image blurring through blind deconvolution. The proposed technique was first evaluated in phantom imaging of fluorescent microspheres that are comparable in size to the underlying capillary vascular structures. The effectiveness of restoring three-dimensional spherical geometry of the microspheres using the estimated distortion function was compared with empirically measured point-spread function. Next, the proposed approach was applied to in vivo vascular imaging of mouse skeletal muscle to reduce the image distortion of the capillary structures. We show that the proposed method effectively improve the image quality and reduce spatially varying distortion that occurs in large field-of-view deep-tissue vascular dataset. The proposed method will help in qualitative interpretation and quantitative analysis of vascular structures from fluorescence microscopy images. PMID:26224257

Hybrid Imaging Labels: Providing the Link Between Mass Spectrometry-Based Molecular Pathology and Theranostics

PubMed Central

Buckle, Tessa; van der Wal, Steffen; van Malderen, Stijn J.M.; Müller, Larissa; Kuil, Joeri; van Unen, Vincent; Peters, Ruud J.B.; van Bemmel, Margaretha E.M.; McDonnell, Liam A.; Velders, Aldrik H.; Koning, Frits; Vanhaeke, Frank; van Leeuwen, Fijs W. B.

2017-01-01

Background: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. Methods: A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5). Results: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging. Conclusion: Lanthanide-isotope chelation expands the scope of fluorescent/radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods. PMID:28255355

A matter of collection and detection for intraoperative and noninvasive near-infrared fluorescence molecular imaging: To see or not to see?

PubMed Central

Zhu, Banghe; Rasmussen, John C.; Sevick-Muraca, Eva M.

2014-01-01

Purpose: Although fluorescence molecular imaging is rapidly evolving as a new combinational drug/device technology platform for molecularly guided surgery and noninvasive imaging, there remains no performance standards for efficient translation of “first-in-humans” fluorescent imaging agents using these devices. Methods: The authors employed a stable, solid phantom designed to exaggerate the confounding effects of tissue light scattering and to mimic low concentrations (nM–pM) of near-infrared fluorescent dyes expected clinically for molecular imaging in order to evaluate and compare the commonly used charge coupled device (CCD) camera systems employed in preclinical studies and in human investigational studies. Results: The results show that intensified CCD systems offer greater contrast with larger signal-to-noise ratios in comparison to their unintensified CCD systems operated at clinically reasonable, subsecond acquisition times. Conclusions: Camera imaging performance could impact the success of future “first-in-humans” near-infrared fluorescence imaging agent studies. PMID:24506637

A matter of collection and detection for intraoperative and noninvasive near-infrared fluorescence molecular imaging: To see or not to see?

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

Zhu, Banghe; Rasmussen, John C.; Sevick-Muraca, Eva M., E-mail: Eva.Sevick@uth.tmc.edu

2014-02-15

Purpose: Although fluorescence molecular imaging is rapidly evolving as a new combinational drug/device technology platform for molecularly guided surgery and noninvasive imaging, there remains no performance standards for efficient translation of “first-in-humans” fluorescent imaging agents using these devices. Methods: The authors employed a stable, solid phantom designed to exaggerate the confounding effects of tissue light scattering and to mimic low concentrations (nM–pM) of near-infrared fluorescent dyes expected clinically for molecular imaging in order to evaluate and compare the commonly used charge coupled device (CCD) camera systems employed in preclinical studies and in human investigational studies. Results: The results show thatmore » intensified CCD systems offer greater contrast with larger signal-to-noise ratios in comparison to their unintensified CCD systems operated at clinically reasonable, subsecond acquisition times. Conclusions: Camera imaging performance could impact the success of future “first-in-humans” near-infrared fluorescence imaging agent studies.« less

The use of in vivo fluorescence image sequences to indicate the occurrence and propagation of transient focal depolarizations in cerebral ischemia.

PubMed

Strong, A J; Harland, S P; Meldrum, B S; Whittington, D J

1996-05-01

A method for the detection and tracking of propagated fluorescence transients as indicators of depolarizations in focal cerebral ischemia is described, together with initial results indicating the potential of the method. The cortex of the right cerebral hemisphere was exposed for nonrecovery experiments in five cats anesthetized with chloralose and subjected to permanent middle cerebral artery (MCA) occlusion. Fluorescence with 370-nm excitation (attributed to the degree of reduction of the NAD/H couple) was imaged with an intensified charge-coupled device camera and digitized. Sequences of images representing changes in gray level from a baseline image were examined, together with the time courses of mean gray levels in specified regions of interest. Spontaneous increases in fluorescence occurred, starting most commonly at the edge of areas of core ischemia; they propagated usually throughout the periinfarct zone and resolved to varying degrees and at varying rates, depending on proximity of the locus to the MCA input. When a fluorescence transient reached the anterior cerebral artery territory, its initial polarity reversed from an increase to a decrease in fluorescence. An initial increase in fluorescence in response to the arrival of a transient may characterize cortex that will become infarcted, if pathophysiological changes in the periinfarct zone are allowed to evolve naturally.

Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

PubMed

Bremer, Daniel; Leben, Ruth; Mothes, Ronja; Radbruch, Helena; Niesner, Raluca

2017-04-03

Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Synthesis of Water-Dispersible Mn2+ Functionalized Silicon Nanoparticles under Room Temperature and Atmospheric Pressure for Fluorescence and Magnetic Resonance Dual-Modality Imaging.

PubMed

Dou, Ya-Kun; Chen, Yang; He, Xi-Wen; Li, Wen-You; Li, Yu-Hao; Zhang, Yu-Kui

2017-11-07

Silicon nanoparticles (Si NPs) have been widely used in fluorescence imaging. However, rigorous synthesis conditions and the single modality imaging limit the further development of Si NPs in the field of biomedical imaging. Here, we reported a method for synthesizing water-dispersible Mn 2+ functionalized Si NPs (Mn-Si NPs) under mild experimental conditions for fluorescence and magnetic resonance dual-modality imaging. The whole synthesis process was completed under room temperature and atmospheric pressure, and no special and expensive equipment was required. The synthetic nanoparticles, with favorable pH stability, NaCl stability, photostability, and low toxicity, emitted green fluorescence (512 nm). At the same time, the nanoparticles also demonstrated excellent magnetic resonance imaging ability. In vitro, their T 1 -weighted magnetic resonance imaging effect was obvious, and the value of longitudinal relaxation degree r 1 reached 4.25 mM -1 s -1 . On the basis of their good biocompatibility, Mn-Si NPs were successfully used for the fluorescence imaging as well as magnetic resonance imaging in vivo.

Multispectral Live-Cell Imaging.

PubMed

Cohen, Sarah; Valm, Alex M; Lippincott-Schwartz, Jennifer

2018-06-01

Fluorescent proteins and vital dyes are invaluable tools for studying dynamic processes within living cells. However, the ability to distinguish more than a few different fluorescent reporters in a single sample is limited by the spectral overlap of available fluorophores. Here, we present a protocol for imaging live cells labeled with six fluorophores simultaneously. A confocal microscope with a spectral detector is used to acquire images, and linear unmixing algorithms are applied to identify the fluorophores present in each pixel of the image. We describe the application of this method to visualize the dynamics of six different organelles, and to quantify the contacts between organelles. However, this method can be used to image any molecule amenable to tagging with a fluorescent probe. Thus, multispectral live-cell imaging is a powerful tool for systems-level analysis of cellular organization and dynamics. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

A simple one-step method to prepare fluorescent carbon dots and their potential application in non-invasive glioma imaging

NASA Astrophysics Data System (ADS)

Ruan, Shaobo; Qian, Jun; Shen, Shun; Zhu, Jianhua; Jiang, Xinguo; He, Qin; Gao, Huile

2014-08-01

Fluorescent carbon dots (CD) possess impressive potential in bioimaging because of their low photobleaching, absence of optical blinking and good biocompatibility. However, their relatively short excitation/emission wavelengths restrict their application in in vivo imaging. In the present study, a kind of CD was prepared by a simple heat treatment method using glycine as the only precursor. The diameter of CD was lower than 5 nm, and the highest emission wavelength was 500 nm. However, at 600 nm, there was still a relatively strong fluorescent emission, suggesting CD could be used for in vivo imaging. Additionally, several experiments demonstrated that CD possessed good serum stability and low cytotoxicity. In vitro, CD could be taken up into C6 glioma cells in a time- and concentration-dependent manner, with both endosomes and mitochondria involved. In vivo, CD could be used for non-invasive glioma imaging because of its high accumulation in the glioma site of the brain, which was demonstrated by both in vivo imaging and ex vivo tissue imaging. Furthermore, the fluorescent distribution in tissue slices also showed CD distributed in glioma with high intensity, while with a low intensity in normal brain tissue. In conclusion, CD were prepared using a simple method with relatively long excitation and emission wavelengths and could be used for non-invasive glioma imaging.Fluorescent carbon dots (CD) possess impressive potential in bioimaging because of their low photobleaching, absence of optical blinking and good biocompatibility. However, their relatively short excitation/emission wavelengths restrict their application in in vivo imaging. In the present study, a kind of CD was prepared by a simple heat treatment method using glycine as the only precursor. The diameter of CD was lower than 5 nm, and the highest emission wavelength was 500 nm. However, at 600 nm, there was still a relatively strong fluorescent emission, suggesting CD could be used for in vivo imaging. Additionally, several experiments demonstrated that CD possessed good serum stability and low cytotoxicity. In vitro, CD could be taken up into C6 glioma cells in a time- and concentration-dependent manner, with both endosomes and mitochondria involved. In vivo, CD could be used for non-invasive glioma imaging because of its high accumulation in the glioma site of the brain, which was demonstrated by both in vivo imaging and ex vivo tissue imaging. Furthermore, the fluorescent distribution in tissue slices also showed CD distributed in glioma with high intensity, while with a low intensity in normal brain tissue. In conclusion, CD were prepared using a simple method with relatively long excitation and emission wavelengths and could be used for non-invasive glioma imaging. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr02657h

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.

The combination design for open and endoscopic surgery using fluorescence molecular imaging technology

NASA Astrophysics Data System (ADS)

Mao, Yamin; Jiang, Shixin; Ye, Jinzuo; An, Yu; Yang, Xin; Chi, Chongwei; Tian, Jie

2015-03-01

For clinical surgery, it is still a challenge to objectively determine tumor margins during surgery. With the development of medical imaging technology, fluorescence molecular imaging (FMI) method can provide real-time intraoperative tumor margin information. Furthermore, surgical navigation system based on FMI technology plays an important role for the aid of surgeons' precise tumor margin decision. However, detection depth is the most limitation exists in the FMI technique and the method convenient for either macro superficial detection or micro deep tissue detection is needed. In this study, we combined advantages of both open surgery and endoscopic imaging systems with FMI technology. Indocyanine green (ICG) experiments were performed to confirm the feasibility of fluorescence detection in our system. Then, the ICG signal was photographed in the detection area with our system. When the system connected with endoscope lens, the minimum quantity of ICG detected by our system was 0.195 ug. For aspect of C mount lens, the sensitivity of ICG detection with our system was 0.195ug. Our experiments results proved that it was feasible to detect fluorescence images with this combination method. Our system shows great potential in the clinical applications of precise dissection of various tumors

Active mask segmentation of fluorescence microscope images.

PubMed

Srinivasa, Gowri; Fickus, Matthew C; Guo, Yusong; Linstedt, Adam D; Kovacević, Jelena

2009-08-01

We propose a new active mask algorithm for the segmentation of fluorescence microscope images of punctate patterns. It combines the (a) flexibility offered by active-contour methods, (b) speed offered by multiresolution methods, (c) smoothing offered by multiscale methods, and (d) statistical modeling offered by region-growing methods into a fast and accurate segmentation tool. The framework moves from the idea of the "contour" to that of "inside and outside," or masks, allowing for easy multidimensional segmentation. It adapts to the topology of the image through the use of multiple masks. The algorithm is almost invariant under initialization, allowing for random initialization, and uses a few easily tunable parameters. Experiments show that the active mask algorithm matches the ground truth well and outperforms the algorithm widely used in fluorescence microscopy, seeded watershed, both qualitatively, as well as quantitatively.

Rapid and prodium iodide-compatible optical clearing method for brain tissue based on sugar/sugar-alcohol

NASA Astrophysics Data System (ADS)

Yu, Tingting; Qi, Yisong; Wang, Jianru; Feng, Wei; Xu, Jianyi; Zhu, Jingtan; Yao, Yingtao; Gong, Hui; Luo, Qingming; Zhu, Dan

2016-08-01

The developed optical clearing methods show great potential for imaging of large-volume tissues, but these methods present some nonnegligible limitations such as complexity of implementation and long incubation times. In this study, we tried to screen out rapid optical clearing agents by means of molecular dynamical simulation and experimental demonstration. According to the optical clearing potential of sugar and sugar-alcohol, we further evaluated the improvement in the optical clearing efficacy of mouse brain samples, imaging depth, fluorescence preservation, and linear deformation. The results showed that drops of sorbitol, sucrose, and fructose could quickly make the mouse brain sample transparent within 1 to 2 min, and induce about threefold enhancement in imaging depth. The former two could evidently enhance the fluorescence intensity of green fluorescent protein (GFP) and prodium iodide (PI) nuclear dye. Fructose could significantly increase the fluorescence intensity of PI, but slightly decrease the fluorescence intensity of GFP. Even though the three agents caused some shrinkage in samples, the contraction in horizontal and longitudinal directions are almost the same.

A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images

PubMed Central

Afshar, Yaser; Sbalzarini, Ivo F.

2016-01-01

Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 1010 pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

PubMed

Afshar, Yaser; Sbalzarini, Ivo F

2016-01-01

Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10) pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments.

Imaging Dot Patterns for Measuring Gossamer Space Structures

NASA Technical Reports Server (NTRS)

Dorrington, A. A.; Danehy, P. M.; Jones, T. W.; Pappa, R. S.; Connell, J. W.

2005-01-01

A paper describes a photogrammetric method for measuring the changing shape of a gossamer (membrane) structure deployed in outer space. Such a structure is typified by a solar sail comprising a transparent polymeric membrane aluminized on its Sun-facing side and coated black on the opposite side. Unlike some prior photogrammetric methods, this method does not require an artificial light source or the attachment of retroreflectors to the gossamer structure. In a basic version of the method, the membrane contains a fluorescent dye, and the front and back coats are removed in matching patterns of dots. The dye in the dots absorbs some sunlight and fluoresces at a longer wavelength in all directions, thereby enabling acquisition of high-contrast images from almost any viewing angle. The fluorescent dots are observed by one or more electronic camera(s) on the Sun side, the shade side, or both sides. Filters that pass the fluorescent light and suppress most of the solar spectrum are placed in front of the camera(s) to increase the contrast of the dots against the background. The dot image(s) in the camera(s) are digitized, then processed by use of commercially available photogrammetric software.

Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology

NASA Astrophysics Data System (ADS)

Wei, Linpeng; Chen, Ye; Yin, Chengbo; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

2017-04-01

Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

Semi-automated discrimination of retinal pigmented epithelial cells in two-photon fluorescence images of mouse retinas.

PubMed

Alexander, Nathan S; Palczewska, Grazyna; Palczewski, Krzysztof

2015-08-01

Automated image segmentation is a critical step toward achieving a quantitative evaluation of disease states with imaging techniques. Two-photon fluorescence microscopy (TPM) has been employed to visualize the retinal pigmented epithelium (RPE) and provide images indicating the health of the retina. However, segmentation of RPE cells within TPM images is difficult due to small differences in fluorescence intensity between cell borders and cell bodies. Here we present a semi-automated method for segmenting RPE cells that relies upon multiple weak features that differentiate cell borders from the remaining image. These features were scored by a search optimization procedure that built up the cell border in segments around a nucleus of interest. With six images used as a test, our method correctly identified cell borders for 69% of nuclei on average. Performance was strongly dependent upon increasing retinosome content in the RPE. TPM image analysis has the potential of providing improved early quantitative assessments of diseases affecting the RPE.

Aluminum surface corrosion and the mechanism of inhibitors using pH and metal ion selective imaging fiber bundles.

PubMed

Szunerits, Sabine; Walt, David R

2002-02-15

The localized corrosion behavior of a galvanic aluminum copper couple was investigated by in situ fluorescence imaging with a fiber-optic imaging sensor. Three different, but complementary methods were used for visualizing remote corrosion sites, mapping the topography of the metal surface, and measuring local chemical concentrations of H+, OH-, and Al3+. The first method is based on a pH-sensitive imaging fiber, where the fluorescent dye SNAFL was covalently attached to the fiber's distal end. Fluorescence images were acquired as a function of time at different areas of the galvanic couple. In a second method, the fluorescent dye morin was immobilized on the fiber-optic imaging sensor, which allowed the in situ localization of corrosion processes on pure aluminum to be visualized over time by monitoring the release of Al3+. The development of fluorescence on the aluminum surface defined the areas associated with the anodic dissolution of aluminum. We also investigated the inhibition of corrosion of pure aluminum by CeCl3 and 8-hydroxyquinoline. The decrease in current, the decrease in the number of active sites on the aluminum surface, and the faster surface passivation are all consistent indications that cerium chloride and 8-hydroxyquinoline inhibit corrosion effectively. From the number and extent of corrosion sites and the release of aluminum ions monitored with the fiber, it was shown that 8-hydroxyquinoline is a more effective inhibitor than cerium chloride.

Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging.

PubMed

Peck, Evan M; Battles, Paul M; Rice, Douglas R; Roland, Felicia M; Norquest, Kathryn A; Smith, Bradley D

2016-05-18

A programmable pre-assembly method is described and shown to produce near-infrared fluorescent molecular probes with tunable multivalent binding properties. The modular assembly process threads one or two copies of a tetralactam macrocycle onto a fluorescent PEGylated squaraine scaffold containing a complementary number of docking stations. Appended to the macrocycle periphery are multiple copies of a ligand that is known to target a biomarker. The structure and high purity of each threaded complex was determined by independent spectrometric methods and also by gel electrophoresis. Especially helpful were diagnostic red-shift and energy transfer features in the absorption and fluorescence spectra. The threaded complexes were found to be effective multivalent molecular probes for fluorescence microscopy and in vivo fluorescence imaging of living subjects. Two multivalent probes were prepared and tested for targeting of bone in mice. A pre-assembled probe with 12 bone-targeting iminodiacetate ligands produced more bone accumulation than an analogous pre-assembled probe with six iminodiacetate ligands. Notably, there was no loss in probe fluorescence at the bone target site after 24 h in the living animal, indicating that the pre-assembled fluorescent probe maintained very high mechanical and chemical stability on the skeletal surface. The study shows how this versatile pre-assembly method can be used in a parallel combinatorial manner to produce libraries of near-infrared fluorescent multivalent molecular probes for different types of imaging and diagnostic applications, with incremental structural changes in the number of targeting groups, linker lengths, linker flexibility, and degree of PEGylation.

Development of fluorescence based handheld imaging devices for food safety inspection

NASA Astrophysics Data System (ADS)

Lee, Hoyoung; Kim, Moon S.; Chao, Kuanglin; Lefcourt, Alan M.; Chan, Diane E.

2013-05-01

For sanitation inspection in food processing environment, fluorescence imaging can be a very useful method because many organic materials reveal unique fluorescence emissions when excited by UV or violet radiation. Although some fluorescence-based automated inspection instrumentation has been developed for food products, there remains a need for devices that can assist on-site inspectors performing visual sanitation inspection of the surfaces of food processing/handling equipment. This paper reports the development of an inexpensive handheld imaging device designed to visualize fluorescence emissions and intended to help detect the presence of fecal contaminants, organic residues, and bacterial biofilms at multispectral fluorescence emission bands. The device consists of a miniature camera, multispectral (interference) filters, and high power LED illumination. With WiFi communication, live inspection images from the device can be displayed on smartphone or tablet devices. This imaging device could be a useful tool for assessing the effectiveness of sanitation procedures and for helping processors to minimize food safety risks or determine potential problem areas. This paper presents the design and development including evaluation and optimization of the hardware components of the imaging devices.

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

PubMed

Yang, Yang; Guan, Xiangming

2017-05-01

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

NASA Astrophysics Data System (ADS)

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-02-01

The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

Novel fusion for hybrid optical/microcomputed tomography imaging based on natural light surface reconstruction and iterated closest point

NASA Astrophysics Data System (ADS)

Ning, Nannan; Tian, Jie; Liu, Xia; Deng, Kexin; Wu, Ping; Wang, Bo; Wang, Kun; Ma, Xibo

2014-02-01

In mathematics, optical molecular imaging including bioluminescence tomography (BLT), fluorescence tomography (FMT) and Cerenkov luminescence tomography (CLT) are concerned with a similar inverse source problem. They all involve the reconstruction of the 3D location of a single/multiple internal luminescent/fluorescent sources based on 3D surface flux distribution. To achieve that, an accurate fusion between 2D luminescent/fluorescent images and 3D structural images that may be acquired form micro-CT, MRI or beam scanning is extremely critical. However, the absence of a universal method that can effectively convert 2D optical information into 3D makes the accurate fusion challengeable. In this study, to improve the fusion accuracy, a new fusion method for dual-modality tomography (luminescence/fluorescence and micro-CT) based on natural light surface reconstruction (NLSR) and iterated closest point (ICP) was presented. It consisted of Octree structure, exact visual hull from marching cubes and ICP. Different from conventional limited projection methods, it is 360° free-space registration, and utilizes more luminescence/fluorescence distribution information from unlimited multi-orientation 2D optical images. A mouse mimicking phantom (one XPM-2 Phantom Light Source, XENOGEN Corporation) and an in-vivo BALB/C mouse with implanted one luminescent light source were used to evaluate the performance of the new fusion method. Compared with conventional fusion methods, the average error of preset markers was improved by 0.3 and 0.2 pixels from the new method, respectively. After running the same 3D internal light source reconstruction algorithm of the BALB/C mouse, the distance error between the actual and reconstructed internal source was decreased by 0.19 mm.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

NASA Astrophysics Data System (ADS)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05839b

Comparative study on fluorescence spectra of Chinese medicine north and south isatis root granules

NASA Astrophysics Data System (ADS)

Liang, Lan; He, Qing; Chen, Zhenqiang; Zhu, Siqi

2016-03-01

Since the spectral imaging technology emerged, it has gained a lot of application achievements in the military field, precision agriculture and biomedical science. When the fluorescence spectrum imaging first applied to the detection of the feature resource of Chinese herbal medicine, the characteristics of holistic and ambiguity made it a new approach to the traditional Chinese medicine testing. In this paper, we applied this method to study the Chinese medicine north and south isatis root granules by comparing their fluorescence spectra. Using cluster analysis, the results showed that the north and south Banlangen can not be divided by ascription. And these indicate that there is a large difference in the quality of Banlangen granules on the market, and fluorescence spectrum imaging method can be used in monitoring the quality of radix isatidis granules.

A study on a portable fluorescence imaging system

NASA Astrophysics Data System (ADS)

Chang, Han-Chao; Wu, Wen-Hong; Chang, Chun-Li; Huang, Kuo-Cheng; Chang, Chung-Hsing; Chiu, Shang-Chen

2011-09-01

The fluorescent reaction is that an organism or dye, excited by UV light (200-405 nm), emits a specific frequency of light; the light is usually a visible or near infrared light (405-900 nm). During the UV light irradiation, the photosensitive agent will be induced to start the photochemical reaction. In addition, the fluorescence image can be used for fluorescence diagnosis and then photodynamic therapy can be given to dental diseases and skin cancer, which has become a useful tool to provide scientific evidence in many biomedical researches. However, most of the methods on acquiring fluorescence biology traces are still stay in primitive stage, catching by naked eyes and researcher's subjective judgment. This article presents a portable camera to obtain the fluorescence image and to make up a deficit from observer competence and subjective judgment. Furthermore, the portable camera offers the 375nm UV-LED exciting light source for user to record fluorescence image and makes the recorded image become persuasive scientific evidence. In addition, when the raising the rate between signal and noise, the signal processing module will not only amplify the fluorescence signal up to 70 %, but also decrease the noise significantly from environmental light on bill and nude mouse testing.

Common fluorescent proteins for single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

2015-07-01

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

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

Analysis of cholesterol trafficking with fluorescent probes

PubMed Central

Maxfield, Frederick R.; Wüstner, Daniel

2013-01-01

Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly. PMID:22325611

Folic acid-targeted magnetic Tb-doped CeF3 fluorescent nanoparticles as bimodal probes for cellular fluorescence and magnetic resonance imaging.

PubMed

Ma, Zhi-Ya; Liu, Yu-Ping; Bai, Ling-Yu; An, Jie; Zhang, Lin; Xuan, Yang; Zhang, Xiao-Shuai; Zhao, Yuan-Di

2015-10-07

Magnetic fluorescent nanoparticles (NPs) have great potential applications for diagnostics, imaging and therapy. We developed a facile polyol method to synthesize multifunctional Fe3O4@CeF3:Tb@CeF3 NPs with small size (<20 nm), high water solubility and good biocompatibility. The NPs were modified by ligand exchange reactions with citric acid (CA) to obtain carboxyl-functionalized NPs (Fe3O4@CeF3:Tb@CeF3-COOH). Folic acid (FA) as an affinity ligand was then covalently conjugated onto NPs to yield Fe3O4@CeF3:Tb@CeF3-FA NPs. They were then applied as multimodal imaging agents for simultaneous in vitro targeted fluorescence imaging and magnetic resonance imaging (MRI) of HeLa cells with overexpressed folate receptors (FR). The results indicated that these NPs had strong luminescence and enhanced T2-weighted MR contrast and would be promising candidates as multimodal probes for both fluorescence and MRI imaging.

Fluorescence Molecular Tomography: Principles and Potential for Pharmaceutical Research

PubMed Central

Stuker, Florian; Ripoll, Jorge; Rudin, Markus

2011-01-01

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue's optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development. PMID:24310495

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

1995-11-28

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

1995-01-01

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

Optical imaging for the diagnosis of oral cancer and oral potentially malignant disorders

NASA Astrophysics Data System (ADS)

Yoshida, K.

2016-03-01

Optical Imaging is being conducted as a therapeutic non-invasive. Many kinds of the light source are selected for this purpose. Recently the oral cancer screening is conducted by using light-induced tissue autofluorescence examination such as several kinds of handheld devices. However, the mechanism of its action is still not clear. Therefore basic experimental research was conducted. One of auto fluorescence Imaging (AFI) device, VELscopeTM and near-infrared (NIR) fluorescence imaging using ICG-labeled antibody as a probe were compared using oral squamous cell carcinoma (OSCC) mouse models. The experiments revealed that intracutaneous tumor was successfully visualized as low density image by VELscopeTM and high density image by NIR image. In addition, VELscopeTM showed higher sensitivity and lower specificity than that of NIR fluorescence imaging and the sensitivity of identification of carcinoma areas with the VELscopeTM was good results. However, further more studies were needed to enhance the screening and diagnostic uses, sensitivity and specificity for detecting malignant lesions and differentiation from premalignant or benign lesions. Therefore, additional studies were conducted using a new developed near infrared (NIR) fluorescence imaging method targeting podoplanine (PDPN) which consists of indocyanine green (ICG)-labeled anti-human podoplanin antibody as a probe and IVIS imaging system or a handy realtime ICG imaging device that is overexpressed in oral malignant neoplasm to improve imaging for detection of early oral malignant neoplasm. Then evaluated for its sensitivity and specificity for detection of oral malignant neoplasm in xenografted mice model and compared with VELscopeTM. The results revealed that ICG fluorescence imaging method and VELscopeTM had the almost the same sensitivity for detection of oral malignant neoplasm. The current topics of optical imaging about oral malignant neoplasm were reviewed.

Fluorescence multispectral imaging-based diagnostic system for atherosclerosis.

PubMed

Ho, Cassandra Su Lyn; Horiuchi, Toshikatsu; Taniguchi, Hiroaki; Umetsu, Araya; Hagisawa, Kohsuke; Iwaya, Keiichi; Nakai, Kanji; Azmi, Amalina; Zulaziz, Natasha; Azhim, Azran; Shinomiya, Nariyoshi; Morimoto, Yuji

2016-08-20

Composition of atherosclerotic arterial walls is rich in lipids such as cholesterol, unlike normal arterial walls. In this study, we aimed to utilize this difference to diagnose atherosclerosis via multispectral fluorescence imaging, which allows for identification of fluorescence originating from the substance in the arterial wall. The inner surface of extracted arteries (rabbit abdominal aorta, human coronary artery) was illuminated by 405 nm excitation light and multispectral fluorescence images were obtained. Pathological examination of human coronary artery samples were carried out and thickness of arteries were calculated by measuring combined media and intima thickness. The fluorescence spectra in atherosclerotic sites were different from those in normal sites. Multiple regions of interest (ROI) were selected within each sample and a ratio between two fluorescence intensity differences (where each intensity difference is calculated between an identifier wavelength and a base wavelength) from each ROI was determined, allowing for discrimination of atherosclerotic sites. Fluorescence intensity and thickness of artery were found to be significantly correlated. These results indicate that multispectral fluorescence imaging provides qualitative and quantitative evaluations of atherosclerosis and is therefore a viable method of diagnosing the disease.

Segmentation and Morphometric Analysis of Cells from Fluorescence Microscopy Images of Cytoskeletons

PubMed Central

Ujihara, Yoshihiro; Nakamura, Masanori; Miyazaki, Hiroshi; Wada, Shigeo

2013-01-01

We developed a method to reconstruct cell geometry from confocal fluorescence microscopy images of the cytoskeleton. In the method, region growing was implemented twice. First, it was applied to the extracellular regions to differentiate them from intracellular noncytoskeletal regions, which both appear black in fluorescence microscopy imagery, and then to cell regions for cell identification. Analysis of morphological parameters revealed significant changes in cell shape associated with cytoskeleton disruption, which offered insight into the mechanical role of the cytoskeleton in maintaining cell shape. The proposed segmentation method is promising for investigations on cell morphological changes with respect to internal cytoskeletal structures. PMID:23762186

Segmentation and morphometric analysis of cells from fluorescence microscopy images of cytoskeletons.

PubMed

Ujihara, Yoshihiro; Nakamura, Masanori; Miyazaki, Hiroshi; Wada, Shigeo

2013-01-01

We developed a method to reconstruct cell geometry from confocal fluorescence microscopy images of the cytoskeleton. In the method, region growing was implemented twice. First, it was applied to the extracellular regions to differentiate them from intracellular noncytoskeletal regions, which both appear black in fluorescence microscopy imagery, and then to cell regions for cell identification. Analysis of morphological parameters revealed significant changes in cell shape associated with cytoskeleton disruption, which offered insight into the mechanical role of the cytoskeleton in maintaining cell shape. The proposed segmentation method is promising for investigations on cell morphological changes with respect to internal cytoskeletal structures.

Fluorescently labeled bevacizumab in human breast cancer: defining the classification threshold

NASA Astrophysics Data System (ADS)

Koch, Maximilian; de Jong, Johannes S.; Glatz, Jürgen; Symvoulidis, Panagiotis; Lamberts, Laetitia E.; Adams, Arthur L. L.; Kranendonk, Mariëtte E. G.; Terwisscha van Scheltinga, Anton G. T.; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N.; Schröder, Carolien P.; Jorritsma-Smit, Annelies; Linssen, Matthijs D.; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B.; Elias, Sjoerd G.; Oliveira, Sabrina; Witkamp, Arjen J.; Mali, Willem P. Th. M.; Van der Wall, Elsken; Garcia-Allende, P. Beatriz; van Diest, Paul J.; de Vries, Elisabeth G. E.; Walch, Axel; van Dam, Gooitzen M.; Ntziachristos, Vasilis

2017-07-01

In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging.

Anti-epidermal growth factor receptor (anti-EGFR) antibody conjugated fluorescent nanoparticles probe for breast cancer imaging

NASA Astrophysics Data System (ADS)

Hun, Xu; Zhang, Zhujun

2009-10-01

Fluorescent nanoparticles (FNs) with unique optical properties may be useful as biosensors in living cancer cell imaging and cancer targeting. In this study, anti-EGFR antibody conjugated fluorescent nanoparticles (FNs) (anti-EGFR antibody conjugated FNs) probe was used to detect breast cancer cells. FNs with excellent character such as non-toxicity and photostability were first synthesized with a simple, cost-effective and environmentally friendly modified Stőber synthesis method, and then successfully modified with anti-EGFR antibody. This kind of fluorescence probe based on the anti-EGFR antibody conjugated FNs has been used to detect breast cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the anti-EGFR antibody conjugated FNs can effectively recognize breast cancer cells and exhibited good sensitivity and exceptional photostability, which would provide a novel way for the diagnosis and curative effect observation of breast cancer cells and offer a new method in detecting EGFR.

Comparison of parameter-adapted segmentation methods for fluorescence micrographs.

PubMed

Held, Christian; Palmisano, Ralf; Häberle, Lothar; Hensel, Michael; Wittenberg, Thomas

2011-11-01

Interpreting images from fluorescence microscopy is often a time-consuming task with poor reproducibility. Various image processing routines that can help investigators evaluate the images are therefore useful. The critical aspect for a reliable automatic image analysis system is a robust segmentation algorithm that can perform accurate segmentation for different cell types. In this study, several image segmentation methods were therefore compared and evaluated in order to identify the most appropriate segmentation schemes that are usable with little new parameterization and robustly with different types of fluorescence-stained cells for various biological and biomedical tasks. The study investigated, compared, and enhanced four different methods for segmentation of cultured epithelial cells. The maximum-intensity linking (MIL) method, an improved MIL, a watershed method, and an improved watershed method based on morphological reconstruction were used. Three manually annotated datasets consisting of 261, 817, and 1,333 HeLa or L929 cells were used to compare the different algorithms. The comparisons and evaluations showed that the segmentation performance of methods based on the watershed transform was significantly superior to the performance of the MIL method. The results also indicate that using morphological opening by reconstruction can improve the segmentation of cells stained with a marker that exhibits the dotted surface of cells. Copyright © 2011 International Society for Advancement of Cytometry.

A fluorescent imaging technique for quantifying spray deposits on plant leaves

USDA-ARS?s Scientific Manuscript database

Because of the unique characteristics of electrostatically-charged sprays, use of traditional methods to quantify deposition from these sprays has been challenging. A new fluorescent imaging technique was developed to quantify spray deposits from electrostatically-charged sprays on natural plant lea...

ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging

PubMed Central

Kurihara, Daisuke; Mizuta, Yoko; Sato, Yoshikatsu; Higashiyama, Tetsuya

2015-01-01

Imaging techniques for visualizing and analyzing precise morphology and gene expression patterns are essential for understanding biological processes during development in all organisms. With the aid of chemical screening, we developed a clearing method using chemical solutions, termed ClearSee, for deep imaging of morphology and gene expression in plant tissues. ClearSee rapidly diminishes chlorophyll autofluorescence while maintaining fluorescent protein stability. By adjusting the refractive index mismatch, whole-organ and whole-plant imaging can be performed by both confocal and two-photon excitation microscopy in ClearSee-treated samples. Moreover, ClearSee is applicable to multicolor imaging of fluorescent proteins to allow structural analysis of multiple gene expression. Given that ClearSee is compatible with staining by chemical dyes, the technique is useful for deep imaging in conjunction with genetic markers and for plant species not amenable to transgenic approaches. This method is useful for whole imaging for intact morphology and will help to accelerate the discovery of new phenomena in plant biological research. PMID:26493404

Live CLEM imaging to analyze nuclear structures at high resolution.

PubMed

Haraguchi, Tokuko; Osakada, Hiroko; Koujin, Takako

2015-01-01

Fluorescence microscopy (FM) and electron microscopy (EM) are powerful tools for observing molecular components in cells. FM can provide temporal information about cellular proteins and structures in living cells. EM provides nanometer resolution images of cellular structures in fixed cells. We have combined FM and EM to develop a new method of correlative light and electron microscopy (CLEM), called "Live CLEM." In this method, the dynamic behavior of specific molecules of interest is first observed in living cells using fluorescence microscopy (FM) and then cellular structures in the same cell are observed using electron microscopy (EM). Following image acquisition, FM and EM images are compared to enable the fluorescent images to be correlated with the high-resolution images of cellular structures obtained using EM. As this method enables analysis of dynamic events involving specific molecules of interest in the context of specific cellular structures at high resolution, it is useful for the study of nuclear structures including nuclear bodies. Here we describe Live CLEM that can be applied to the study of nuclear structures in mammalian cells.

In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Gray, Daniel C.; Merigan, William; Wolfing, Jessica I.; Gee, Bernard P.; Porter, Jason; Dubra, Alfredo; Twietmeyer, Ted H.; Ahamd, Kamran; Tumbar, Remy; Reinholz, Fred; Williams, David R.

2006-08-01

The ability to resolve single cells noninvasively in the living retina has important applications for the study of normal retina, diseased retina, and the efficacy of therapies for retinal disease. We describe a new instrument for high-resolution, in vivo imaging of the mammalian retina that combines the benefits of confocal detection, adaptive optics, multispectral, and fluorescence imaging. The instrument is capable of imaging single ganglion cells and their axons through retrograde transport in ganglion cells of fluorescent dyes injected into the monkey lateral geniculate nucleus (LGN). In addition, we demonstrate a method involving simultaneous imaging in two spectral bands that allows the integration of very weak signals across many frames despite inter-frame movement of the eye. With this method, we are also able to resolve the smallest retinal capillaries in fluorescein angiography and the mosaic of retinal pigment epithelium (RPE) cells with lipofuscin autofluorescence.

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.

Improved detection of soma location and morphology in fluorescence microscopy images of neurons.

PubMed

Kayasandik, Cihan Bilge; Labate, Demetrio

2016-12-01

Automated detection and segmentation of somas in fluorescent images of neurons is a major goal in quantitative studies of neuronal networks, including applications of high-content-screenings where it is required to quantify multiple morphological properties of neurons. Despite recent advances in image processing targeted to neurobiological applications, existing algorithms of soma detection are often unreliable, especially when processing fluorescence image stacks of neuronal cultures. In this paper, we introduce an innovative algorithm for the detection and extraction of somas in fluorescent images of networks of cultured neurons where somas and other structures exist in the same fluorescent channel. Our method relies on a new geometrical descriptor called Directional Ratio and a collection of multiscale orientable filters to quantify the level of local isotropy in an image. To optimize the application of this approach, we introduce a new construction of multiscale anisotropic filters that is implemented by separable convolution. Extensive numerical experiments using 2D and 3D confocal images show that our automated algorithm reliably detects somas, accurately segments them, and separates contiguous ones. We include a detailed comparison with state-of-the-art existing methods to demonstrate that our algorithm is extremely competitive in terms of accuracy, reliability and computational efficiency. Our algorithm will facilitate the development of automated platforms for high content neuron image processing. A Matlab code is released open-source and freely available to the scientific community. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of pre-processing techniques for fluorescence microscopy images of cells labeled for actin.

PubMed

Muralidhar, Gautam S; Channappayya, Sumohana S; Slater, John H; Blinka, Ellen M; Bovik, Alan C; Frey, Wolfgang; Markey, Mia K

2008-11-06

Automated analysis of fluorescence microscopy images of endothelial cells labeled for actin is important for quantifying changes in the actin cytoskeleton. The current manual approach is laborious and inefficient. The goal of our work is to develop automated image analysis methods, thereby increasing cell analysis throughput. In this study, we present preliminary results on comparing different algorithms for cell segmentation and image denoising.

Green Synthesis of Bifunctional Fluorescent Carbon Dots from Garlic for Cellular Imaging and Free Radical Scavenging.

PubMed

Zhao, Shaojing; Lan, Minhuan; Zhu, Xiaoyue; Xue, Hongtao; Ng, Tsz-Wai; Meng, Xiangmin; Lee, Chun-Sing; Wang, Pengfei; Zhang, Wenjun

2015-08-12

Nitrogen and sulfur codoped carbon dots (CDs) were prepared from garlic by a hydrothermal method. The as-prepared CDs possess good water dispersibility, strong blue fluorescence emission with a fluorescent quantum yield of 17.5%, and excellent photo and pH stabilities. It is also demonstrated that the fluorescence of CDs are resistant to the interference of metal ions, biomolecules, and high ionic strength environments. Combining with low cytotoxicity properties, CDs could be used as an excellent fluorescent probe for cellular multicolor imaging. Moreover, the CDs were also demonstrated to exhibit favorable radical scavenging activity.

Deep-tissue focal fluorescence imaging with digitally time-reversed ultrasound-encoded light

PubMed Central

Wang, Ying Min; Judkewitz, Benjamin; DiMarzio, Charles A.; Yang, Changhuei

2012-01-01

Fluorescence imaging is one of the most important research tools in biomedical sciences. However, scattering of light severely impedes imaging of thick biological samples beyond the ballistic regime. Here we directly show focusing and high-resolution fluorescence imaging deep inside biological tissues by digitally time-reversing ultrasound-tagged light with high optical gain (~5×105). We confirm the presence of a time-reversed optical focus along with a diffuse background—a corollary of partial phase conjugation—and develop an approach for dynamic background cancellation. To illustrate the potential of our method, we image complex fluorescent objects and tumour microtissues at an unprecedented depth of 2.5 mm in biological tissues at a lateral resolution of 36 μm×52 μm and an axial resolution of 657 μm. Our results set the stage for a range of deep-tissue imaging applications in biomedical research and medical diagnostics. PMID:22735456

Novel fluorescence molecular imaging of chemotherapy-induced intestinal apoptosis

NASA Astrophysics Data System (ADS)

Levin, Galit; Shirvan, Anat; Grimberg, Hagit; Reshef, Ayelet; Yogev-Falach, Merav; Cohen, Avi; Ziv, Ilan

2009-09-01

Chemotherapy-induced enteropathy (CIE) is one of the most serious complications of anticancer therapy, and tools for its early detection and monitoring are highly needed. We report on a novel fluorescence method for detection of CIE, based on molecular imaging of the related apoptotic process. The method comprises systemic intravenous administration of the ApoSense fluorescent biomarker (N,N'-didansyl-L-cystine DDC) in vivo and subsequent fluorescence imaging of the intestinal mucosa. In the reported proof-of-concept studies, mice were treated with either taxol+cyclophosphamide or doxil. DDC was administered in vivo at various time points after drug administration, and tracer uptake by ileum tissue was subsequently evaluated by ex vivo fluorescent microscopy. Chemotherapy caused marked and selective uptake of DDC in ileal epithelial cells, in correlation with other hallmarks of apoptosis (i.e., DNA fragmentation and Annexin-V binding). Induction of DDC uptake occurred early after chemotherapy, and its temporal profile was parallel to that of the apoptotic process, as assessed histologically. DDC may therefore serve as a useful tool for detection of CIE. Future potential integration of this method with fluorescent endoscopic techniques, or development of radio-labeled derivatives of DDC for emission tomography, may advance early diagnosis and monitoring of this severe adverse effect of chemotherapy.

Multi-Modal Imaging in a Mouse Model of Orthotopic Lung Cancer

PubMed Central

Patel, Priya; Kato, Tatsuya; Ujiie, Hideki; Wada, Hironobu; Lee, Daiyoon; Hu, Hsin-pei; Hirohashi, Kentaro; Ahn, Jin Young; Zheng, Jinzi; Yasufuku, Kazuhiro

2016-01-01

Background Investigation of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine green (ICG) and iohexol, for real-time near infrared (NIR) fluorescence and computed tomography (CT) image-guided surgery in an orthotopic lung cancer model in nude mice. Methods CF800 was intravenously administered into 13 mice bearing the H460 orthotopic human lung cancer. At 48 h post-injection (peak imaging agent accumulation time point), ex vivo NIR and CT imaging was performed. A clinical NIR imaging system (SPY®, Novadaq) was used to measure fluorescence intensity of tumor and lung. Tumor-to-background-ratios (TBR) were calculated in inflated and deflated states. The mean Hounsfield unit (HU) of lung tumor was quantified using the CT data set and a semi-automated threshold-based method. Histological evaluation using H&E, the macrophage marker F4/80 and the endothelial cell marker CD31, was performed, and compared to the liposomal fluorescence signal obtained from adjacent tissue sections Results The fluorescence TBR measured when the lung is in the inflated state (2.0 ± 0.58) was significantly greater than in the deflated state (1.42 ± 0.380 (n = 7, p<0.003). Mean fluorescent signal in tumor was highly variable across samples, (49.0 ± 18.8 AU). CT image analysis revealed greater contrast enhancement in lung tumors (a mean increase of 110 ± 57 HU) when CF800 is administered compared to the no contrast enhanced tumors (p = 0.0002). Conclusion Preliminary data suggests that the high fluorescence TBR and CT tumor contrast enhancement provided by CF800 may have clinical utility in localization of lung cancer during CT and NIR image-guided surgery. PMID:27584018

Single pulse two-photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate and an all fiber based setup

NASA Astrophysics Data System (ADS)

Eibl, Matthias; Karpf, Sebastian; Hakert, Hubertus; Weng, Daniel; Pfeiffer, Tom; Kolb, Jan Philip; Huber, Robert

2017-07-01

Newly developed microscopy methods have the goal to give researches in bio-molecular science a better understanding of processes ongoing on a cellular level. Especially two-photon excited fluorescence (TPEF) microscopy is a readily applied and widespread modality. Compared to one photon fluorescence imaging, it is possible to image not only the surface but also deeper lying structures. Together with fluorescence lifetime imaging (FLIM), which provides information on the chemical composition of a specimen, deeper insights on a molecular level can be gained. However, the need for elaborate light sources for TPEF and speed limitations for FLIM hinder an even wider application. In this contribution, we present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is perfectly suited for fiber delivery as typically limiting non-linear effects like self-phase or cross-phase modulation (SPM, XPM) are negligible. Furthermore, compared to the typically applied femtosecond pulses, our longer pulses produce much more fluorescence photons per single shot. In this paper, we show that this higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate our system, we acquired FLIM images of a dye solution with single exponential behavior to assess the accuracy of our lifetime determination and also FLIM images of a plant stem at a pixel rate of 1 MHz to show the speed performance of our single pulse two-photon FLIM (SP-FLIM) system.

Automatic tracking of cells for video microscopy in patch clamp experiments

PubMed Central

2014-01-01

Background Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. Methods Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). Results We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. Conclusion The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices. PMID:24946774

Fluorescence hyperspectral imaging technique for foreign substance detection on fresh-cut lettuce.

PubMed

Mo, Changyeun; Kim, Giyoung; Kim, Moon S; Lim, Jongguk; Cho, Hyunjeong; Barnaby, Jinyoung Yang; Cho, Byoung-Kwan

2017-09-01

Non-destructive methods based on fluorescence hyperspectral imaging (HSI) techniques were developed to detect worms on fresh-cut lettuce. The optimal wavebands for detecting the worms were investigated using the one-way ANOVA and correlation analyses. The worm detection imaging algorithms, RSI-I (492-626)/492 , provided a prediction accuracy of 99.0%. The fluorescence HSI techniques indicated that the spectral images with a pixel size of 1 × 1 mm had the best classification accuracy for worms. The overall results demonstrate that fluorescence HSI techniques have the potential to detect worms on fresh-cut lettuce. In the future, we will focus on developing a multi-spectral imaging system to detect foreign substances such as worms, slugs and earthworms on fresh-cut lettuce. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

[Development of an original computer program FISHMet: use for molecular cytogenetic diagnosis and genome mapping by fluorescent in situ hybridization (FISH)].

PubMed

Iurov, Iu B; Khazatskiĭ, I A; Akindinov, V A; Dovgilov, L V; Kobrinskiĭ, B A; Vorsanova, S G

2000-08-01

Original software FISHMet has been developed and tried for improving the efficiency of diagnosis of hereditary diseases caused by chromosome aberrations and for chromosome mapping by fluorescent in situ hybridization (FISH) method. The program allows creation and analysis of pseudocolor chromosome images and hybridization signals in the Windows 95 system, allows computer analysis and editing of the results of pseudocolor hybridization in situ, including successive imposition of initial black-and-white images created using fluorescent filters (blue, green, and red), and editing of each image individually or of a summary pseudocolor image in BMP, TIFF, and JPEG formats. Components of image computer analysis system (LOMO, Leitz Ortoplan, and Axioplan fluorescent microscopes, COHU 4910 and Sanyo VCB-3512P CCD cameras, Miro-Video, Scion LG-3 and VG-5 image capture maps, and Pentium 100 and Pentium 200 computers) and specialized software for image capture and visualization (Scion Image PC and Video-Cup) have been used with good results in the study.

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

Automated detection of fluorescent cells in in-resin fluorescence sections for integrated light and electron microscopy.

PubMed

Delpiano, J; Pizarro, L; Peddie, C J; Jones, M L; Griffin, L D; Collinson, L M

2018-04-26

Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Dynamic Assessment of the Endothelialization of Tissue-Engineered Blood Vessels Using an Optical Coherence Tomography Catheter-Based Fluorescence Imaging System

PubMed Central

Gurjarpadhye, Abhijit Achyut; DeWitt, Matthew R.; Xu, Yong; Wang, Ge; Rylander, Marissa Nichole

2015-01-01

Background: Lumen endothelialization of bioengineered vascular scaffolds is essential to maintain small-diameter graft patency and prevent thrombosis postimplantation. Unfortunately, nondestructive imaging methods to visualize this dynamic process are lacking, thus slowing development and clinical translation of these potential tissue-engineering approaches. To meet this need, a fluorescence imaging system utilizing a commercial optical coherence tomography (OCT) catheter was designed to visualize graft endothelialization. Methods: C7 DragonFly™ intravascular OCT catheter was used as a channel for delivery and collection of excitation and emission spectra. Poly-dl-lactide (PDLLA) electrospun scaffolds were seeded with endothelial cells (ECs). Seeded cells were exposed to Calcein AM before imaging, causing the living cells to emit green fluorescence in response to blue laser. By positioning the catheter tip precisely over a specimen using high-fidelity electromechanical components, small regions of the specimen were excited selectively. The resulting fluorescence intensities were mapped on a two-dimensional digital grid to generate spatial distribution of fluorophores at single-cell-level resolution. Fluorescence imaging of endothelialization on glass and PDLLA scaffolds was performed using the OCT catheter-based imaging system as well as with a commercial fluorescence microscope. Cell coverage area was calculated for both image sets for quantitative comparison of imaging techniques. Tubular PDLLA scaffolds were maintained in a bioreactor on seeding with ECs, and endothelialization was monitored over 5 days using the OCT catheter-based imaging system. Results: No significant difference was observed in images obtained using our imaging system to those acquired with the fluorescence microscope. Cell area coverage calculated using the images yielded similar values. Nondestructive imaging of endothelialization on tubular scaffolds showed cell proliferation with cell coverage area increasing from 15±4% to 89±6% over 5 days. Conclusion: In this study, we showed the capability of an OCT catheter-based imaging system to obtain single-cell resolution and to quantify endothelialization in tubular electrospun scaffolds. We also compared the resulting images with traditional microscopy, showing high fidelity in image capability. This imaging system, used in conjunction with OCT, could potentially be a powerful tool for in vitro optimization of scaffold cellularization, ensuring long-term graft patency postimplantation. PMID:25539889

Fabrication of Indocyanine Green and 2H, 3H-perfluoropentane loaded microbubbles for fluorescence and ultrasound imaging

NASA Astrophysics Data System (ADS)

He, Yutong; Wu, Qiang; Ma, Rong; Chang, Shufang; Shao, Pengfei; Xu, Ronald

2016-03-01

As a near-infrared (NIR) fluorescence dye, Indocyanine Green (ICG) has not gained broader clinical applications, owing to its multiple limitations such as concentration-dependent aggregation, low fluorescence quantum yield, poor physicochemical stability and rapid elimination from the body. In the meanwhile, 2H,3H-perfluoropentane (H-PFP) has been widely studied in ultrasound imaging as a vehicle for targeted delivery of contrast agents and drugs. We synthesized a novel dual-modal fluorescence and ultrasound contrast agent by encapsulating ICG and H-PFP in lipid microbubbles using a liquid-driven coaxial flow focusing (LDCFF) process. Uniform microbubbles with the sizes ranging from 1-10um and great ICG loading efficiency was achieved by this method. Our benchtop experiments showed that ICG/H-PFP microbubbles exhibited less aggregation, increased fluorescence intensity and more stable photostability compared to free ICG aqueous solution. Our phantom experiments demonstrated that ICG/H-PFP microbubbles enhanced the imaging contrasts in fluorescence imaging and ultrasonography. Our animal experiments indicated that ICG/H-PFP microbubbles extended the ICG life time and facilitated dual mode fluorescence and ultrasound imaging in vivo.

Video-rate hyperspectral two-photon fluorescence microscopy for in vivo imaging

NASA Astrophysics Data System (ADS)

Deng, Fengyuan; Ding, Changqin; Martin, Jerald C.; Scarborough, Nicole M.; Song, Zhengtian; Eakins, Gregory S.; Simpson, Garth J.

2018-02-01

Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam's position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.

Hessian-based quantitative image analysis of host-pathogen confrontation assays.

PubMed

Cseresnyes, Zoltan; Kraibooj, Kaswara; Figge, Marc Thilo

2018-03-01

Host-fungus interactions have gained a lot of interest in the past few decades, mainly due to an increasing number of fungal infections that are often associated with a high mortality rate in the absence of effective therapies. These interactions can be studied at the genetic level or at the functional level via imaging. Here, we introduce a new image processing method that quantifies the interaction between host cells and fungal invaders, for example, alveolar macrophages and the conidia of Aspergillus fumigatus. The new technique relies on the information content of transmitted light bright field microscopy images, utilizing the Hessian matrix eigenvalues to distinguish between unstained macrophages and the background, as well as between macrophages and fungal conidia. The performance of the new algorithm was measured by comparing the results of our method with that of an alternative approach that was based on fluorescence images from the same dataset. The comparison shows that the new algorithm performs very similarly to the fluorescence-based version. Consequently, the new algorithm is able to segment and characterize unlabeled cells, thus reducing the time and expense that would be spent on the fluorescent labeling in preparation for phagocytosis assays. By extending the proposed method to the label-free segmentation of fungal conidia, we will be able to reduce the need for fluorescence-based imaging even further. Our approach should thus help to minimize the possible side effects of fluorescence labeling on biological functions. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Fluorescence lifetime imaging and Fourier transform infrared spectroscopy of Michelangelo's David.

PubMed

Comelli, Daniela; Valentini, Gianluca; Cubeddu, Rinaldo; Toniolo, Lucia

2005-09-01

We developed a combined procedure for the analysis of works of art based on a portable system for fluorescence imaging integrated with analytical measurements on microsamples. The method allows us to localize and identify organic and inorganic compounds present on the surface of artworks. The fluorescence apparatus measures the temporal and spectral features of the fluorescence emission, excited by ultraviolet (UV) laser pulses. The kinetic of the emission is studied through a fluorescence lifetime imaging system, while an optical multichannel analyzer measures the fluorescence spectra of selected points. The chemical characterization of the compounds present on the artistic surfaces is then performed by means of analytical measurements on microsamples collected with the assistance of the fluorescence maps. The previous concepts have been successfully applied to study the contaminants on the surface of Michelangelo's David. The fluorescence analysis combined with Fourier transform infrared (FT-IR) measurements revealed the presence of beeswax, which permeates most of the statue surface, and calcium oxalate deposits mainly arranged in vertical patterns and related to rain washing.

HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer

NASA Astrophysics Data System (ADS)

Wang, Can; Bao, Chenchen; Liang, Shujing; Zhang, Lingxia; Fu, Hualin; Wang, Yutian; Wang, Kan; Li, Chao; Deng, Min; Liao, Qiande; Ni, Jian; Cui, Daxiang

2014-05-01

The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.

Three-dimensional refractive index and fluorescence tomography using structured illumination (Conference Presentation)

NASA Astrophysics Data System (ADS)

Park, GwangSik; Shin, SeungWoo; Kim, Kyoohyun; Park, YongKeun

2017-02-01

Optical diffraction tomography (ODT) has been an emerging optical technique for label-free imaging of three-dimensional (3-D) refractive index (RI) distribution of biological samples. ODT employs interferometric microscopy for measuring multiple holograms of samples with various incident angles, from which the Fourier diffraction theorem reconstructs the 3-D RI distribution of samples from retrieved complex optical fields. Since the RI value is linearly proportional to the protein concentration of biological samples where the proportional coefficient is called as refractive index increment (RII), reconstructed 3-D RI tomograms provide precise structural and biochemical information of individual biological samples. Because most proteins have similar RII value, however, ODT has limited molecular specificity, especially for imaging eukaryotic cells having various types of proteins and subcellular organelles. Here, we present an ODT system combined with structured illumination microscopy which can measure the 3-D RI distribution of biological samples as well as 3-D super-resolution fluorescent images in the same optical setup. A digital micromirror device (DMD) controls the incident angle of the illumination beam for tomogram reconstruction, and the same DMD modulates the structured illumination pattern of the excitation beam for super-resolution fluorescent imaging. We first validate the proposed method for simultaneous optical diffraction tomographic imaging and super-resolution fluorescent imaging of fluorescent beads. The proposed method is also exploited for various biological samples.

Open source tools for fluorescent imaging.

PubMed

Hamilton, Nicholas A

2012-01-01

As microscopy becomes increasingly automated and imaging expands in the spatial and time dimensions, quantitative analysis tools for fluorescent imaging are becoming critical to remove both bottlenecks in throughput as well as fully extract and exploit the information contained in the imaging. In recent years there has been a flurry of activity in the development of bio-image analysis tools and methods with the result that there are now many high-quality, well-documented, and well-supported open source bio-image analysis projects with large user bases that cover essentially every aspect from image capture to publication. These open source solutions are now providing a viable alternative to commercial solutions. More importantly, they are forming an interoperable and interconnected network of tools that allow data and analysis methods to be shared between many of the major projects. Just as researchers build on, transmit, and verify knowledge through publication, open source analysis methods and software are creating a foundation that can be built upon, transmitted, and verified. Here we describe many of the major projects, their capabilities, and features. We also give an overview of the current state of open source software for fluorescent microscopy analysis and the many reasons to use and develop open source methods. Copyright © 2012 Elsevier Inc. All rights reserved.

Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures.

PubMed

Tsunematsu, Hiroto; Uyeda, Akiko; Yamamoto, Nobuhiko; Sugo, Noriyuki

2017-08-01

CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

An automated protocol for performance benchmarking a widefield fluorescence microscope.

PubMed

Halter, Michael; Bier, Elianna; DeRose, Paul C; Cooksey, Gregory A; Choquette, Steven J; Plant, Anne L; Elliott, John T

2014-11-01

Widefield fluorescence microscopy is a highly used tool for visually assessing biological samples and for quantifying cell responses. Despite its widespread use in high content analysis and other imaging applications, few published methods exist for evaluating and benchmarking the analytical performance of a microscope. Easy-to-use benchmarking methods would facilitate the use of fluorescence imaging as a quantitative analytical tool in research applications, and would aid the determination of instrumental method validation for commercial product development applications. We describe and evaluate an automated method to characterize a fluorescence imaging system's performance by benchmarking the detection threshold, saturation, and linear dynamic range to a reference material. The benchmarking procedure is demonstrated using two different materials as the reference material, uranyl-ion-doped glass and Schott 475 GG filter glass. Both are suitable candidate reference materials that are homogeneously fluorescent and highly photostable, and the Schott 475 GG filter glass is currently commercially available. In addition to benchmarking the analytical performance, we also demonstrate that the reference materials provide for accurate day to day intensity calibration. Published 2014 Wiley Periodicals Inc. Published 2014 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

Adaptive thresholding image series from fluorescence confocal scanning laser microscope using orientation intensity profiles

NASA Astrophysics Data System (ADS)

Feng, Judy J.; Ip, Horace H.; Cheng, Shuk H.

2004-05-01

Many grey-level thresholding methods based on histogram or other statistic information about the interest image such as maximum entropy and so on have been proposed in the past. However, most methods based on statistic analysis of the images concerned little about the characteristics of morphology of interest objects, which sometimes could provide very important indication which can help to find the optimum threshold, especially for those organisms which have special texture morphologies such as vasculature, neuro-network etc. in medical imaging. In this paper, we propose a novel method for thresholding the fluorescent vasculature image series recorded from Confocal Scanning Laser Microscope. After extracting the basic orientation of the slice of vessels inside a sub-region partitioned from the images, we analysis the intensity profiles perpendicular to the vessel orientation to get the reasonable initial threshold for each region. Then the threshold values of those regions near the interest one both in x-y and optical directions have been referenced to get the final result of thresholds of the region, which makes the whole stack of images look more continuous. The resulting images are characterized by suppressing both noise and non-interest tissues conglutinated to vessels, while improving the vessel connectivities and edge definitions. The value of the method for idealized thresholding the fluorescence images of biological objects is demonstrated by a comparison of the results of 3D vascular reconstruction.

A Ratiometric Threshold for Determining Presence of Cancer During Fluorescence-guided Surgery

PubMed Central

Warram, Jason M; de Boer, Esther; Moore, Lindsay S.; Schmalbach, Cecelia E; Withrow, Kirk P; Carroll, William R; Richman, Joshua S; Morlandt, Anthony B; Brandwein-Gensler, Margaret; Rosenthal, Eben L

2015-01-01

Background&Objective Fluorescence-guided imaging to assist in identification of malignant margins has the potential to dramatically improve oncologic surgery. However a standardized method for quantitative assessment of disease-specific fluorescence has not been investigated. Introduced here is a ratiometric threshold derived from mean fluorescent tissue intensity that can be used to semi-quantitatively delineate tumor from normal tissue. Methods Open-field and a closed-field imaging devices were used to quantify fluorescence in punch biopsy tissues sampled from primary tumors collected during a phase 1 trial evaluating the safety of cetuximab-IRDye800 in patients (n=11) undergoing surgical intervention for head and neck cancer. Fluorescence ratios were calculated using mean fluorescence intensity (MFI) from punch biopsy normalized by MFI of patient-matched tissues. Ratios were compared to pathological assessment and a ratiometric threshold was established to predict presence of cancer. Results During open-field imaging using an intraoperative device, the threshold for muscle normalized tumor fluorescence was found to be 2.7, which produced a sensitivity of 90.5% and specificity of 78.6% for delineating disease tissue. The skin-normalized threshold generated greater sensitivity (92.9%) and specificity (81.0%). Conclusion Successful implementation of a semi-quantitative threshold can provide a scientific methodology for delineating disease from normal tissue during fluorescence-guided resection of cancer. PMID:26074273

A novel method to visually determine the intracellular pH of xenografted tumor in vivo by utilizing fluorescent protein as an indicator.

PubMed

Tanaka, Shotaro; Harada, Hiroshi; Hiraoka, Masahiro

2015-09-04

The alkalization of intracellular pH (pHin) advances together with enhancement of aerobic glycolysis within tumor cells (the Warburg effect), and that is responsible for the progression of tumor malignancy together with hypoxia and angiogenesis. But how they correlate each other during tumor growth is poorly understood, partly due to the lack of suitable imaging methods. In present study, we propose a novel method to visually determine the pHin of tumor xenograft model from fluorescent image ratios. We utilized tandemly-linked two fluorescent proteins as a pH indicator; yellow fluorescent protein (YFP, pH sensitive) as an indicator, and red fluorescent protein (RFP, pH insensitive) as a reference. This method can eliminate the influence of optical factors from tissue as well as of the diverse expression level of pH indicator in the grafted cells. In addition, that can be operated by filter-based fluorescent imagers that are generally used in small animal study. The efficacy of the pH indicator, RFP-YFP, was confirmed by studies using recombinant protein in vitro and HeLa cells expressing RFP-YFP in vivo. Furthermore, we prepared nude mice subcutaneously xenografted HeLa cells expressing RFP-YFP cells as tumor model. The image ratios (YFP/RFP) of the tumor at the day 5 after surgery clearly showed the heterogeneous distribution of diverse pHin cells in the tumor tissue. Concomitantly acquired angiography using near-infrared fluorescence (680 nm for emission) also indicated that the relative alkaline pHin cells located in the region far from tumor vessels in which tumor aerobic glycolysis would be facilitated by progression of hypoxia and nutrient starvation. Applying the present method for a multi-wavelength imaging concerning pO2 and/or nutrient starvation states in addition to pHin and angiogenesis would provide valuable information about complicated alteration of tumoral cell states during tumorigenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Improving the quantitative accuracy of optical-emission computed tomography by incorporating an attenuation correction: application to HIF1 imaging

NASA Astrophysics Data System (ADS)

Kim, E.; Bowsher, J.; Thomas, A. S.; Sakhalkar, H.; Dewhirst, M.; Oldham, M.

2008-10-01

Optical computed tomography (optical-CT) and optical-emission computed tomography (optical-ECT) are new techniques for imaging the 3D structure and function (including gene expression) of whole unsectioned tissue samples. This work presents a method of improving the quantitative accuracy of optical-ECT by correcting for the 'self'-attenuation of photons emitted within the sample. The correction is analogous to a method commonly applied in single-photon-emission computed tomography reconstruction. The performance of the correction method was investigated by application to a transparent cylindrical gelatin phantom, containing a known distribution of attenuation (a central ink-doped gelatine core) and a known distribution of fluorescing fibres. Attenuation corrected and uncorrected optical-ECT images were reconstructed on the phantom to enable an evaluation of the effectiveness of the correction. Significant attenuation artefacts were observed in the uncorrected images where the central fibre appeared ~24% less intense due to greater attenuation from the surrounding ink-doped gelatin. This artefact was almost completely removed in the attenuation-corrected image, where the central fibre was within ~4% of the others. The successful phantom test enabled application of attenuation correction to optical-ECT images of an unsectioned human breast xenograft tumour grown subcutaneously on the hind leg of a nude mouse. This tumour cell line had been genetically labelled (pre-implantation) with fluorescent reporter genes such that all viable tumour cells expressed constitutive red fluorescent protein and hypoxia-inducible factor 1 transcription-produced green fluorescent protein. In addition to the fluorescent reporter labelling of gene expression, the tumour microvasculature was labelled by a light-absorbing vasculature contrast agent delivered in vivo by tail-vein injection. Optical-CT transmission images yielded high-resolution 3D images of the absorbing contrast agent, and revealed highly inhomogeneous vasculature perfusion within the tumour. Optical-ECT emission images yielded high-resolution 3D images of the fluorescent protein distribution in the tumour. Attenuation-uncorrected optical-ECT images showed clear loss of signal in regions of high attenuation, including regions of high perfusion, where attenuation is increased by increased vascular ink stain. Application of attenuation correction showed significant changes in an apparent expression of fluorescent proteins, confirming the importance of the attenuation correction. In conclusion, this work presents the first development and application of an attenuation correction for optical-ECT imaging. The results suggest that successful attenuation correction for optical-ECT is feasible and is essential for quantitatively accurate optical-ECT imaging.

A three-step reconstruction method for fluorescence molecular tomography based on compressive sensing

NASA Astrophysics Data System (ADS)

Zhu, Yansong; Jha, Abhinav K.; Dreyer, Jakob K.; Le, Hanh N. D.; Kang, Jin U.; Roland, Per E.; Wong, Dean F.; Rahmim, Arman

2017-02-01

Fluorescence molecular tomography (FMT) is a promising tool for real time in vivo quantification of neurotransmission (NT) as we pursue in our BRAIN initiative effort. However, the acquired image data are noisy and the reconstruction problem is ill-posed. Further, while spatial sparsity of the NT effects could be exploited, traditional compressive-sensing methods cannot be directly applied as the system matrix in FMT is highly coherent. To overcome these issues, we propose and assess a three-step reconstruction method. First, truncated singular value decomposition is applied on the data to reduce matrix coherence. The resultant image data are input to a homotopy-based reconstruction strategy that exploits sparsity via l1 regularization. The reconstructed image is then input to a maximum-likelihood expectation maximization (MLEM) algorithm that retains the sparseness of the input estimate and improves upon the quantitation by accurate Poisson noise modeling. The proposed reconstruction method was evaluated in a three-dimensional simulated setup with fluorescent sources in a cuboidal scattering medium with optical properties simulating human brain cortex (reduced scattering coefficient: 9.2 cm-1, absorption coefficient: 0.1 cm-1 and tomographic measurements made using pixelated detectors. In different experiments, fluorescent sources of varying size and intensity were simulated. The proposed reconstruction method provided accurate estimates of the fluorescent source intensity, with a 20% lower root mean square error on average compared to the pure-homotopy method for all considered source intensities and sizes. Further, compared with conventional l2 regularized algorithm, overall, the proposed method reconstructed substantially more accurate fluorescence distribution. The proposed method shows considerable promise and will be tested using more realistic simulations and experimental setups.

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

PubMed

Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

2017-07-01

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Background fluorescence estimation and vesicle segmentation in live cell imaging with conditional random fields.

PubMed

Pécot, Thierry; Bouthemy, Patrick; Boulanger, Jérôme; Chessel, Anatole; Bardin, Sabine; Salamero, Jean; Kervrann, Charles

2015-02-01

Image analysis applied to fluorescence live cell microscopy has become a key tool in molecular biology since it enables to characterize biological processes in space and time at the subcellular level. In fluorescence microscopy imaging, the moving tagged structures of interest, such as vesicles, appear as bright spots over a static or nonstatic background. In this paper, we consider the problem of vesicle segmentation and time-varying background estimation at the cellular scale. The main idea is to formulate the joint segmentation-estimation problem in the general conditional random field framework. Furthermore, segmentation of vesicles and background estimation are alternatively performed by energy minimization using a min cut-max flow algorithm. The proposed approach relies on a detection measure computed from intensity contrasts between neighboring blocks in fluorescence microscopy images. This approach permits analysis of either 2D + time or 3D + time data. We demonstrate the performance of the so-called C-CRAFT through an experimental comparison with the state-of-the-art methods in fluorescence video-microscopy. We also use this method to characterize the spatial and temporal distribution of Rab6 transport carriers at the cell periphery for two different specific adhesion geometries.

Bessel beam fluorescence lifetime tomography of live embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Peng, Leilei

2016-03-01

Optical tomography allows isotropic 3D imaging of embryos. Scanning-laser optical tomography (SLOT) has superior light collecting efficiency than wide-field optical tomography, making it ideal for fluorescence imaging of live embryos. We previously reported an imaging system that combines SLOT with a novel Fourier-multiplexed fluorescence lifetime imaging (FmFLIM) technique named FmFLIM-SLOT. FmFLIM-SLOT performs multiplexed FLIM-FRET readout of multiple FRET sensors in live embryos. Here we report a recent effort on improving the spatial resolution of the FmFLIM-SLOT system in order to image complex biochemical processes in live embryos at the cellular level. Optical tomography has to compromise between resolution and the depth of view. In SLOT, the commonly-used focused Gaussian beam diverges quickly from the focal plane, making it impossible to achieve high resolution imaging in a large volume specimen. We thus introduce Bessel beam laser-scanning tomography, which illuminates the sample with a spatial-light-modulator-generated Bessel beam that has an extended focal depth. The Bessel beam is scanned across the whole specimen. Fluorescence projection images are acquired at equal angular intervals as the sample rotates. Reconstruction artifacts due to annular-rings of the Bessel beam are removed by a modified 3D filtered back projection algorithm. Furthermore, in combination of Fourier-multiplexing fluorescence lifetime imaging (FmFLIM) method, the Bessel FmFLIM-SLOT system is capable of perform 3D lifetime imaging of live embryos at cellular resolution. The system is applied to in-vivo imaging of transgenic Zebrafish embryos. Results prove that Bessel FmFLIM-SLOT is a promising imaging method in development biology research.

MULTISCALE TENSOR ANISOTROPIC FILTERING OF FLUORESCENCE MICROSCOPY FOR DENOISING MICROVASCULATURE.

PubMed

Prasath, V B S; Pelapur, R; Glinskii, O V; Glinsky, V V; Huxley, V H; Palaniappan, K

2015-04-01

Fluorescence microscopy images are contaminated by noise and improving image quality without blurring vascular structures by filtering is an important step in automatic image analysis. The application of interest here is to automatically extract the structural components of the microvascular system with accuracy from images acquired by fluorescence microscopy. A robust denoising process is necessary in order to extract accurate vascular morphology information. For this purpose, we propose a multiscale tensor with anisotropic diffusion model which progressively and adaptively updates the amount of smoothing while preserving vessel boundaries accurately. Based on a coherency enhancing flow with planar confidence measure and fused 3D structure information, our method integrates multiple scales for microvasculature preservation and noise removal membrane structures. Experimental results on simulated synthetic images and epifluorescence images show the advantage of our improvement over other related diffusion filters. We further show that the proposed multiscale integration approach improves denoising accuracy of different tensor diffusion methods to obtain better microvasculature segmentation.

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

PubMed

Siegel, Nisan; Brooker, Gary

2014-09-22

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

USDA-ARS?s Scientific Manuscript database

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

Fluorescence hyperspectral imaging technique for the foreign substance detection on fresh-cut lettuce

USDA-ARS?s Scientific Manuscript database

Nondestructive methods based on fluorescence hyperspectral imaging (HSI) techniques were developed in order to detect worms on fresh-cut lettuce. The optimal wavebands for detecting worms on fresh-cut lettuce were investigated using the one-way ANOVA analysis and correlation analysis. The worm detec...

Ultrasound guided fluorescence molecular tomography with improved quantification by an attenuation compensated born-normalization and in vivo preclinical study of cancer

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

Li, Baoqiang; Berti, Romain; Abran, Maxime

2014-05-15

Ultrasound imaging, having the advantages of low-cost and non-invasiveness over MRI and X-ray CT, was reported by several studies as an adequate complement to fluorescence molecular tomography with the perspective of improving localization and quantification of fluorescent molecular targets in vivo. Based on the previous work, an improved dual-modality Fluorescence-Ultrasound imaging system was developed and then validated in imaging study with preclinical tumor model. Ultrasound imaging and a profilometer were used to obtain the anatomical prior information and 3D surface, separately, to precisely extract the tissue boundary on both sides of sample in order to achieve improved fluorescence reconstruction. Furthermore,more » a pattern-based fluorescence reconstruction on the detection side was incorporated to enable dimensional reduction of the dataset while keeping the useful information for reconstruction. Due to its putative role in the current imaging geometry and the chosen reconstruction technique, we developed an attenuation compensated Born-normalization method to reduce the attenuation effects and cancel off experimental factors when collecting quantitative fluorescence datasets over large area. Results of both simulation and phantom study demonstrated that fluorescent targets could be recovered accurately and quantitatively using this reconstruction mechanism. Finally, in vivo experiment confirms that the imaging system associated with the proposed image reconstruction approach was able to extract both functional and anatomical information, thereby improving quantification and localization of molecular targets.« less

Dental fluorosis in populations from Chiang Mai, Thailand with different fluoride exposures - Paper 2: The ability of fluorescence imaging to detect differences in fluorosis prevalence and severity for different fluoride intakes from water

PubMed Central

2012-01-01

Background To assess the ability of fluorescence imaging to detect a dose response relationship between fluorosis severity and different levels of fluoride in water supplies compared to remote photographic scoring in selected populations participating in an observational, epidemiological survey in Chiang Mai, Thailand. Methods Subjects were male and female lifetime residents aged 8-13 years. For each child the fluoride content of cooking water samples (CWS) was assessed to create categorical intervals of water fluoride concentration. Fluorescence images were taken of the maxillary central incisors and analyzed for dental fluorosis using two different software techniques. Output metrics for the fluorescence imaging techniques were compared to TF scores from blinded photographic scores obtained from the survey. Results Data from 553 subjects were available. Both software analysis techniques demonstrated significant correlations with the photographic scores. The metrics for area effected by fluorosis and the overall fluorescence loss had the strongest association with the photographic TF score (Spearman’s rho 0.664 and 0.652 respectively). Both software techniques performed well for comparison of repeat fluorescence images with ICC values of 0.95 and 0.85 respectively. Conclusions This study supports the potential use of fluorescence imaging for the objective quantification of dental fluorosis. Fluorescence imaging was able to discriminate between populations with different fluoride exposures on a comparable level to remote photographic scoring with acceptable levels of repeatability. PMID:22908997

Synthesis and bio-applications of targeted magnetic-fluorescent composite nanoparticles

NASA Astrophysics Data System (ADS)

Xia, Hui; Tong, Ruijie; Song, Yanling; Xiong, Fang; Li, Jiman; Wang, Shichao; Fu, Huihui; Wen, Jirui; Li, Dongze; Zeng, Ye; Zhao, Zhiwei; Wu, Jiang

2017-04-01

Magnetic-fluorescent nanoparticles have a tremendous potential in biology. As the benefits of these materials gained recognition, increasing attention has been given to the conjugation of magnetic-fluorescent nanoparticles with targeting ligands. The magnetic and fluorescent properties of nanoparticles offer several functionalities, including imaging, separation, and visualization, while the presence of a targeting ligand allows for selective cell and tissue targeting. In this review, methods for the synthesis of targeted magnetic-fluorescent nanoparticles are explored, and recent applications of these nanocomposites to the detection and separation of biomolecules, fluorescent and magnetic resonance imaging, and cancer diagnosis and treatment will be summarized. As these materials are further optimized, targeted magnetic-fluorescent nanoparticles hold great promise for the diagnosis and treatment of some diseases.

Self-interference fluorescence microscopy with three-phase detection for depth-resolved confocal epi-fluorescence imaging.

PubMed

Braaf, Boy; de Boer, Johannes F

2017-03-20

Three-dimensional confocal fluorescence imaging of in vivo tissues is challenging due to sample motion and limited imaging speeds. In this paper a novel method is therefore presented for scanning confocal epi-fluorescence microscopy with instantaneous depth-sensing based on self-interference fluorescence microscopy (SIFM). A tabletop epi-fluorescence SIFM setup was constructed with an annular phase plate in the emission path to create a spectral self-interference signal that is phase-dependent on the axial position of a fluorescent sample. A Mach-Zehnder interferometer based on a 3 × 3 fiber-coupler was developed for a sensitive phase analysis of the SIFM signal with three photon-counter detectors instead of a spectrometer. The Mach-Zehnder interferometer created three intensity signals that alternately oscillated as a function of the SIFM spectral phase and therefore encoded directly for the axial sample position. Controlled axial translation of fluorescent microsphere layers showed a linear dependence of the SIFM spectral phase with sample depth over axial image ranges of 500 µm and 80 µm (3.9 × Rayleigh range) for 4 × and 10 × microscope objectives respectively. In addition, SIFM was in good agreement with optical coherence tomography depth measurements on a sample with indocyanine green dye filled capillaries placed at multiple depths. High-resolution SIFM imaging applications are demonstrated for fluorescence angiography on a dye-filled capillary blood vessel phantom and for autofluorescence imaging on an ex vivo fly eye.

A current-assisted CMOS photonic sampler with two taps for fluorescence lifetime sensing

NASA Astrophysics Data System (ADS)

Ingelberts, H.; Kuijk, M.

2016-04-01

Imaging based on fluorescence lifetime is becoming increasingly important in medical and biological applications. State-of- the-art fluorescence lifetime microscopes either use bulky and expensive gated image intensifiers coupled to a CCD or single-photon detectors in a slow scanning setup. Numerous attempts are being made to create compact, cost-effective all- CMOS imagers for fluorescence lifetime sensing. Single-photon avalanche diode (SPAD) imagers can have very good timing resolution and noise characteristics but have low detection efficiency. Another approach is to use CMOS imagers based on demodulation detectors. These imagers can be either very fast or very efficient but it remains a challenge to combine both characteristics. Recently we developed the current-assisted photonic sampler (CAPS) to tackle these problems and in this work, we present a new CAPS with two detection taps that can sample a fluorescence decay in two time windows. In the case of mono-exponential decays, two windows provide enough information to resolve the lifetime. We built an electro-optical setup to characterize the detector and use it for fluorescence lifetime measurements. It consists of a supercontinuum pulsed laser source, an optical system to focus light into the detector and picosecond timing electronics. We describe the structure and operation of the two-tap CAPS and provide basic characterization of the speed performance at multiple wavelengths in the visible and near-infrared spectrum. We also record fluorescence decays of different visible and NIR fluorescent dyes and provide different methods to resolve the fluorescence lifetime.

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).

Development of fluorescent probes based on protection-deprotection of the key functional groups for biological imaging.

PubMed

Tang, Yonghe; Lee, Dayoung; Wang, Jiaoliang; Li, Guanhan; Yu, Jinghua; Lin, Weiying; Yoon, Juyoung

2015-08-07

Recently, the strategy of protection-deprotection of functional groups has been widely employed to design fluorescent probes, as the protection-deprotection of functional groups often induces a marked change in electronic properties. Significant advances have been made in the development of analyte-responsive fluorescent probes based on the protection-deprotection strategy. In this tutorial review, we highlight the representative examples of small-molecule based fluorescent probes for bioimaging, which are operated via the protection-deprotection of key functional groups such as aldehyde, hydroxyl, and amino functional groups reported from 2010 to 2014. The discussion includes the general protection-deprotection methods for aldehyde, hydroxyl, or amino groups, as well as the design strategies, sensing mechanisms, and deprotection modes of the representative fluorescent imaging probes applied to bio-imaging.

Fluorescence-based endoscopic imaging of Thomsen-Friedenreich antigen to improve early detection of colorectal cancer.

PubMed

Sakuma, Shinji; Yu, James Y H; Quang, Timothy; Hiwatari, Ken-Ichiro; Kumagai, Hironori; Kao, Stephanie; Holt, Alex; Erskind, Jalysa; McClure, Richard; Siuta, Michael; Kitamura, Tokio; Tobita, Etsuo; Koike, Seiji; Wilson, Kevin; Richards-Kortum, Rebecca; Liu, Eric; Washington, Kay; Omary, Reed; Gore, John C; Pham, Wellington

2015-03-01

Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level. © 2014 UICC.

Full-direct method for imaging pharmacokinetic parameters in dynamic fluorescence molecular tomography

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

Zhang, Guanglei, E-mail: guangleizhang@bjtu.edu.cn; Department of Biomedical Engineering, School of Computer and Information Technology, Beijing Jiaotong University, Beijing 100044; Pu, Huangsheng

2015-02-23

Images of pharmacokinetic parameters (also known as parametric images) in dynamic fluorescence molecular tomography (FMT) can provide three-dimensional metabolic information for biological studies and drug development. However, the ill-posed nature of FMT and the high temporal variation of fluorophore concentration together make it difficult to obtain accurate parametric images in small animals in vivo. In this letter, we present a method to directly reconstruct the parametric images from the boundary measurements based on hybrid FMT/X-ray computed tomography (XCT) system. This method can not only utilize structural priors obtained from the XCT system to mitigate the ill-posedness of FMT but alsomore » make full use of the temporal correlations of boundary measurements to model the high temporal variation of fluorophore concentration. The results of numerical simulation and mouse experiment demonstrate that the proposed method leads to significant improvements in the reconstruction quality of parametric images.« less

Modern Micro and Nanoparticle-Based Imaging Techniques

PubMed Central

Ryvolova, Marketa; Chomoucka, Jana; Drbohlavova, Jana; Kopel, Pavel; Babula, Petr; Hynek, David; Adam, Vojtech; Eckschlager, Tomas; Hubalek, Jaromir; Stiborova, Marie; Kaiser, Jozef; Kizek, Rene

2012-01-01

The requirements for early diagnostics as well as effective treatment of insidious diseases such as cancer constantly increase the pressure on development of efficient and reliable methods for targeted drug/gene delivery as well as imaging of the treatment success/failure. One of the most recent approaches covering both the drug delivery as well as the imaging aspects is benefitting from the unique properties of nanomaterials. Therefore a new field called nanomedicine is attracting continuously growing attention. Nanoparticles, including fluorescent semiconductor nanocrystals (quantum dots) and magnetic nanoparticles, have proven their excellent properties for in vivo imaging techniques in a number of modalities such as magnetic resonance and fluorescence imaging, respectively. In this article, we review the main properties and applications of nanoparticles in various in vitro imaging techniques, including microscopy and/or laser breakdown spectroscopy and in vivo methods such as magnetic resonance imaging and/or fluorescence-based imaging. Moreover the advantages of the drug delivery performed by nanocarriers such as iron oxides, gold, biodegradable polymers, dendrimers, lipid based carriers such as liposomes or micelles are also highlighted. PMID:23202187

Development of an imaging system for the detection of alumina on turbine blades

NASA Astrophysics Data System (ADS)

Greenwell, S. J.; Kell, J.; Day, J. C. C.

2014-03-01

An imaging system capable of detecting alumina on turbine blades by acquiring LED-induced fluorescence images has been developed. Acquiring fluorescence images at adjacent spectral bands allows the system to distinguish alumina from fluorescent surface contaminants. Repair and overhaul processes require that alumina is entirely removed from the blades by grit blasting and chemical stripping. The capability of the system to detect alumina has been investigated with two series of turbine blades provided by Rolls-Royce plc. The results illustrate that the system provides a superior inspection method to visual assessment when ascertaining whether alumina is present on turbine blades during repair and overhaul processes.

Visualisation of blood and lymphatic vessels with increasing exposure time of the detector

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

Kalchenko, V V; Kuznetsov, Yu L; Meglinski, I V

2013-07-31

We describe the laser speckle contrast method for simultaneous noninvasive imaging of blood and lymphatic vessels of living organisms, based on increasing detector exposure time. In contrast to standard methods of fluorescent angiography, this technique of vascular bed imaging and lymphatic and blood vessel demarcation does not employ toxic fluorescent markers. The method is particularly promising with respect to the physiology of the cardiovascular system under in vivo conditions. (laser applications in biology and medicine)

Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

PubMed

Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike

2010-01-01

An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

Nuclear Forensics Applications of Principal Component Analysis on Micro X-ray Fluorescence Images

DTIC Science & Technology

analysis on quantified micro x-ray fluorescence intensity values. This method is then applied to address goals of nuclear forensics . Thefirst...researchers in the development and validation of nuclear forensics methods. A method for determining material homogeneity is developed and demonstrated

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

Facile and eco-friendly synthesis of green fluorescent carbon nanodots for applications in bioimaging, patterning and staining

NASA Astrophysics Data System (ADS)

Shi, Lihong; Li, Yanyan; Li, Xiaofeng; Wen, Xiangping; Zhang, Guomei; Yang, Jun; Dong, Chuan; Shuang, Shaomin

2015-04-01

We report a facile and eco-friendly strategy for the fabrication of green fluorescent carbon nanodots (CDs), and demonstrate their applications for bio-imaging, patterning, and staining. A one-pot hydrothermal method using various plant petals yields bright green-emitting CDs, providing an easy way for the production of green fluorescent CDs without the need for a tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. The as-prepared CDs show small size distribution and excellent dispersibility. Their strong green fluorescence is observed when the excitation wavelength is between 430 nm and 490 nm. Moreover, they exhibit high tolerance to various external conditions, such as pH values, external cations, and continuous excitation. Due to minimum toxicity as well as good photoluminescence properties, these CDs can be applied to in vitro and in vivo imaging, patterning, and staining. According to confocal fluorescence imaging of human uterine cervical squamous cell carcinoma cells, CDs penetrate into the cell and enter the cytoplasm and the nucleus. More strikingly, carp is directly fed with CDs for in vivo imaging and shows bright green fluorescence at an excitation wavelength of 470 nm. In addition, the obtained CDs are used as fluorescent inks for drawing luminescence patterns. Finally, we also apply the CDs as a fluorescent dye. Interestingly, the absorbent filter paper with staining emits dramatic fluorescence under 470 nm excitation.We report a facile and eco-friendly strategy for the fabrication of green fluorescent carbon nanodots (CDs), and demonstrate their applications for bio-imaging, patterning, and staining. A one-pot hydrothermal method using various plant petals yields bright green-emitting CDs, providing an easy way for the production of green fluorescent CDs without the need for a tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. The as-prepared CDs show small size distribution and excellent dispersibility. Their strong green fluorescence is observed when the excitation wavelength is between 430 nm and 490 nm. Moreover, they exhibit high tolerance to various external conditions, such as pH values, external cations, and continuous excitation. Due to minimum toxicity as well as good photoluminescence properties, these CDs can be applied to in vitro and in vivo imaging, patterning, and staining. According to confocal fluorescence imaging of human uterine cervical squamous cell carcinoma cells, CDs penetrate into the cell and enter the cytoplasm and the nucleus. More strikingly, carp is directly fed with CDs for in vivo imaging and shows bright green fluorescence at an excitation wavelength of 470 nm. In addition, the obtained CDs are used as fluorescent inks for drawing luminescence patterns. Finally, we also apply the CDs as a fluorescent dye. Interestingly, the absorbent filter paper with staining emits dramatic fluorescence under 470 nm excitation. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr00783f

Green preparation of carbon dots with mangosteen pulp for the selective detection of Fe3+ ions and cell imaging

NASA Astrophysics Data System (ADS)

Yang, Rui; Guo, Xiangfeng; Jia, Lihua; Zhang, Yu; Zhao, Zhenlong; Lonshakov, Fedor

2017-11-01

A simple method was developed in the synthesis of fluorescent carbon dots (referred to as M-CDs), calcined treatment of mangosteen pulp in air, without the assistance of any chemical reagent. The M-CDs possess good-solubility, satisfactory chemical stability and can be applied as the fluorescent temperature probe. More strikingly, the fluorescence of M-CDs can be fleetly and selectively quenched by Fe3+ ions. The phenomenon was used to develop a fluorescent method for facile detection of Fe3+ with a linear range of 0-0.18 mM and a detection limit of 52 nM. Eventually, the M-CDs were applied for cell imaging, demonstrating their potential toward diverse applications.

High throughput analysis of samples in flowing liquid

DOEpatents

Ambrose, W. Patrick; Grace, W. Kevin; Goodwin, Peter M.; Jett, James H.; Orden, Alan Van; Keller, Richard A.

2001-01-01

Apparatus and method enable imaging multiple fluorescent sample particles in a single flow channel. A flow channel defines a flow direction for samples in a flow stream and has a viewing plane perpendicular to the flow direction. A laser beam is formed as a ribbon having a width effective to cover the viewing plane. Imaging optics are arranged to view the viewing plane to form an image of the fluorescent sample particles in the flow stream, and a camera records the image formed by the imaging optics.

Automatic segmentation of fluorescence lifetime microscopy images of cells using multiresolution community detection--a first study.

PubMed

Hu, D; Sarder, P; Ronhovde, P; Orthaus, S; Achilefu, S; Nussinov, Z

2014-01-01

Inspired by a multiresolution community detection based network segmentation method, we suggest an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells in a first pilot investigation on two selected images. The image processing problem is framed as identifying segments with respective average FLTs against the background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network defined using image pixels as the nodes and similarity between the FLTs of the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments, and high network resolution leads to smaller segments. Furthermore, using the proposed method, the mean-square error in estimating the FLT segments in a FLIM image was found to consistently decrease with increasing resolution of the corresponding network. The multiresolution community detection method appeared to perform better than a popular spectral clustering-based method in performing FLIM image segmentation. At high resolution, the spectral segmentation method introduced noisy segments in its output, and it was unable to achieve a consistent decrease in mean-square error with increasing resolution. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Automatic Segmentation of Fluorescence Lifetime Microscopy Images of Cells Using Multi-Resolution Community Detection -A First Study

PubMed Central

Hu, Dandan; Sarder, Pinaki; Ronhovde, Peter; Orthaus, Sandra; Achilefu, Samuel; Nussinov, Zohar

2014-01-01

Inspired by a multi-resolution community detection (MCD) based network segmentation method, we suggest an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells in a first pilot investigation on two selected images. The image processing problem is framed as identifying segments with respective average FLTs against the background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network defined using image pixels as the nodes and similarity between the FLTs of the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments, and high network resolution leads to smaller segments. Further, using the proposed method, the mean-square error (MSE) in estimating the FLT segments in a FLIM image was found to consistently decrease with increasing resolution of the corresponding network. The MCD method appeared to perform better than a popular spectral clustering based method in performing FLIM image segmentation. At high resolution, the spectral segmentation method introduced noisy segments in its output, and it was unable to achieve a consistent decrease in MSE with increasing resolution. PMID:24251410

Joint L1 and Total Variation Regularization for Fluorescence Molecular Tomography

PubMed Central

Dutta, Joyita; Ahn, Sangtae; Li, Changqing; Cherry, Simon R.; Leahy, Richard M.

2012-01-01

Fluorescence molecular tomography (FMT) is an imaging modality that exploits the specificity of fluorescent biomarkers to enable 3D visualization of molecular targets and pathways in vivo in small animals. Owing to the high degree of absorption and scattering of light through tissue, the FMT inverse problem is inherently illconditioned making image reconstruction highly susceptible to the effects of noise and numerical errors. Appropriate priors or penalties are needed to facilitate reconstruction and to restrict the search space to a specific solution set. Typically, fluorescent probes are locally concentrated within specific areas of interest (e.g., inside tumors). The commonly used L2 norm penalty generates the minimum energy solution, which tends to be spread out in space. Instead, we present here an approach involving a combination of the L1 and total variation norm penalties, the former to suppress spurious background signals and enforce sparsity and the latter to preserve local smoothness and piecewise constancy in the reconstructed images. We have developed a surrogate-based optimization method for minimizing the joint penalties. The method was validated using both simulated and experimental data obtained from a mouse-shaped phantom mimicking tissue optical properties and containing two embedded fluorescent sources. Fluorescence data was collected using a 3D FMT setup that uses an EMCCD camera for image acquisition and a conical mirror for full-surface viewing. A range of performance metrics were utilized to evaluate our simulation results and to compare our method with the L1, L2, and total variation norm penalty based approaches. The experimental results were assessed using Dice similarity coefficients computed after co-registration with a CT image of the phantom. PMID:22390906

Differential laser-induced perturbation spectroscopy and fluorescence imaging for biological and materials sensing

NASA Astrophysics Data System (ADS)

Burton, Dallas Jonathan

The field of laser-based diagnostics has been a topic of research in various fields, more specifically for applications in environmental studies, military defense technologies, and medicine, among many others. In this dissertation, a novel laser-based optical diagnostic method, differential laser-induced perturbation spectroscopy (DLIPS), has been implemented in a spectroscopy mode and expanded into an imaging mode in combination with fluorescence techniques. The DLIPS method takes advantage of deep ultraviolet (UV) laser perturbation at sub-ablative energy fluences to photochemically cleave bonds and alter fluorescence signal response before and after perturbation. The resulting difference spectrum or differential image adds more information about the target specimen, and can be used in combination with traditional fluorescence techniques for detection of certain materials, characterization of many materials and biological specimen, and diagnosis of various human skin conditions. The differential aspect allows for mitigation of patient or sample variation, and has the potential to develop into a powerful, noninvasive optical sensing tool. The studies in this dissertation encompass efforts to continue the fundamental research on DLIPS including expansion of the method to an imaging mode. Five primary studies have been carried out and presented. These include the use of DLIPS in a spectroscopy mode for analysis of nitrogen-based explosives on various substrates, classification of Caribbean fruit flies versus Caribbean fruit flies that have been irradiated with gamma rays, and diagnosis of human skin cancer lesions. The nitrogen-based explosives and Caribbean fruit flies have been analyzed with the DLIPS scheme using the imaging modality, providing complementary information to the spectroscopic scheme. In each study, a comparison between absolute fluorescence signals and DLIPS responses showed that DLIPS statistically outperformed traditional fluorescence techniques with regards to regression error and classification.

Homing peptide guiding optical molecular imaging for the diagnosis of bladder cancer

NASA Astrophysics Data System (ADS)

Yang, Xiao-feng; Pang, Jian-zhi; Liu, Jie-hao; Zhao, Yang; Jia, Xing-you; Li, Jun; Liu, Reng-xin; Wang, Wei; Fan, Zhen-wei; Zhang, Zi-qiang; Yan, San-hua; Luo, Jun-qian; Zhang, Xiao-lei

2014-11-01

Background: The limitations of primary transurethral resection of bladder tumor (TURBt) have led the residual tumors rates as high as 75%. The intraoperative fluorescence imaging offers a great potential for improving TURBt have been confirmed. So we aim to distinguish the residual tumors and normal mucosa using fluorescence molecular imaging formed by conjugated molecule of the CSNRDARRC bladder cancer homing peptide with fluorescent dye. The conjugated molecule was abbreviated FIuo-ACP. In our study, we will research the image features of FIuo-ACP probe targeted bladder cancer for fluorescence molecular imaging diagnosis for bladder cancer in vivo and ex vivo. Methods: After the FIuo-ACP probe was synthetized, the binding sites, factors affecting binding rates, the specificity and the targeting of Fluo-ACP labeled with bladder cancer cells were studied respectively by laser scanning confocal microscope (LSCM), immunofluorescence and multispectral fluorescence ex vivo optical molecular imaging system. Results: The binding sites were located in nucleus and the binding rates were correlated linearly with the dose of probe and the grade of pathology. Moreover, the probe has a binding specificity with bladder cancer in vivo and ex vivo. Tumor cells being labeled by the Fluo-ACP, bright green spots were observed under LSCM. The tissue samples and tumor cells can be labeled and identified by fluorescence microscope. Optical molecular imaging of xenograft tumor tissues was exhibited as fluorescent spots under EMCCD. Conclusion: The CSNRDARRC peptides might be a useful bladder cancer targeting vector. The FIuo-ACP molecular probe was suitable for fluorescence molecular imaging diagnosis for bladder cancer in vivo and ex vivo.

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.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

PubMed

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-12-14

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections.

PubMed

Wegner, Kyle A; Keikhosravi, Adib; Eliceiri, Kevin W; Vezina, Chad M

2017-08-01

The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.

Computer Reconstruction of Plant Growth and Chlorophyll Fluorescence Emission in Three Spatial Dimensions

PubMed Central

Bellasio, Chandra; Olejníčková, Julie; Tesař, Radek; Šebela, David; Nedbal, Ladislav

2012-01-01

Plant leaves grow and change their orientation as well their emission of chlorophyll fluorescence in time. All these dynamic plant properties can be semi-automatically monitored by a 3D imaging system that generates plant models by the method of coded light illumination, fluorescence imaging and computer 3D reconstruction. Here, we describe the essentials of the method, as well as the system hardware. We show that the technique can reconstruct, with a high fidelity, the leaf size, the leaf angle and the plant height. The method fails with wilted plants when leaves overlap obscuring their true area. This effect, naturally, also interferes when the method is applied to measure plant growth under water stress. The method is, however, very potent in capturing the plant dynamics under mild stress and without stress. The 3D reconstruction is also highly effective in correcting geometrical factors that distort measurements of chlorophyll fluorescence emission of naturally positioned plant leaves. PMID:22368511

Computer reconstruction of plant growth and chlorophyll fluorescence emission in three spatial dimensions.

PubMed

Bellasio, Chandra; Olejníčková, Julie; Tesař, Radek; Sebela, David; Nedbal, Ladislav

2012-01-01

Plant leaves grow and change their orientation as well their emission of chlorophyll fluorescence in time. All these dynamic plant properties can be semi-automatically monitored by a 3D imaging system that generates plant models by the method of coded light illumination, fluorescence imaging and computer 3D reconstruction. Here, we describe the essentials of the method, as well as the system hardware. We show that the technique can reconstruct, with a high fidelity, the leaf size, the leaf angle and the plant height. The method fails with wilted plants when leaves overlap obscuring their true area. This effect, naturally, also interferes when the method is applied to measure plant growth under water stress. The method is, however, very potent in capturing the plant dynamics under mild stress and without stress. The 3D reconstruction is also highly effective in correcting geometrical factors that distort measurements of chlorophyll fluorescence emission of naturally positioned plant leaves.

High speed fluorescence imaging with compressed ultrafast photography

NASA Astrophysics Data System (ADS)

Thompson, J. V.; Mason, J. D.; Beier, H. T.; Bixler, J. N.

2017-02-01

Fluorescent lifetime imaging is an optical technique that facilitates imaging molecular interactions and cellular functions. Because the excited lifetime of a fluorophore is sensitive to its local microenvironment,1, 2 measurement of fluorescent lifetimes can be used to accurately detect regional changes in temperature, pH, and ion concentration. However, typical state of the art fluorescent lifetime methods are severely limited when it comes to acquisition time (on the order of seconds to minutes) and video rate imaging. Here we show that compressed ultrafast photography (CUP) can be used in conjunction with fluorescent lifetime imaging to overcome these acquisition rate limitations. Frame rates up to one hundred billion frames per second have been demonstrated with compressed ultrafast photography using a streak camera.3 These rates are achieved by encoding time in the spatial direction with a pseudo-random binary pattern. The time domain information is then reconstructed using a compressed sensing algorithm, resulting in a cube of data (x,y,t) for each readout image. Thus, application of compressed ultrafast photography will allow us to acquire an entire fluorescent lifetime image with a single laser pulse. Using a streak camera with a high-speed CMOS camera, acquisition rates of 100 frames per second can be achieved, which will significantly enhance our ability to quantitatively measure complex biological events with high spatial and temporal resolution. In particular, we will demonstrate the ability of this technique to do single-shot fluorescent lifetime imaging of cells and microspheres.

Fluorescence-enhanced optical tomography and nuclear imaging system for small animals

NASA Astrophysics Data System (ADS)

Tan, I.-Chih; Lu, Yujie; Darne, Chinmay; Rasmussen, John C.; Zhu, Banghe; Azhdarinia, Ali; Yan, Shikui; Smith, Anne M.; Sevick-Muraca, Eva M.

2012-03-01

Near-infrared (NIR) fluorescence is an alternative modality for molecular imaging that has been demonstrated in animals and recently in humans. Fluorescence-enhanced optical tomography (FEOT) using continuous wave or frequency domain photon migration techniques could be used to provide quantitative molecular imaging in vivo if it could be validated against "gold-standard," nuclear imaging modalities, using dual-labeled imaging agents. Unfortunately, developed FEOT systems are not suitable for incorporation with CT/PET/SPECT scanners because they utilize benchtop devices and require a large footprint. In this work, we developed a miniaturized fluorescence imaging system installed in the gantry of the Siemens Inveon PET/CT scanner to enable NIR transillumination measurements. The system consists of a CCD camera equipped with NIR sensitive intensifier, a diode laser controlled by a single board compact controller, a 2-axis galvanometer, and RF circuit modules for homodyne detection of the phase and amplitude of fluorescence signals. The performance of the FEOT system was tested and characterized. A mouse-shaped solid phantom of uniform optical properties with a fluorescent inclusion was scanned using CT, and NIR fluorescence images at several projections were collected. The method of high-order approximation to the radioactive transfer equation was then used to reconstruct the optical images. Dual-labeled agents were also used on a tumor bearing mouse to validate the results of the FEOT against PET/CT image. The results showed that the location of the fluorophore obtained from the FEOT matches the location of tumor obtained from the PET/CT images. Besides validation of FEOT, this hybrid system could allow multimodal molecular imaging (FEOT/PET/CT) for small animal imaging.

Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Ahmed, Syeed Ehsan

Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level.

Near-Infrared Imaging for the Assessment of Anastomotic Patency, Thrombosis, and Reperfusion in Microsurgery: A Pilot Study in a Porcine Model

PubMed Central

Vargas, Christina R.; Nguyen, John T.; Ashitate, Yoshitomo; Silvestre, Jason; Venugopal, Vivek; Neacsu, Florin; Kettenring, Frank; Frangioni, John V.; Gioux, Sylvain; Lee, Bernard T.

2015-01-01

Background Advances in microsurgical techniques have increased the use of free tissue transfer. Methods of intraoperative flap perfusion assessment, however, still rely primarily on subjective evaluation of traditional clinical parameters. Anastomotic thrombosis, if not expeditiously identified and revised, can result in flap loss with significant associated morbidity. This study aims to evaluate the use of near-infrared (NIR) fluorescence imaging in the assessment of microsurgical anastomotic patency, thrombosis, and vascular revision. Materials and Methods A model of pedicle thrombosis was created using bilateral abdominal flaps isolated on deep superior epigastric vascular pedicles in four Yorkshire pigs. Following flap elevation, microvascular arterial and venous anastomoses were performed unilaterally, preserving an intact contralateral control flap. Thrombosis was induced at the arterial anastomosis site using ferric chloride, and both flaps imaged using NIR fluorescence angiography. The thrombosed vascular segments were subsequently excised and new anastomoses performed to restore flow. Follow-up imaging of both flaps was then obtained to confirm patency using fluorescence imaging technology. Results Pedicled abdominal flaps were created and successful anastomotic thrombosis was induced unilaterally in each pig. Fluorescence imaging technology identified large decreases in tissue perfusion of the thrombosed flap within 2 minutes. After successful revision anastomosis, NIR imaging demonstrated dramatic increase in flow to the reconstructed flap, but intensity did not return to pre-thrombosis levels. Conclusions Early identification of anastomotic thrombosis is important in successful free tissue transfer. Real-time, intraoperative evaluation of flap perfusion, anastomotic thrombosis, and successful revision can be performed using NIR fluorescence imaging. PMID:25571855

Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

PubMed Central

Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R.

2013-01-01

Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821

Sentinel lymph nodes detection with an imaging system using Patent Blue V dye as fluorescent tracer

NASA Astrophysics Data System (ADS)

Tellier, F.; Steibel, J.; Chabrier, R.; Rodier, J. F.; Pourroy, G.; Poulet, P.

2013-03-01

Sentinel lymph node biopsy is the gold standard to detect metastatic invasion from primary breast cancer. This method can help patients avoid full axillary chain dissection, thereby decreasing the risk of morbidity. We propose an alternative to the traditional isotopic method, to detect and map the sentinel lymph nodes. Indeed, Patent Blue V is the most widely used dye in clinical routine for the visual detection of sentinel lymph nodes. A Recent study has shown the possibility of increasing the fluorescence quantum yield of Patent Blue V, when it is bound to human serum albumin. In this study we present a preclinical fluorescence imaging system to detect sentinel lymph nodes labeled with this fluorescent tracer. The setup is composed of a black and white CCD camera and two laser sources. One excitation source with a laser emitting at 635 nm and a second laser at 785 nm to illuminate the region of interest. The prototype is operated via a laptop. Preliminary experiments permitted to determine the device sensitivity in the μmol.L-1 range as regards the detection of PBV fluorescence signals. We also present a preclinical evaluation performed on Lewis rats, during which the fluorescence imaging setup detected the accumulation and fixation of the fluorescent dye on different nodes through the skin.

Online multispectral fluorescence lifetime values estimation and overlay onto tissue white-light video frames

NASA Astrophysics Data System (ADS)

Gorpas, Dimitris; Ma, Dinglong; Bec, Julien; Yankelevich, Diego R.; Marcu, Laura

2016-03-01

Fluorescence lifetime imaging has been shown to be a robust technique for biochemical and functional characterization of tissues and to present great potential for intraoperative tissue diagnosis and guidance of surgical procedures. We report a technique for real-time mapping of fluorescence parameters (i.e. lifetime values) onto the location from where the fluorescence measurements were taken. This is achieved by merging a 450 nm aiming beam generated by a diode laser with the excitation light in a single delivery/collection fiber and by continuously imaging the region of interest with a color CMOS camera. The interrogated locations are then extracted from the acquired frames via color-based segmentation of the aiming beam. Assuming a Gaussian profile of the imaged aiming beam, the segmentation results are fitted to ellipses that are dynamically scaled at the full width of three automatically estimated thresholds (50%, 75%, 90%) of the Gaussian distribution's maximum value. This enables the dynamic augmentation of the white-light video frames with the corresponding fluorescence decay parameters. A fluorescence phantom and fresh tissue samples were used to evaluate this method with motorized and hand-held scanning measurements. At 640x512 pixels resolution the area of interest augmented with fluorescence decay parameters can be imaged at an average 34 frames per second. The developed method has the potential to become a valuable tool for real-time display of optical spectroscopy data during continuous scanning applications that subsequently can be used for tissue characterization and diagnosis.

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography).

PubMed

Siegel, Nisan; Storrie, Brian; Bruce, Marc; Brooker, Gary

2015-02-07

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material1

PubMed Central

Bell, Karen; Mitchell, Steve; Paultre, Danae; Posch, Markus; Oparka, Karl

2013-01-01

Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology. PMID:23457228

Endoscopic fluorescence imaging for early assessment of anastomotic recurrence of Crohn's disease

NASA Astrophysics Data System (ADS)

Mordon, Serge R.; Maunoury, Vincent; Geboes, K.; Klein, Olivier; Desreumaux, P.; Debaert, A.; Colombel, Jean-Frederic

1999-02-01

Crohn's disease is an inflammatory bowel disease of unknown etiology. The mechanism of the initial mucosal alterations is still unclear: ulcerations overlying lymphoid follicles and/or vasculitis have been proposed as the early lesions. We have developed a new and original method combining endoscopy of fluorescence angiography for identifying the early pathological lesions, occurring in the neo-terminal ileum after right ileocolonic resection. The patient population consisted of 10 subjects enrolled in a prospective protocol of endoscopic follow-up at 3 and 12 months after surgery. Fluorescence imaging showed small spots giving a bright fluorescence distributed singly in mucosa which appeared normal in routine endoscopy. Histopathological examination demonstrated that the fluorescence of small spots originated from small, usually superficial, erosive lesions. In several cases, these erosive lesions occurred over lymphoid follicles. Endoscopic fluorescence imaging provides a suitable means of investigating the initial aspect of the Crohn's disease process in displaying some correlative findings between fluorescent aspects and early pathological mucosal alterations.

Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds

NASA Astrophysics Data System (ADS)

Wu, Tsai-Jung; Tzeng, Yan-Kai; Chang, Wei-Wei; Cheng, Chi-An; Kuo, Yung; Chien, Chin-Hsiang; Chang, Huan-Cheng; Yu, John

2013-09-01

Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45-CD54+CD157+ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resolution. Fluorescent nanodiamond labelling did not eliminate the cells' properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labelled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after intravenous transplantation.

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

PubMed Central

Kopek, Benjamin G.; Shtengel, Gleb; Xu, C. Shan; Clayton, David A.; Hess, Harald F.

2012-01-01

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to “colorize” detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging. PMID:22474357

Determining the distribution of probes between different subcellular locations through automated unmixing of subcellular patterns.

PubMed

Peng, Tao; Bonamy, Ghislain M C; Glory-Afshar, Estelle; Rines, Daniel R; Chanda, Sumit K; Murphy, Robert F

2010-02-16

Many proteins or other biological macromolecules are localized to more than one subcellular structure. The fraction of a protein in different cellular compartments is often measured by colocalization with organelle-specific fluorescent markers, requiring availability of fluorescent probes for each compartment and acquisition of images for each in conjunction with the macromolecule of interest. Alternatively, tailored algorithms allow finding particular regions in images and quantifying the amount of fluorescence they contain. Unfortunately, this approach requires extensive hand-tuning of algorithms and is often cell type-dependent. Here we describe a machine-learning approach for estimating the amount of fluorescent signal in different subcellular compartments without hand tuning, requiring only the acquisition of separate training images of markers for each compartment. In testing on images of cells stained with mixtures of probes for different organelles, we achieved a 93% correlation between estimated and expected amounts of probes in each compartment. We also demonstrated that the method can be used to quantify drug-dependent protein translocations. The method enables automated and unbiased determination of the distributions of protein across cellular compartments, and will significantly improve imaging-based high-throughput assays and facilitate proteome-scale localization efforts.

Automatic Cell Segmentation in Fluorescence Images of Confluent Cell Monolayers Using Multi-object Geometric Deformable Model.

PubMed

Yang, Zhen; Bogovic, John A; Carass, Aaron; Ye, Mao; Searson, Peter C; Prince, Jerry L

2013-03-13

With the rapid development of microscopy for cell imaging, there is a strong and growing demand for image analysis software to quantitatively study cell morphology. Automatic cell segmentation is an important step in image analysis. Despite substantial progress, there is still a need to improve the accuracy, efficiency, and adaptability to different cell morphologies. In this paper, we propose a fully automatic method for segmenting cells in fluorescence images of confluent cell monolayers. This method addresses several challenges through a combination of ideas. 1) It realizes a fully automatic segmentation process by first detecting the cell nuclei as initial seeds and then using a multi-object geometric deformable model (MGDM) for final segmentation. 2) To deal with different defects in the fluorescence images, the cell junctions are enhanced by applying an order-statistic filter and principal curvature based image operator. 3) The final segmentation using MGDM promotes robust and accurate segmentation results, and guarantees no overlaps and gaps between neighboring cells. The automatic segmentation results are compared with manually delineated cells, and the average Dice coefficient over all distinguishable cells is 0.88.

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy

PubMed Central

Siegel, Nisan; Brooker, Gary

2014-01-01

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called “CINCH”. PMID:25321701

In vivo assessment of wound re-epithelialization by UV fluorescence excitation imaging

NASA Astrophysics Data System (ADS)

Wang, Ying; Ortega-Martinez, Antonio; Padilla-Martinez, Juan Pablo; Williams, Maura; Farinelli, William; Anderson, R. R.; Franco, Walfre

2017-02-01

Background and Objectives: We have previously demonstrated the efficacy of a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds in vitro. This system can image highly-proliferating cellular processes (295/340 nm excitation/emission wavelengths) to study epithelialization in a cultured wound model. The objective of the current work is to evaluate the suitability of u-FEI for monitoring wound re-epithelialization in vivo. Study Design: Full-thickness wounds were created in the tail of rats and imaged weekly using u-FEI at 295/340nm excitation/emission wavelengths. Histology was used to investigate the correlation between the spatial distribution and intensity of fluorescence and the extent of wound epithelialization. In addition, the expression of the nuclear protein Ki67 was used to confirm the association between the proliferation of keratinocyte cells and the intensity of fluorescence. Results: Keratinocytes forming neo-epidermis exhibited higher fluorescence intensity than the keratinocytes not involved in re-epithelialization. In full-thickness wounds the fluorescence first appeared at the wound edge where keratinocytes initiated the epithelialization process. Fluorescence intensity increased towards the center as the keratinocytes partially covered the wound. As the wound healed, fluorescence decreased at the edges and was present only at the center as the keratinocytes completely covered the wound at day 21. Histology demonstrated that changes in fluorescence intensity from the 295/340nm band corresponded to newly formed epidermis. Conclusions: u-FEI at 295/340nm allows visualization of proliferating keratinocyte cells during re-epithelialization of wounds in vivo, potentially providing a quantitative, objective and simple method for evaluating wound closure in the clinic.

Enabling Histopathological Annotations on Immunofluorescent Images through Virtualization of Hematoxylin and Eosin.

PubMed

Lahiani, Amal; Klaiman, Eldad; Grimm, Oliver

2018-01-01

Medical diagnosis and clinical decisions rely heavily on the histopathological evaluation of tissue samples, especially in oncology. Historically, classical histopathology has been the gold standard for tissue evaluation and assessment by pathologists. The most widely and commonly used dyes in histopathology are hematoxylin and eosin (H&E) as most malignancies diagnosis is largely based on this protocol. H&E staining has been used for more than a century to identify tissue characteristics and structures morphologies that are needed for tumor diagnosis. In many cases, as tissue is scarce in clinical studies, fluorescence imaging is necessary to allow staining of the same specimen with multiple biomarkers simultaneously. Since fluorescence imaging is a relatively new technology in the pathology landscape, histopathologists are not used to or trained in annotating or interpreting these images. To allow pathologists to annotate these images without the need for additional training, we designed an algorithm for the conversion of fluorescence images to brightfield H&E images. In this algorithm, we use fluorescent nuclei staining to reproduce the hematoxylin information and natural tissue autofluorescence to reproduce the eosin information avoiding the necessity to specifically stain the proteins or intracellular structures with an additional fluorescence stain. Our method is based on optimizing a transform function from fluorescence to H&E images using least mean square optimization. It results in high quality virtual H&E digital images that can easily and efficiently be analyzed by pathologists. We validated our results with pathologists by making them annotate tumor in real and virtual H&E whole slide images and we obtained promising results. Hence, we provide a solution that enables pathologists to assess tissue and annotate specific structures based on multiplexed fluorescence images.

Temporal and spatial binning of TCSPC data to improve signal-to-noise ratio and imaging speed

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Beier, Hope T.

2016-03-01

Time-correlated single photon counting (TCSPC) is the most robust method for fluorescence lifetime imaging using laser scanning microscopes. However, TCSPC is inherently slow making it ineffective to capture rapid events due to the single photon product per laser pulse causing extensive acquisition time limitations and the requirement of low fluorescence emission efficiency to avoid bias of measurement towards short lifetimes. Furthermore, thousands of photons per pixel are required for traditional instrument response deconvolution and fluorescence lifetime exponential decay estimation. Instrument response deconvolution and fluorescence exponential decay estimation can be performed in several ways including iterative least squares minimization and Laguerre deconvolution. This paper compares the limitations and accuracy of these fluorescence decay analysis techniques to accurately estimate double exponential decays across many data characteristics including various lifetime values, lifetime component weights, signal-to-noise ratios, and number of photons detected. Furthermore, techniques to improve data fitting, including binning data temporally and spatially, are evaluated as methods to improve decay fits and reduce image acquisition time. Simulation results demonstrate that binning temporally to 36 or 42 time bins, improves accuracy of fits for low photon count data. Such a technique reduces the required number of photons for accurate component estimation if lifetime values are known, such as for commercial fluorescent dyes and FRET experiments, and improve imaging speed 10-fold.

System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents

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

Demos, Stavros; Levenson, Richard

The present disclosure relates to a method for analyzing tissue specimens. In one implementation the method involves obtaining a tissue sample and exposing the sample to one or more fluorophores as contrast agents to enhance contrast of subcellular compartments of the tissue sample. The tissue sample is illuminated by an ultraviolet (UV) light having a wavelength between about 200 nm to about 400 nm, with the wavelength being selected to result in penetration to only a specified depth below a surface of the tissue sample. Inter-image operations between images acquired under different imaging parameters allow for improvement of the imagemore » quality via removal of unwanted image components. A microscope may be used to image the tissue sample and provide the image to an image acquisition system that makes use of a camera. The image acquisition system may create a corresponding image that is transmitted to a display system for processing and display.« less

Volumetric Two-photon Imaging of Neurons Using Stereoscopy (vTwINS)

PubMed Central

Song, Alexander; Charles, Adam S.; Koay, Sue Ann; Gauthier, Jeff L.; Thiberge, Stephan Y.; Pillow, Jonathan W.; Tank, David W.

2017-01-01

Two-photon laser scanning microscopy of calcium dynamics using fluorescent indicators is a widely used imaging method for large scale recording of neural activity in vivo. Here we introduce volumetric Two-photon Imaging of Neurons using Stereoscopy (vTwINS), a volumetric calcium imaging method that employs an elongated, V-shaped point spread function to image a 3D brain volume. Single neurons project to spatially displaced “image pairs” in the resulting 2D image, and the separation distance between images is proportional to depth in the volume. To demix the fluorescence time series of individual neurons, we introduce a novel orthogonal matching pursuit algorithm that also infers source locations within the 3D volume. We illustrate vTwINS by imaging neural population activity in mouse primary visual cortex and hippocampus. Our results demonstrate that vTwINS provides an effective method for volumetric two-photon calcium imaging that increases the number of neurons recorded while maintaining a high frame-rate. PMID:28319111

Laser-Based Flowfield Imaging in a Lean Premixed Prevaporized Sector Combustor

NASA Technical Reports Server (NTRS)

Hicks, Yolanda R.; Locke, Randy J.; Anderson, Robert C.

2005-01-01

OH and fuel planar laser-induced fluorescence (PLIF) is used qualitatively in this study to observe the flame structure resultant from different fuel injector dome configurations within the 3-cup sector combustor test rig. The fluorescence images are compared with some computational fluid dynamics (CFD) results. Interferences in obtaining OH fluorescence signals due to the emission of other species are assessed. NO PLIF images are presented and compared to gas analysis results. The comparison shows that PLIF NO can be an excellent method for measuring NO in the flame. Additionally, we present flow visualization of the molecular species C2.

Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

DOEpatents

Mayer-Cumblidge, M. Uljana; Cao, Haishi

2013-01-15

A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

Cyanine-based probe\\tag-peptide pair for fluorescence protein imaging and fluorescence protein imaging methods

DOEpatents

Mayer-Cumblidge, M Uljana [Richland, WA; Cao, Haishi [Richland, WA

2010-08-17

A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

Brain morphology imaging by 3D microscopy and fluorescent Nissl staining.

PubMed

Lazutkin, A A; Komissarova, N V; Toptunov, D M; Anokhin, K V

2013-07-01

Modern optical methods (multiphoton and light-sheet fluorescent microscopy) allow 3D imaging of large specimens of the brain with cell resolution. It is therefore essential to refer the resultant 3D pictures of expression of transgene, protein, and other markers in the brain to the corresponding structures in the atlas. This implies counterstaining of specimens with morphological dyes. However, there are no methods for contrasting large samples of the brain without their preliminary slicing. We have developed a method for fluorescent Nissl staining of whole brain samples. 3D reconstructions of specimens of the hippocampus, olfactory bulbs, and cortex were created. The method can be used for morphological control and evaluation of the effects of various factors on the brain using 3D microscopy technique.

A synthesis of fluorescent starch based on carbon nanoparticles for fingerprints detection

NASA Astrophysics Data System (ADS)

Li, Hongren; Guo, Xingjia; Liu, Jun; Li, Feng

2016-10-01

A pyrolysis method for synthesizing carbon nanoparticles (CNPs) were developed by using malic acid and ammonium oxalate as raw materials. The incorporation of a minor amount of carbon nanoparticles into starch powder imparts remarkable color-tunability. Based on this phenomenon, an environment friendly fluorescent starch powder for detecting latent fingerprints in non-porous surfaces was prepared. The fingerprints on different non-porous surfaces developed with this powder showed very good fluorescent images under ultraviolet excitation. The method using fluorescent starch powder as fluorescent marks is simple, rapid and green. Experimental results illustrated the effectiveness of proposed methods, enabling its practical applications in forensic sciences.

Characteristics of subgingival calculus detection by multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Tung, Oi-Hong; Lee, Shyh-Yuan; Lai, Yu-Lin; Chen, How-Foo

2011-06-01

Subgingival calculus has been recognized as a major cause of periodontitis, which is one of the main chronic infectious diseases of oral cavities and a principal cause of tooth loss in humans. Bacteria deposited in subgingival calculus or plaque cause gingival inflammation, function deterioration, and then periodontitis. However, subgingival calculus within the periodontal pocket is a complicated and potentially delicate structure to be detected with current dental armamentaria, namely dental x-rays and dental probes. Consequently, complete removal of subgingival calculus remains a challenge to periodontal therapies. In this study, the detection of subgingival calculus employing a multiphoton autofluorescence imaging method was characterized in comparison with a one-photon confocal fluorescence imaging technique. Feasibility of such a system was studied based on fluorescence response of gingiva, healthy teeth, and calculus with and without gingiva covered. The multiphoton fluorescence technology perceived the tissue-covered subgingival calculus that cannot be observed by the one-photon confocal fluorescence method.

Dead-time correction for high-throughput fluorescence lifetime imaging microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Enderlein, Joerg; Ruhlandt, Daja; Chithik, Anna; Ebrecht, René; Wouters, Fred S.; Gregor, Ingo

2016-02-01

Fluorescence lifetime microscopy has become an important method of bioimaging, allowing not only to record intensity and spectral, but also lifetime information across an image. One of the most widely used methods of FLIM is based on Time-Correlated Single Photon Counting (TCSPC). In TCSPC, one determines this curve by exciting molecules with a periodic train of short laser pulses, and then measuring the time delay between the first recorded fluorescence photon after each exciting laser pulse. An important technical detail of TCSPC measurements is the fact that the delay times between excitation laser pulses and resulting fluorescence photons are always measured between a laser pulse and the first fluorescence photon which is detected after that pulse. At high count rates, this leads to so-called pile-up: ``early'' photons eclipse long-delay photons, resulting in heavily skewed TCSPC histograms. To avoid pile-up, a rule of thumb is to perform TCSPC measurements at photon count rates which are at least hundred times smaller than the laser-pulse excitation rate. The downside of this approach is that the fluorescence-photon count-rate is restricted to a value below one hundredth of the laser-pulse excitation-rate, reducing the overall speed with which a fluorescence signal can be measured. We present a new data evaluation method which provides pile-up corrected fluorescence decay estimates from TCSPC measurements at high count rates, and we demonstrate our method on FLIM of fluorescently labeled cells.

Passive and active targeting of quantum dots for whole-body fluorescence imaging of breast cancer xenografts.

PubMed

Balalaeva, Irina V; Zdobnova, Tatiana A; Krutova, Irina V; Brilkina, Anna A; Lebedenko, Ekaterina N; Deyev, Sergey M

2012-11-01

Far-red and near-infrared fluorescent quantum dots (QDs) have become advancing contrast agents for efficient whole-body tumor imaging. In this study, we investigated the possibility of the vital fluorescence imaging of tumor using two contrast agents on the basis of QDs: bioinert QDs coated with polyethyleneglycol and QDs bound with anti-HER2/neu scFv antibodies. HER2/neu-positive breast cancer tumor xenografts in nude mice were used as a model. It was shown that both bioinert and tumor-targeted QD probes can be successfully applied for visualization of the tumor using in vivo imaging method, but fluorescent signal of QD-4D5scFv in tumors was considerably stronger than that of QD-PEG. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

One-pot synthesis of polyamines improved magnetism and fluorescence Fe3O4-carbon dots hybrid NPs for dual modal imaging.

PubMed

Li, Bo; Wang, Xudong; Guo, Yali; Iqbal, Anam; Dong, Yaping; Li, Wu; Liu, Weisheng; Qin, Wenwu; Chen, Shizhen; Zhou, Xin; Yang, Yunhuang

2016-04-07

A one-step hydrothermal method was developed to fabricate Fe3O4-carbon dots (Fe3O4-CDs) magnetic-fluorescent hybrid nanoparticles (NPs). Ferric ammonium citrate (FAC) was used as a cheap and nontoxic iron precursor and as the carbon source. Moreover, triethylenetetramine (TETA) was used to improve the adhesive strength of CDs on Fe3O4 and the fluorescence intensity of CDs. The prepared water-soluble hybrid NPs not only exhibit excellent superparamagnetic properties (Ms = 56.8 emu g(-1)), but also demonstrate excitation-independent photoluminescence for down-conversion and up-conversion at 445 nm. Moreover, the prepared water-soluble Fe3O4-CDs hybrid NPs have a dual modal imaging ability for both magnetic resonance imaging (MRI) and fluorescence imaging.

A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

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

Jung, Kyung oh; Biomedical Sciences, Seoul National University College of Medicine; Cancer Research Institute, Seoul National University College of Medicine

Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and {sup 64}Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescencemore » signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.« less

Automatic choroid cells segmentation and counting based on approximate convexity and concavity of chain code in fluorescence microscopic image

NASA Astrophysics Data System (ADS)

Lu, Weihua; Chen, Xinjian; Zhu, Weifang; Yang, Lei; Cao, Zhaoyuan; Chen, Haoyu

2015-03-01

In this paper, we proposed a method based on the Freeman chain code to segment and count rhesus choroid-retinal vascular endothelial cells (RF/6A) automatically for fluorescence microscopy images. The proposed method consists of four main steps. First, a threshold filter and morphological transform were applied to reduce the noise. Second, the boundary information was used to generate the Freeman chain codes. Third, the concave points were found based on the relationship between the difference of the chain code and the curvature. Finally, cells segmentation and counting were completed based on the characteristics of the number of the concave points, the area and shape of the cells. The proposed method was tested on 100 fluorescence microscopic cell images, and the average true positive rate (TPR) is 98.13% and the average false positive rate (FPR) is 4.47%, respectively. The preliminary results showed the feasibility and efficiency of the proposed method.

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.

Correlative cryogenic tomography of cells using light and soft x-rays

PubMed Central

Smith, Elizabeth A.; Cinquin, Bertrand P.; Do, Myan; McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

2013-01-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryo-preserved state. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited to correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. PMID:24355261

Analytic solutions to modelling exponential and harmonic functions using Chebyshev polynomials: fitting frequency-domain lifetime images with photobleaching.

PubMed

Malachowski, George C; Clegg, Robert M; Redford, Glen I

2007-12-01

A novel approach is introduced for modelling linear dynamic systems composed of exponentials and harmonics. The method improves the speed of current numerical techniques up to 1000-fold for problems that have solutions of multiple exponentials plus harmonics and decaying components. Such signals are common in fluorescence microscopy experiments. Selective constraints of the parameters being fitted are allowed. This method, using discrete Chebyshev transforms, will correctly fit large volumes of data using a noniterative, single-pass routine that is fast enough to analyse images in real time. The method is applied to fluorescence lifetime imaging data in the frequency domain with varying degrees of photobleaching over the time of total data acquisition. The accuracy of the Chebyshev method is compared to a simple rapid discrete Fourier transform (equivalent to least-squares fitting) that does not take the photobleaching into account. The method can be extended to other linear systems composed of different functions. Simulations are performed and applications are described showing the utility of the method, in particular in the area of fluorescence microscopy.

Red fluorescence imaging for dental plaque detection and quantification: pilot study

NASA Astrophysics Data System (ADS)

Liu, Zhao; Gomez, Juliana; Khan, Soniya; Peru, Debbie; Ellwood, Roger

2017-09-01

The red fluorescence of dental plaque originating from porphyrins in oral bacteria may allow visualization, detection, and scoring of plaque without disclosing agents. Two studies were conducted. The first included 24 healthy participants who abstained from oral hygiene for 24 h. Dental plaque was collected from tooth surfaces, and a 10% solution was prepared. These were scanned by a molecular spectrometer to identify the optimum excitation and emission wavelengths of plaque for developing a red fluorescence imaging system. Fourteen healthy subjects completed the second study. After a washout period (1 week), participants had a prophylaxis at baseline and abstained from oral hygiene during the study. They were monitored using the fluorescence imaging system at baseline, 24 h, and 48 h. A dentist clinically assessed plaque after disclosing and on red fluorescence images. Three descriptors were extracted from images and a RUSBoost classifier derived computer fluorescence scores through cross-validation. Red fluorescence plaque levels increased during the 48-h accumulation. Plaque progression was identified by dentist assessment and computer analysis, presenting significant differences between visits at tooth and subject levels (p<0.05). Moderate correlations showed between clinical plaque and red fluorescence plaque (r=0.62 dentist, r=0.55 computer). The best agreement was observed when disclosing plaque threshold at level 2, for both dentist evaluation (sensitivity 71.1%, specificity 67.7%, accuracy 70.2%) and computer classification (sensitivity 68.4%, specificity 62.9%, accuracy 67.1%). Given the correlation with clinical diagnosis, red fluorescence imaging shows its potential for providing an objective and promising method for proper oral hygiene assessment.

A novel Kalman filter based video image processing scheme for two-photon fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sun, Wenqing; Huang, Xia; Li, Chunqiang; Xiao, Chuan; Qian, Wei

2016-03-01

Two-photon fluorescence microscopy (TPFM) is a perfect optical imaging equipment to monitor the interaction between fast moving viruses and hosts. However, due to strong unavoidable background noises from the culture, videos obtained by this technique are too noisy to elaborate this fast infection process without video image processing. In this study, we developed a novel scheme to eliminate background noises, recover background bacteria images and improve video qualities. In our scheme, we modified and implemented the following methods for both host and virus videos: correlation method, round identification method, tree-structured nonlinear filters, Kalman filters, and cell tracking method. After these procedures, most of noises were eliminated and host images were recovered with their moving directions and speed highlighted in the videos. From the analysis of the processed videos, 93% bacteria and 98% viruses were correctly detected in each frame on average.

Intraoperative spatial frequency domain diffuse optical tomography with indo-cyanine green (ICG) fluorescence contrast (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chong, Sang Hoon; Parthasarathy, Ashwin B.; Kavuri, Venkaiah C.; Moscatelli, Frank A.; Singhal, Sunil; Yodh, Arjun G.

2017-02-01

Surgical resection is the most effective treatment strategy for solid tumors, but complete removal of the tumor is critical for post-surgical recovery/long-term survival and is dependent on correct identification of the tumor margin and accurate excision of microscopic residual tumor in the surgical field. Fluorescence image guided surgery is an emerging technique that has shown promise for intraoperative location of tumors and tumor margins. Current versions of such intraoperative fluorescence imaging, however, are generally limited to 2D near-surface images, i.e., without information about tumor depth. Here we present an intraoperative fluorescence imaging system for 3D volumetric imaging of tumors; the system uses spatial frequency domain diffuse optical tomography with an analytic inversion reconstruction method. The new instrument can derive depth-sensitive 3D tumor images at depths up to 1 cm, and it employs compact epi-imaging and illumination suitable for the operating room, with quasi-real-time image reconstruction for surgical visualization. We present experimental results with FDA-approved Indocynanine Green using an extensive array of tissue phantoms and in a pilot in-vivo study.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent.

PubMed

Schwenck, Johannes; Maier, Florian C; Kneilling, Manfred; Wiehr, Stefan; Fuchs, Kerstin

2017-05-08

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models. Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

PubMed Central

Viles, C L; Sieracki, M E

1992-01-01

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured. Images PMID:1610183

Sentinel lymph node biopsy under fluorescent indocyanin green guidance: Initial experience

PubMed Central

Aydoğan, Fatih; Arıkan, Akif Enes; Aytaç, Erman; Velidedeoğlu, Mehmet; Yılmaz, Mehmet Halit; Sager, Muhammet Sait; Çelik, Varol; Uras, Cihan

2016-01-01

Objective: Sentinel lymph node biopsy can be applied by using either blue dye or radionuclide method or both in breast cancer. Fluorescent imaging with indocyanine green is a new defined method. This study evaluates the applicability of sentinel lymph node biopsy via fluorescent indocyanine green. Material and Methods: IC-VIEW (Pulsion Medical Systems AG, Munich, Germany) infrared visualization system was used for imaging. Two mL of indocyanine green was injected to visualize sentinel lymph nodes. After injection, subcutaneous lymphatics were traced and sentinel lymph nodes were found with simultaneous imaging. Sentinel lymph nodes were excised under fluorescent light guidance, and excised lymph nodes were examined histopathologically. Patients with sentinel lymph node metastases underwent axillary dissection. Results: Four patients with sentinel lymph node biopsy due to breast cancer were included in the study. Sentinel lymph nodes were visualized with indocyanine green in all patients. The median number of excised sentinel lymph node was 2 (2–3). Two patients with lymph node metastasis underwent axillary dissection. No metastasis was detected in lymph nodes other than the sentinel nodes in patients with axillary dissection. There was no complication during and after the operation related to the method. Conclusion: According to our limited experience, sentinel lymph node biopsy under fluorescent indocyanine green guidance, which has an advantage of simultaneous visualization, is technically feasible. PMID:26985159

Morphological spot counting from stacked images for automated analysis of gene copy numbers by fluorescence in situ hybridization.

PubMed

Grigoryan, Artyom M; Dougherty, Edward R; Kononen, Juha; Bubendorf, Lukas; Hostetter, Galen; Kallioniemi, Olli

2002-01-01

Fluorescence in situ hybridization (FISH) is a molecular diagnostic technique in which a fluorescent labeled probe hybridizes to a target nucleotide sequence of deoxyribose nucleic acid. Upon excitation, each chromosome containing the target sequence produces a fluorescent signal (spot). Because fluorescent spot counting is tedious and often subjective, automated digital algorithms to count spots are desirable. New technology provides a stack of images on multiple focal planes throughout a tissue sample. Multiple-focal-plane imaging helps overcome the biases and imprecision inherent in single-focal-plane methods. This paper proposes an algorithm for global spot counting in stacked three-dimensional slice FISH images without the necessity of nuclei segmentation. It is designed to work in complex backgrounds, when there are agglomerated nuclei, and in the presence of illumination gradients. It is based on the morphological top-hat transform, which locates intensity spikes on irregular backgrounds. After finding signals in the slice images, the algorithm groups these together to form three-dimensional spots. Filters are employed to separate legitimate spots from fluorescent noise. The algorithm is set in a comprehensive toolbox that provides visualization and analytic facilities. It includes simulation software that allows examination of algorithm performance for various image and algorithm parameter settings, including signal size, signal density, and the number of slices.

Dynamic optical projection of acquired luminescence for aiding oncologic surgery

NASA Astrophysics Data System (ADS)

Sarder, Pinaki; Gullicksrud, Kyle; Mondal, Suman; Sudlow, Gail P.; Achilefu, Samuel; Akers, Walter J.

2013-12-01

Optical imaging enables real-time visualization of intrinsic and exogenous contrast within biological tissues. Applications in human medicine have demonstrated the power of fluorescence imaging to enhance visualization in dermatology, endoscopic procedures, and open surgery. Although few optical contrast agents are available for human medicine at this time, fluorescence imaging is proving to be a powerful tool in guiding medical procedures. Recently, intraoperative detection of fluorescent molecular probes that target cell-surface receptors has been reported for improvement in oncologic surgery in humans. We have developed a novel system, optical projection of acquired luminescence (OPAL), to further enhance real-time guidance of open oncologic surgery. In this method, collected fluorescence intensity maps are projected onto the imaged surface rather than via wall-mounted display monitor. To demonstrate proof-of-principle for OPAL applications in oncologic surgery, lymphatic transport of indocyanine green was visualized in live mice for intraoperative identification of sentinel lymph nodes. Subsequently, peritoneal tumors in a murine model of breast cancer metastasis were identified using OPAL after systemic administration of a tumor-selective fluorescent molecular probe. These initial results clearly show that OPAL can enhance adoption and ease-of-use of fluorescence imaging in oncologic procedures relative to existing state-of-the-art intraoperative imaging systems.

Fluorescent carbon nanoparticles derived from natural materials of mango fruit for bio-imaging probes

NASA Astrophysics Data System (ADS)

Jeong, Chan Jin; Roy, Arup Kumer; Kim, Sung Han; Lee, Jung-Eun; Jeong, Ji Hoon; Insik; Park, Sung Young

2014-11-01

Water soluble fluorescent carbon nanoparticles (FCP) obtained from a single natural source, mango fruit, were developed as unique materials for non-toxic bio-imaging with different colors and particle sizes. The prepared FCPs showed blue (FCP-B), green (FCP-G) and yellow (FCP-Y) fluorescence, derived by the controlled carbonization method. The FCPs demonstrated hydrodynamic diameters of 5-15 nm, holding great promise for clinical applications. The biocompatible FCPs demonstrated great potential in biological fields through the results of in vitro imaging and in vivo biodistribution. Using intravenously administered FCPs with different colored particles, we precisely defined the clearance and biodistribution, showing rapid and efficient urinary excretion for safe elimination from the body. These findings therefore suggest the promising possibility of using natural sources for producing fluorescent materials.Water soluble fluorescent carbon nanoparticles (FCP) obtained from a single natural source, mango fruit, were developed as unique materials for non-toxic bio-imaging with different colors and particle sizes. The prepared FCPs showed blue (FCP-B), green (FCP-G) and yellow (FCP-Y) fluorescence, derived by the controlled carbonization method. The FCPs demonstrated hydrodynamic diameters of 5-15 nm, holding great promise for clinical applications. The biocompatible FCPs demonstrated great potential in biological fields through the results of in vitro imaging and in vivo biodistribution. Using intravenously administered FCPs with different colored particles, we precisely defined the clearance and biodistribution, showing rapid and efficient urinary excretion for safe elimination from the body. These findings therefore suggest the promising possibility of using natural sources for producing fluorescent materials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr04805a

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.

RAMTaB: Robust Alignment of Multi-Tag Bioimages

PubMed Central

Raza, Shan-e-Ahmed; Humayun, Ahmad; Abouna, Sylvie; Nattkemper, Tim W.; Epstein, David B. A.; Khan, Michael; Rajpoot, Nasir M.

2012-01-01

Background In recent years, new microscopic imaging techniques have evolved to allow us to visualize several different proteins (or other biomolecules) in a visual field. Analysis of protein co-localization becomes viable because molecules can interact only when they are located close to each other. We present a novel approach to align images in a multi-tag fluorescence image stack. The proposed approach is applicable to multi-tag bioimaging systems which (a) acquire fluorescence images by sequential staining and (b) simultaneously capture a phase contrast image corresponding to each of the fluorescence images. To the best of our knowledge, there is no existing method in the literature, which addresses simultaneous registration of multi-tag bioimages and selection of the reference image in order to maximize the overall overlap between the images. Methodology/Principal Findings We employ a block-based method for registration, which yields a confidence measure to indicate the accuracy of our registration results. We derive a shift metric in order to select the Reference Image with Maximal Overlap (RIMO), in turn minimizing the total amount of non-overlapping signal for a given number of tags. Experimental results show that the Robust Alignment of Multi-Tag Bioimages (RAMTaB) framework is robust to variations in contrast and illumination, yields sub-pixel accuracy, and successfully selects the reference image resulting in maximum overlap. The registration results are also shown to significantly improve any follow-up protein co-localization studies. Conclusions For the discovery of protein complexes and of functional protein networks within a cell, alignment of the tag images in a multi-tag fluorescence image stack is a key pre-processing step. The proposed framework is shown to produce accurate alignment results on both real and synthetic data. Our future work will use the aligned multi-channel fluorescence image data for normal and diseased tissue specimens to analyze molecular co-expression patterns and functional protein networks. PMID:22363510

Concentration Measurements in a Cold Flow Model Annular Combustor Using Laser Induced Fluorescence

NASA Technical Reports Server (NTRS)

Morgan, Douglas C.

1996-01-01

A nonintrusive concentration measurement method is developed for determining the concentration distribution in a complex flow field. The measurement method consists of marking a liquid flow with a water soluble fluorescent dye. The dye is excited by a two dimensional sheet of laser light. The fluorescent intensity is shown to be proportional to the relative concentration level. The fluorescent field is recorded on a video cassette recorder through a video camera. The recorded images are analyzed with image processing hardware and software to obtain intensity levels. Mean and root mean square (rms) values are calculated from these intensity levels. The method is tested on a single round turbulent jet because previous concentration measurements have been made on this configuration by other investigators. The previous results were used to comparison to qualify the current method. These comparisons showed that this method provides satisfactory results. 'Me concentration measurement system was used to measure the concentrations in the complex flow field of a model gas turbine annular combustor. The model annular combustor consists of opposing primary jets and an annular jet which discharges perpendicular to the primary jets. The mixing between the different jet flows can be visualized from the calculated mean and rms profiles. Concentration field visualization images obtained from the processing provide further qualitative information about the flow field.

Quantification of ligand density and stoichiometry on the surface of liposomes using single-molecule fluorescence imaging.

PubMed

Belfiore, Lisa; Spenkelink, Lisanne M; Ranson, Marie; van Oijen, Antoine M; Vine, Kara L

2018-05-28

Despite the longstanding existence of liposome technology in drug delivery applications, there have been no ligand-directed liposome formulations approved for clinical use to date. This lack of translation is due to several factors, one of which is the absence of molecular tools for the robust quantification of ligand density on the surface of liposomes. We report here for the first time the quantification of proteins attached to the surface of small unilamellar liposomes using single-molecule fluorescence imaging. Liposomes were surface-functionalized with fluorescently labeled human proteins previously validated to target the cancer cell surface biomarkers plasminogen activator inhibitor-2 (PAI-2) and trastuzumab (TZ, Herceptin®). These protein-conjugated liposomes were visualized using a custom-built wide-field fluorescence microscope with single-molecule sensitivity. By counting the photobleaching steps of the fluorescently labeled proteins, we calculated the number of attached proteins per liposome, which was 11 ± 4 proteins for single-ligand liposomes. Imaging of dual-ligand liposomes revealed stoichiometries of the two attached proteins in accordance with the molar ratios of protein added during preparation. Preparation of PAI-2/TZ dual-ligand liposomes via two different methods revealed that the post-insertion method generated liposomes with a more equal representation of the two differently sized proteins, demonstrating the ability of this preparation method to enable better control of liposome protein densities. We conclude that the single-molecule imaging method presented here is an accurate and reliable quantification tool for determining ligand density and stoichiometry on the surface of liposomes. This method has the potential to allow for comprehensive characterization of novel ligand-directed liposomes that should facilitate the translation of these nanotherapies through to the clinic. Copyright © 2018 Elsevier B.V. All rights reserved.

In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles.

PubMed

Gu, Luo; Hall, David J; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J; Howell, Stephen B; Sailor, Michael J

2013-01-01

Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (<10 ns) emission signals from organic chromophores or tissue autofluorescence. Here using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by >50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.

Decoding of quantum dots encoded microbeads using a hyperspectral fluorescence imaging method.

PubMed

Liu, Yixi; Liu, Le; He, Yonghong; Zhu, Liang; Ma, Hui

2015-05-19

We presented a decoding method of quantum dots encoded microbeads with its fluorescence spectra using line scan hyperspectral fluorescence imaging (HFI) method. A HFI method was developed to attain both the spectra of fluorescence signal and the spatial information of the encoded microbeads. A decoding scheme was adopted to decode the spectra of multicolor microbeads acquired by the HFI system. Comparison experiments between the HFI system and the flow cytometer were conducted. The results showed that the HFI system has higher spectrum resolution; thus, more channels in spectral dimension can be used. The HFI system detection and decoding experiment with the single-stranded DNA (ssDNA) immobilized multicolor beads was done, and the result showed the efficiency of the HFI system. Surface modification of the microbeads by use of the polydopamine was characterized by the scanning electron microscopy and ssDNA immobilization was characterized by the laser confocal microscope. These results indicate that the designed HFI system can be applied to practical biological and medical applications.

Sentinel lymph node biopsy under fluorescent indocyanin green guidance: Initial experience.

PubMed

Aydoğan, Fatih; Arıkan, Akif Enes; Aytaç, Erman; Velidedeoğlu, Mehmet; Yılmaz, Mehmet Halit; Sager, Muhammet Sait; Çelik, Varol; Uras, Cihan

2016-01-01

Sentinel lymph node biopsy can be applied by using either blue dye or radionuclide method or both in breast cancer. Fluorescent imaging with indocyanine green is a new defined method. This study evaluates the applicability of sentinel lymph node biopsy via fluorescent indocyanine green. IC-VIEW (Pulsion Medical Systems AG, Munich, Germany) infrared visualization system was used for imaging. Two mL of indocyanine green was injected to visualize sentinel lymph nodes. After injection, subcutaneous lymphatics were traced and sentinel lymph nodes were found with simultaneous imaging. Sentinel lymph nodes were excised under fluorescent light guidance, and excised lymph nodes were examined histopathologically. Patients with sentinel lymph node metastases underwent axillary dissection. Four patients with sentinel lymph node biopsy due to breast cancer were included in the study. Sentinel lymph nodes were visualized with indocyanine green in all patients. The median number of excised sentinel lymph node was 2 (2-3). Two patients with lymph node metastasis underwent axillary dissection. No metastasis was detected in lymph nodes other than the sentinel nodes in patients with axillary dissection. There was no complication during and after the operation related to the method. According to our limited experience, sentinel lymph node biopsy under fluorescent indocyanine green guidance, which has an advantage of simultaneous visualization, is technically feasible.

Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo

NASA Astrophysics Data System (ADS)

Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V.; Roberts, Michael S.

2013-02-01

There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

A Novel Water-Soluble Fluorescence Probe with Wash-Free Cellular Imaging Capacity Based on AIE Characteristics.

PubMed

Qian, Yunxia; Liu, Hongmei; Tan, Haijian; Yang, Qingmin; Zhang, Shuchen; Han, Lingui; Yi, Xuegang; Huo, Li; Zhao, Hongchi; Wu, Yonggang; Bai, Libin; Ba, Xinwu

2017-05-01

A potential real-time imaging water-soluble fluorescent polymer (P3) is facilely prepared via one-pot method. For P3, tetraphenylethene unit serves as the fluorescent unit, poly(acryloyl ethylene diamine) (a kind of polyelectrolyte) with specific degree of polymerization acts as water-soluble part. 1 H-NMR, gel permeation chromatography (GPC), UV-vis spectroscopy, photoluminescence (PL), and confocal laser scanning microscopy are undertaken to characterize the structure and property of P3. The results of wash-free cellular imaging show that the signal-to-noise ratio is high as the concentration of P3 is 50 μg mL -1 . In addition, the pH-responsive and Cd 2+ -responsive are also investigated in this paper. The results coming from pH-responsive show that P3 solution displays significant fluorescence under near neutral. And the result from the cellular imaging shows that intracellular fluorescence intensity enhances with the augment of concentration of Cd 2+ , which reveals that P3 can give a hint to resolve the dilemma of traditional fluorescent dyes used as living cellular fluorescent probe. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo.

PubMed

Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V; Roberts, Michael S

2013-02-01

There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

Fast methods for analysis of neurotransmitters from single cell and monitoring their releases in central nervous system by capillary electrophoresis, fluorescence microscopy and luminescence imaging

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

Wang, Ziqiang

1999-12-10

Fast methods for separation and detection of important neurotransmitters and the releases in central nervous system (CNS) were developed. Enzyme based immunoassay combined with capillary electrophoresis was used to analyze the contents of amino acid neurotransmitters from single neuron cells. The release of amino acid neurotransmitters from neuron cultures was monitored by laser induced fluorescence imaging method. The release and signal transduction of adenosine triphosphate (ATP) in CNS was studied with sensitive luminescence imaging method. A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Detection was based onmore » monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. The detection limit of glutamate is down to 10 -8 M level, which is 1 order of magnitude lower than the previously reported detection limit based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most of amino acids. The glutamate content in single human erythrocyte and baby rat brain neurons were determined with this method and results agreed well with literature values.« less

Long Term Non-Invasive Imaging of Embryonic Stem Cells Using Reporter Genes

PubMed Central

Sun, Ning; Lee, Andrew; Wu, Joseph C.

2013-01-01

Development of non-invasive and accurate methods to track cell fate following delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. Here we describe a protocol for the in vivo monitoring of stem cell survival, proliferation, and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase) reporter genes using lentiviral transduction. We used fluorescence activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography imaging to track them in vivo, and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny. PMID:19617890

Fluorescence intensity positivity classification of Hep-2 cells images using fuzzy logic

NASA Astrophysics Data System (ADS)

Sazali, Dayang Farzana Abang; Janier, Josefina Barnachea; May, Zazilah Bt.

2014-10-01

Indirect Immunofluorescence (IIF) is a good standard used for antinuclear autoantibody (ANA) test using Hep-2 cells to determine specific diseases. Different classifier algorithm methods have been proposed in previous works however, there still no valid set as a standard to classify the fluorescence intensity. This paper presents the use of fuzzy logic to classify the fluorescence intensity and to determine the positivity of the Hep-2 cell serum samples. The fuzzy algorithm involves the image pre-processing by filtering the noises and smoothen the image, converting the red, green and blue (RGB) color space of images to luminosity layer, chromaticity layer "a" and "b" (LAB) color space where the mean value of the lightness and chromaticity layer "a" was extracted and classified by using fuzzy logic algorithm based on the standard score ranges of antinuclear autoantibody (ANA) fluorescence intensity. Using 100 data sets of positive and intermediate fluorescence intensity for testing the performance measurements, the fuzzy logic obtained an accuracy of intermediate and positive class as 85% and 87% respectively.

Ratiometric Fluorescence Azide-Alkyne Cycloaddition for Live Mammalian Cell Imaging.

PubMed

Fu, Hongxia; Li, Yanru; Sun, Lingbo; He, Pan; Duan, Xinrui

2015-11-17

Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules.

Fluorescence laminar optical tomography for brain imaging: system implementation and performance evaluation.

PubMed

Azimipour, Mehdi; Sheikhzadeh, Mahya; Baumgartner, Ryan; Cullen, Patrick K; Helmstetter, Fred J; Chang, Woo-Jin; Pashaie, Ramin

2017-01-01

We present our effort in implementing a fluorescence laminar optical tomography scanner which is specifically designed for noninvasive three-dimensional imaging of fluorescence proteins in the brains of small rodents. A laser beam, after passing through a cylindrical lens, scans the brain tissue from the surface while the emission signal is captured by the epi-fluorescence optics and is recorded using an electron multiplication CCD sensor. Image reconstruction algorithms are developed based on Monte Carlo simulation to model light–tissue interaction and generate the sensitivity matrices. To solve the inverse problem, we used the iterative simultaneous algebraic reconstruction technique. The performance of the developed system was evaluated by imaging microfabricated silicon microchannels embedded inside a substrate with optical properties close to the brain as a tissue phantom and ultimately by scanning brain tissue in vivo. Details of the hardware design and reconstruction algorithms are discussed and several experimental results are presented. The developed system can specifically facilitate neuroscience experiments where fluorescence imaging and molecular genetic methods are used to study the dynamics of the brain circuitries.

Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.

PubMed

Rizk, Aurélien; Paul, Grégory; Incardona, Pietro; Bugarski, Milica; Mansouri, Maysam; Niemann, Axel; Ziegler, Urs; Berger, Philipp; Sbalzarini, Ivo F

2014-03-01

Detection and quantification of fluorescently labeled molecules in subcellular compartments is a key step in the analysis of many cell biological processes. Pixel-wise colocalization analyses, however, are not always suitable, because they do not provide object-specific information, and they are vulnerable to noise and background fluorescence. Here we present a versatile protocol for a method named 'Squassh' (segmentation and quantification of subcellular shapes), which is used for detecting, delineating and quantifying subcellular structures in fluorescence microscopy images. The workflow is implemented in freely available, user-friendly software. It works on both 2D and 3D images, accounts for the microscope optics and for uneven image background, computes cell masks and provides subpixel accuracy. The Squassh software enables both colocalization and shape analyses. The protocol can be applied in batch, on desktop computers or computer clusters, and it usually requires <1 min and <5 min for 2D and 3D images, respectively. Basic computer-user skills and some experience with fluorescence microscopy are recommended to successfully use the protocol.

Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

PubMed

Nothdurft, R; Sarder, P; Bloch, S; Culver, J; Achilefu, S

2012-08-01

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

Fluorescence Lifetime Imaging Microscopy Using Near-Infrared Contrast Agents

PubMed Central

Nothdurft, Ralph; Sarder, Pinaki; Bloch, Sharon; Culver, Joseph; Achilefu, Samuel

2013-01-01

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labeled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes’ relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. PMID:22788550

Qualitative and Quantitative Analysis of Histone Deacetylases in Kidney Tissue Sections.

PubMed

Ververis, Katherine; Marzully, Selly; Samuel, Chrishan S; Hewitson, Tim D; Karagiannis, Tom C

2016-01-01

Fluorescent microscope imaging technologies are increasing in their applications and are being used on a wide scale. However methods used to quantify the level of fluorescence intensity are often not utilized-perhaps given the result may be immediately seen, quantification of the data may not seem necessary. However there are a number of reasons given to quantify fluorescent images including the importance of removing potential bias in the data upon observation as well as quantification of large numbers of images gives statistical power to detect subtle changes in experiments. In addition discreet localization of a protein could be detected without selection bias that may not be detectable by eye. Such data will be deemed useful when detecting the levels of HDAC enzymes within cells in order to develop more effective HDAC inhibitor compounds for use against multiple diseased states. Hence, we discuss a methodology devised to analyze fluorescent images using Image J to detect the mean fluorescence intensity of the 11 metal-dependent HDAC enzymes using murine kidney tissue sections as an example.

Dual-modal three-dimensional imaging of single cells with isometric high resolution using an optical projection tomography microscope

NASA Astrophysics Data System (ADS)

Miao, Qin; Rahn, J. Richard; Tourovskaia, Anna; Meyer, Michael G.; Neumann, Thomas; Nelson, Alan C.; Seibel, Eric J.

2009-11-01

The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 μm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.

Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish

PubMed Central

Kumar, Sunil; Lockwood, Nicola; Ramel, Marie-Christine; Correia, Teresa; Ellis, Matthew; Alexandrov, Yuriy; Andrews, Natalie; Patel, Rachel; Bugeon, Laurence; Dallman, Margaret J.; Brandner, Sebastian; Arridge, Simon; Katan, Matilda; McGinty, James; Frankel, Paul; French, Paul M.W.

2016-01-01

We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This “mesoscopic” imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models. PMID:27259259

Characterization of E coli biofim formations on baby spinach leaf surfaces using hyperspectral fluorescence imaging

NASA Astrophysics Data System (ADS)

Cho, Hyunjeong; Baek, Insuck; Oh, Mirae; Kim, Sungyoun; Lee, Hoonsoo; Kim, Moon S.

2017-05-01

Bacterial biofilm formed by pathogens on fresh produce surfaces is a food safety concern because the complex extracellular matrix in the biofilm structure reduces the reduction and removal efficacies of washing and sanitizing processes such as chemical or irradiation treatments. Therefore, a rapid and nondestructive method to identify pathogenic biofilm on produce surfaces is needed to ensure safe consumption of fresh, raw produce. This research aimed to evaluate the feasibility of hyperspectral fluorescence imaging for detecting Escherichia.coli (ATCC 25922) biofilms on baby spinach leaf surfaces. Samples of baby spinach leaves were immersed and inoculated with five different levels (from 2.6x104 to 2.6x108 CFU/mL) of E.coli and stored at 4°C for 24 h and 48 h to induce biofilm formation. Following the two treatment days, individual leaves were gently washed to remove excess liquid inoculums from the leaf surfaces and imaged with a hyperspectral fluorescence imaging system equipped with UV-A (365 nm) and violet (405 nm) excitation sources to evaluate a spectral-image-based method for biofilm detection. The imaging results with the UV-A excitation showed that leaves even at early stages of biofilm formations could be differentiated from the control leaf surfaces. This preliminary investigation demonstrated the potential of fluorescence imaging techniques for detection of biofilms on leafy green surfaces.

Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells.

PubMed

Hebert, Benedict; Costantino, Santiago; Wiseman, Paul W

2005-05-01

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.

Lq -Lp optimization for multigrid fluorescence tomography of small animals using simplified spherical harmonics

NASA Astrophysics Data System (ADS)

Edjlali, Ehsan; Bérubé-Lauzière, Yves

2018-01-01

We present the first Lq -Lp optimization scheme for fluorescence tomographic imaging. This is then applied to small animal imaging. Fluorescence tomography is an ill-posed, and in full generality, a nonlinear problem that seeks to image the 3D concentration distribution of a fluorescent agent inside a biological tissue. Standard candidates for regularization to deal with the ill-posedness of the image reconstruction problem include L1 and L2 regularization. In this work, a general Lq -Lp regularization framework (Lq discrepancy function - Lp regularization term) is introduced for fluorescence tomographic imaging. A method to calculate the gradient for this general framework is developed which allows evaluating the performance of different cost functions/regularization schemes in solving the fluorescence tomographic problem. The simplified spherical harmonics approximation is used to accurately model light propagation inside the tissue. Furthermore, a multigrid mesh is utilized to decrease the dimension of the inverse problem and reduce the computational cost of the solution. The inverse problem is solved iteratively using an lm-BFGS quasi-Newton optimization method. The simulations are performed under different scenarios of noisy measurements. These are carried out on the Digimouse numerical mouse model with the kidney being the target organ. The evaluation of the reconstructed images is performed both qualitatively and quantitatively using several metrics including QR, RMSE, CNR, and TVE under rigorous conditions. The best reconstruction results under different scenarios are obtained with an L1.5 -L1 scheme with premature termination of the optimization process. This is in contrast to approaches commonly found in the literature relying on L2 -L2 schemes.

Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone

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

Moester, Martiene J.C.; Schoeman, Monique A.E.; Oudshoorn, Ineke B.

2014-01-03

Highlights: •We validate a simple and fast method of quantification of in vitro mineralization. •Fluorescently labeled agents can detect calcium deposits in the mineralized matrix of cell cultures. •Fluorescent signals of the probes correlated with Alizarin Red S staining. -- Abstract: Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and ismore » therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments. KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21 days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.« less

Near-infrared optical imaging of nucleic acid nanocarriers in vivo.

PubMed

Rome, Claire; Gravier, Julien; Morille, Marie; Divita, Gilles; Bolcato-Bellemin, Anne-Laure; Josserand, Véronique; Coll, Jean-Luc

2013-01-01

Noninvasive, real-time optical imaging methods are well suited to follow the in vivo distribution of nucleic acid nanocarriers, their dissociation, and the resulting gene expression or inhibition. Indeed, most small animal imaging devices perform bioluminescence and fluorescence measurements without moving the animal, allowing a simple, rapid, and cost-effective method of investigation of several parameters at a time, in longitudinal experiments that can last for days or weeks.Here we help the reader in choosing adapted near-infrared (NIR) fluorophores or pairs of fluorophores for Förster resonance energy transfer assays, imaging of reporter genes, as well as nanocarriers for in vivo gene and siRNA delivery. In addition, we present the labeling methods of these macromolecules and of their payload and the protocols to detect them using bioluminescence and NIR fluorescence imaging in mice.

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.

Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes.

PubMed

Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

2015-02-11

Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

Quantitative Measurement of Protease-Activity with Correction of Probe Delivery and Tissue Absorption Effects

PubMed Central

Salthouse, Christopher D.; Reynolds, Fred; Tam, Jenny M.; Josephson, Lee; Mahmood, Umar

2009-01-01

Proteases play important roles in a variety of pathologies from heart disease to cancer. Quantitative measurement of protease activity is possible using a novel spectrally matched dual fluorophore probe and a small animal lifetime imager. The recorded fluorescence from an activatable fluorophore, one that changes its fluorescent amplitude after biological target interaction, is also influenced by other factors including imaging probe delivery and optical tissue absorption of excitation and emission light. Fluorescence from a second spectrally matched constant (non-activatable) fluorophore on each nanoparticle platform can be used to correct for both probe delivery and tissue absorption. The fluorescence from each fluorophore is separated using fluorescence lifetime methods. PMID:20161242

Crystallization Kinetics of an Amorphous Pharmaceutical Compound Using Fluorescence-Lifetime-Imaging Microscopy.

PubMed

Rautaniemi, Kaisa; Vuorimaa-Laukkanen, Elina; Strachan, Clare J; Laaksonen, Timo

2018-05-07

Pharmaceutical scientists are increasingly interested in amorphous drug formulations especially because of their higher dissolution rates. Consequently, the thorough characterization and analysis of these formulations are becoming more and more important for the pharmaceutical industry. Here, fluorescence-lifetime-imaging microscopy (FLIM) was used to monitor the crystallization of an amorphous pharmaceutical compound, indomethacin. Initially, we identified different solid indomethacin forms, amorphous and γ- and α-crystalline, on the basis of their time-resolved fluorescence. All of the studied indomethacin forms showed biexponential decays with characteristic fluorescence lifetimes and amplitudes. Using this information, the crystallization of amorphous indomethacin upon storage in 60 °C was monitored for 10 days with FLIM. The progress of crystallization was detected as lifetime changes both in the FLIM images and in the fluorescence-decay curves extracted from the images. The fluorescence-lifetime amplitudes were used for quantitative analysis of the crystallization process. We also demonstrated that the fluorescence-lifetime distribution of the sample changed during crystallization, and when the sample was not moved between measuring times, the lifetime distribution could also be used for the analysis of the reaction kinetics. Our results clearly show that FLIM is a sensitive and nondestructive method for monitoring solid-state transformations on the surfaces of fluorescent samples.

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography)

PubMed Central

Siegel, Nisan; Storrie, Brian; Bruce, Marc

2016-01-01

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called “CINCH”. An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution. PMID:26839443

In vivo fluorescence lifetime optical projection tomography

PubMed Central

McGinty, James; Taylor, Harriet B.; Chen, Lingling; Bugeon, Laurence; Lamb, Jonathan R.; Dallman, Margaret J.; French, Paul M. W.

2011-01-01

We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions. PMID:21559145

Nanoparticles for multimodal in vivo imaging in nanomedicine

PubMed Central

Key, Jaehong; Leary, James F

2014-01-01

While nanoparticles are usually designed for targeted drug delivery, they can also simultaneously provide diagnostic information by a variety of in vivo imaging methods. These diagnostic capabilities make use of specific properties of nanoparticle core materials. Near-infrared fluorescent probes provide optical detection of cells targeted by real-time nanoparticle-distribution studies within the organ compartments of live, anesthetized animals. By combining different imaging modalities, we can start with deep-body imaging by magnetic resonance imaging or computed tomography, and by using optical imaging, get down to the resolution required for real-time fluorescence-guided surgery. PMID:24511229

Dual-modality imaging with 99mTc and fluorescent indocyanine green using surface-modified silica nanoparticles for biopsy of the sentinel lymph node: an animal study

PubMed Central

2013-01-01

Background We propose a new approach to facilitate sentinel node biopsy examination by multimodality imaging in which radioactive and near-infrared (NIR) fluorescent nanoparticles depict deeply situated sentinel nodes and fluorescent nodes with anatomical resolution in the surgical field. For this purpose, we developed polyamidoamine (PAMAM)-coated silica nanoparticles loaded with technetium-99m (99mTc) and indocyanine green (ICG). Methods We conducted animal studies to test the feasibility and utility of this dual-modality imaging probe. The mean diameter of the PAMAM-coated silica nanoparticles was 30 to 50 nm, as evaluated from the images of transmission electron microscopy and scanning electron microscopy. The combined labeling with 99mTc and ICG was verified by thin-layer chromatography before each experiment. A volume of 0.1 ml of the nanoparticle solution (7.4 MBq, except for one rat that was injected with 3.7 MBq, and 1 μg of an ICG derivative [ICG-sulfo-OSu]) was injected submucosally into the tongue of six male Wistar rats. Results Scintigraphic images showed increased accumulation of 99mTc in the neck of four of the six rats. Nineteen lymph nodes were identified in the dissected neck of the six rats, and a contact radiographic study showed three nodes with a marked increase in uptake and three nodes with a weak uptake. NIR fluorescence imaging provided real-time clear fluorescent images of the lymph nodes in the neck with anatomical resolution. Six lymph nodes showed weak (+) to strong (+++) fluorescence, whereas other lymph nodes showed no fluorescence. Nodes showing increased radioactivity coincided with the fluorescent nodes. The radioactivity of 15 excised lymph nodes from the four rats was assayed using a gamma well counter. Comparisons of the levels of radioactivity revealed a large difference between the high-fluorescence-intensity group (four lymph nodes; mean, 0.109% ± 0.067%) and the low- or no-fluorescence-intensity group (11 lymph nodes; mean, 0.001% ± 0.000%, p < 0.05). Transmission electron microscopy revealed that small black granules were localized to and dispersed within the cytoplasm of macrophages in the lymph nodes. Conclusion Although further studies are needed to determine the appropriate dose of the dual-imaging nanoparticle probe for effective sensitivity and safety, the results of this animal study revealed a novel method for improved node detection by a dual-modality approach for sentinel lymph node biopsy. PMID:23618132

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Fluorescence Live Cell Imaging

PubMed Central

Ettinger, Andreas

2014-01-01

Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

The possibilities of improvement in the sensitivity of cancer fluorescence diagnostics by computer image processing

NASA Astrophysics Data System (ADS)

Ledwon, Aleksandra; Bieda, Robert; Kawczyk-Krupka, Aleksandra; Polanski, Andrzej; Wojciechowski, Konrad; Latos, Wojciech; Sieron-Stoltny, Karolina; Sieron, Aleksander

2008-02-01

Background: Fluorescence diagnostics uses the ability of tissues to fluoresce after exposition to a specific wavelength of light. The change in fluorescence between normal and progression to cancer allows to see early cancer and precancerous lesions often missed by white light. Aim: To improve by computer image processing the sensitivity of fluorescence images obtained during examination of skin, oral cavity, vulva and cervix lesions, during endoscopy, cystoscopy and bronchoscopy using Xillix ONCOLIFE. Methods: Function of image f(x,y):R2 --> R 3 was transformed from original color space RGB to space in which vector of 46 values refers to every point labeled by defined xy-coordinates- f(x,y):R2 --> R 46. By means of Fisher discriminator vector of attributes of concrete point analalyzed in the image was reduced according to two defined classes defined as pathologic areas (foreground) and healthy areas (background). As a result the highest four fisher's coefficients allowing the greatest separation between points of pathologic (foreground) and healthy (background) areas were chosen. In this way new function f(x,y):R2 --> R 4 was created in which point x,y corresponds with vector Y, H, a*, c II. In the second step using Gaussian Mixtures and Expectation-Maximisation appropriate classificator was constructed. This classificator enables determination of probability that the selected pixel of analyzed image is a pathologically changed point (foreground) or healthy one (background). Obtained map of probability distribution was presented by means of pseudocolors. Results: Image processing techniques improve the sensitivity, quality and sharpness of original fluorescence images. Conclusion: Computer image processing enables better visualization of suspected areas examined by means of fluorescence diagnostics.

LASER BIOLOGY AND MEDICINE: Combination of fluorescence imaging and local spectrophotometry in fluorescence diagnostics of early cancer of larynx and bronchi

NASA Astrophysics Data System (ADS)

Sokolov, Vladimir V.; Filonenko, E. V.; Telegina, L. V.; Boulgakova, N. N.; Smirnov, V. V.

2002-11-01

The results of comparative studies of autofluorescence and 5-ALA-induced fluorescence of protoporphyrin IX, used in the diagnostics of early cancer of larynx and bronchi, are presented. The autofluorescence and 5-ALA-induced fluorescence images of larynx and bronchial tissues are analysed during the endoscopic study. The method of local spectrophotometry is used to verify findings obtained from fluorescence images. It is shown that such a combined approach can be efficiently used to improve the diagnostics of precancer and early cancer, to detect a primary multiple tumours, as well as for the diagnostics of a residual tumour or an early recurrence after the endoscopic, surgery or X-ray treatment. The developed approach allows one to minimise the number of false-positive results and to reduce the number of biopsies, which are commonly used in the white-light bronchoscopy search for occult cancerous loci.

Fluorescence endoscopic imaging for evaluation of gastric mucosal blood flow: a preliminary study

NASA Astrophysics Data System (ADS)

Bocquillon, Nicolas; Mordon, Serge R.; Mathieu, D.; Maunoury, Vincent; Marechal, Xavier-Marie; Neviere, Remi; Wattel, Francis; Chopin, Claude

1999-02-01

Microcirculatory disorders of the gastrointestinal tract appear to be a major compound of the multiple organ dysfunction syndrome secondary to sepsis or septic shock. A better analysis of mucosal hypoperfusion in critically ill patients with sepsis may be helpful for the comprehension of this high mortality-associated syndrome. Fluorescence endoscopy has been recognized as a non-invasive method for both spatial and temporal evaluation of gastrointestinal mucosal perfusion. We performed this imaging technique during routine gastric endoscopy in patients with sepsis criteria. The study included gastric observation and appearance time of gastric fluorescence after an intravenous 10% sodium - fluorescein bolus. Qualitative analysis of high fluorescence areas was compared with mucosal blood flow measurements by laser - Doppler flowmetry. We concluded that the fluorescence endoscopic imaging in critically ill patients with sepsis may reveal spacial and temporal differences in the mucosal microcirculation distribution.

Segmentation and classification of cell cycle phases in fluorescence imaging.

PubMed

Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan

2009-01-01

Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

[Application of Fourier transform profilometry in 3D-surface reconstruction].

PubMed

Shi, Bi'er; Lu, Kuan; Wang, Yingting; Li, Zhen'an; Bai, Jing

2011-08-01

With the improvement of system frame and reconstruction methods in fluorescent molecules tomography (FMT), the FMT technology has been widely used as an important experimental tool in biomedical research. It is necessary to get the 3D-surface profile of the experimental object as the boundary constraints of FMT reconstruction algorithms. We proposed a new 3D-surface reconstruction method based on Fourier transform profilometry (FTP) method under the blue-purple light condition. The slice images were reconstructed using proper image processing methods, frequency spectrum analysis and filtering. The results of experiment showed that the method properly reconstructed the 3D-surface of objects and has the mm-level accuracy. Compared to other methods, this one is simple and fast. Besides its well-reconstructed, the proposed method could help monitor the behavior of the object during the experiment to ensure the correspondence of the imaging process. Furthermore, the method chooses blue-purple light section as its light source to avoid the interference towards fluorescence imaging.

Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein–DNA complexes

PubMed Central

Ebenstein, Yuval; Gassman, Natalie; Kim, Soohong; Weiss, Shimon

2011-01-01

Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA. PMID:19452448

An analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images.

PubMed

Haass-Koffler, Carolina L; Naeemuddin, Mohammad; Bartlett, Selena E

2012-08-31

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology even in complex tissue sections. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenössische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

Near-isotropic 3D optical nanoscopy with photon-limited chromophores

PubMed Central

Tang, Jianyong; Akerboom, Jasper; Vaziri, Alipasha; Looger, Loren L.; Shank, Charles V.

2010-01-01

Imaging approaches based on single molecule localization break the diffraction barrier of conventional fluorescence microscopy, allowing for bioimaging with nanometer resolution. It remains a challenge, however, to precisely localize photon-limited single molecules in 3D. We have developed a new localization-based imaging technique achieving almost isotropic subdiffraction resolution in 3D. A tilted mirror is used to generate a side view in addition to the front view of activated single emitters, allowing their 3D localization to be precisely determined for superresolution imaging. Because both front and side views are in focus, this method is able to efficiently collect emitted photons. The technique is simple to implement on a commercial fluorescence microscope, and especially suitable for biological samples with photon-limited chromophores such as endogenously expressed photoactivatable fluorescent proteins. Moreover, this method is relatively resistant to optical aberration, as it requires only centroid determination for localization analysis. Here we demonstrate the application of this method to 3D imaging of bacterial protein distribution and neuron dendritic morphology with subdiffraction resolution. PMID:20472826

Facile and eco-friendly synthesis of green fluorescent carbon nanodots for applications in bioimaging, patterning and staining.

PubMed

Shi, Lihong; Li, Yanyan; Li, Xiaofeng; Wen, Xiangping; Zhang, Guomei; Yang, Jun; Dong, Chuan; Shuang, Shaomin

2015-04-28

We report a facile and eco-friendly strategy for the fabrication of green fluorescent carbon nanodots (CDs), and demonstrate their applications for bio-imaging, patterning, and staining. A one-pot hydrothermal method using various plant petals yields bright green-emitting CDs, providing an easy way for the production of green fluorescent CDs without the need for a tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. The as-prepared CDs show small size distribution and excellent dispersibility. Their strong green fluorescence is observed when the excitation wavelength is between 430 nm and 490 nm. Moreover, they exhibit high tolerance to various external conditions, such as pH values, external cations, and continuous excitation. Due to minimum toxicity as well as good photoluminescence properties, these CDs can be applied to in vitro and in vivo imaging, patterning, and staining. According to confocal fluorescence imaging of human uterine cervical squamous cell carcinoma cells, CDs penetrate into the cell and enter the cytoplasm and the nucleus. More strikingly, carp is directly fed with CDs for in vivo imaging and shows bright green fluorescence at an excitation wavelength of 470 nm. In addition, the obtained CDs are used as fluorescent inks for drawing luminescence patterns. Finally, we also apply the CDs as a fluorescent dye. Interestingly, the absorbent filter paper with staining emits dramatic fluorescence under 470 nm excitation.

Using Dual Fluorescence Reporting Genes to Establish an In Vivo Imaging Model of Orthotopic Lung Adenocarcinoma in Mice.

PubMed

Lai, Cheng-Wei; Chen, Hsiao-Ling; Yen, Chih-Ching; Wang, Jiun-Long; Yang, Shang-Hsun; Chen, Chuan-Mu

2016-12-01

Lung adenocarcinoma is characterized by a poor prognosis and high mortality worldwide. In this study, we purposed to use the live imaging techniques and a reporter gene that generates highly penetrative near-infrared (NIR) fluorescence to establish a preclinical animal model that allows in vivo monitoring of lung cancer development and provides a non-invasive tool for the research on lung cancer pathogenesis and therapeutic efficacy. A human lung adenocarcinoma cell line (A549), which stably expressed the dual fluorescence reporting gene (pCAG-iRFP-2A-Venus), was used to generate subcutaneous or orthotopic lung cancer in nude mice. Cancer development was evaluated by live imaging via the NIR fluorescent signals from iRFP, and the signals were verified ex vivo by the green fluorescence of Venus from the gross lung. The tumor-bearing mice received miR-16 nucleic acid therapy by intranasal administration to demonstrate therapeutic efficacy in this live imaging system. For the subcutaneous xenografts, the detection of iRFP fluorescent signals revealed delicate changes occurring during tumor growth that are not distinguishable by conventional methods of tumor measurement. For the orthotopic xenografts, the positive correlation between the in vivo iRFP signal from mice chests and the ex vivo green fluorescent signal from gross lung tumors and the results of the suppressed tumorigenesis by miR-16 treatment indicated that lung tumor size can be accurately quantified by the emission of NIR fluorescence. In addition, orthotopic lung tumor localization can be accurately visualized using iRFP fluorescence tomography in vivo, thus revealing the trafficking of lung tumor cells. We introduced a novel dual fluorescence lung cancer model that provides a non-invasive option for preclinical research via the use of NIR fluorescence in live imaging of lung.

Microscale Concentration Measurements Using Laser Light Scattering Methods

NASA Technical Reports Server (NTRS)

Niederhaus, Charles; Miller, Fletcher

2004-01-01

The development of lab-on-a-chip devices for microscale biochemical assays has led to the need for microscale concentration measurements of specific analyses. While fluorescence methods are the current choice, this method requires developing fluorophore-tagged conjugates for each analyte of interest. In addition, fluorescent imaging is also a volume-based method, and can be limiting as smaller detection regions are required.

Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging†

PubMed Central

Digman, Michelle A.; Gratton, Enrico; Storti, Barbara; Beltram, Fabio

2013-01-01

A versatile pH-dependent fluorescent protein was applied to intracellular pH measurements by means of the phasor approach to fluorescence lifetime imaging. By this fit-less method we obtain intracellular pH maps under resting or altered physiological conditions by single-photon confocal or two-photon microscopy. PMID:22517076

Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp.

USDA-ARS?s Scientific Manuscript database

Aims: To simplify the determination of the nuclear condition of the pathogenic Rhizoctonia, which currently needs to be performed either using two fluorescent dyes, thus is more costly and time-consuming, or using only one fluorescent dye, and thus less accurate. Methods and Results: A red primary ...

Novel techniques with multiphoton microscopy: Deep-brain imaging with microprisms, neurometabolism of epilepsy, and counterfeit paper money detection

NASA Astrophysics Data System (ADS)

Chia, Thomas H.

Multiphoton microscopy is a laser-scanning fluorescence imaging method with extraordinary potential. We describe three innovative multiphoton microscopy techniques across various disciplines. Traditional in vivo fluorescence microscopy of the mammalian brain has a limited penetration depth (<400 microm). We present a method of imaging 1 mm deep into mouse neocortex by using a glass microprism to relay the excitation and emission light. This technique enables simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution was sufficient to resolve dendritic spines on layer V GFP neurons. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. Multiphoton fluorescence lifetime imaging (FLIM) of NADH reveals information on neurometabolism. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic coenzyme, has a lifetime dependent on enzymatic binding. A novel NADH FLIM algorithm is presented that produces images showing spatially distinct NADH fluorescence lifetimes in mammalian brain slices. This program provides advantages over traditional FLIM processing of multi-component lifetime data. We applied this technique to a GFP-GFAP pilocarpine mouse model of temporal lobe epilepsy. Results indicated significant changes in the neurometabolism of astrocytes and neuropil in the cell and dendritic layers of the hippocampus when compared to control tissue. Data obtained with NADH FLIM were subsequently interpreted based on the abnormal activity reported in epileptic tissue. Genuine U.S. Federal Reserve Notes have a consistent, two-component intrinsic fluorescence lifetime. This allows for detection of counterfeit paper money because of its significant differences in fluorescence lifetime when compared to genuine paper money. We used scanning multiphoton laser excitation to sample a ˜4 mm2 region from 54 genuine Reserve Notes. Three types of counterfeit samples were tested. Four out of the nine counterfeit samples fit to a one-component decay. Five out of nine counterfeit samples fit to a two-component model, but are identified as counterfeit due to significant deviations in the longer lifetime component compared to genuine bills.

A novel method for enhancing the lateral resolution and image SNR in confocal microscopy

NASA Astrophysics Data System (ADS)

Chen, Youhua; Zhu, Dazhao; Fang, Yue; Kuang, Cuifang; Liu, Xu

2017-12-01

There is always a tradeoff between the resolution and the signal-to-noise ratio (SNR) in confocal microscopy. In particular, the pinhole size is very important for maintaining a balance between them. In this paper, we propose a method for improving the lateral resolution and image SNR in confocal microscopy without making any changes to the hardware. By using the fluorescence emission difference (FED) approach, we divide the images acquired by different pinhole sizes into one image acquired by the central pinhole and several images acquired by ring-shaped pinholes. Then, they are added together with the deconvolution method. Simulation and experimental results for fluorescent particles and cells show that our method can achieve a far better resolution than a large pinhole and a higher SNR than a small pinhole. Moreover, our method can improve the performance of classic confocal laser scanning microscopy (CLSM) to a certain extent, especially CLSM with a continuously variable pinhole.

Demonstration of the lack of cytotoxicity of unmodified and folic acid modified graphene oxide quantum dots, and their application to fluorescence lifetime imaging of HaCaT cells.

PubMed

Goreham, Renee V; Schroeder, Kathryn L; Holmes, Amy; Bradley, Siobhan J; Nann, Thomas

2018-01-24

The authors describe the synthesis of water-soluble and fluorescent graphene oxide quantum dots via acid exfoliation of graphite nanoparticles. The resultant graphene oxide quantum dots (GoQDs) were then modified with folic acid. Folic acid receptors are overexpressed in cancer cells and hence can bind to functionalized graphene oxide quantum dots. On excitation at 305 nm, the GoQDs display green fluorescence with a peak wavelength at ~520 nm. The modified GoQDs are non-toxic to macrophage cells even after prolonged exposure and high concentrations. Fluorescence lifetime imaging and multiphoton microscopy was used (in combination) to image HeCaT cells exposed to GoQDs, resulting in a superior method for bioimaging. Graphical abstract Schematic representation of graphene oxide quantum dots, folic acid modified graphene oxide quantum dots (red), and the use of fluorescence lifetime to discriminate against green auto-fluorescence of HeCaT cells.

Development of a quantitative validation method for forensic investigation of human spermatozoa using a commercial fluorescence staining kit (SPERM HY-LITER™ Express).

PubMed

Takamura, Ayari; Watanabe, Ken; Akutsu, Tomoko

2016-11-01

In investigations of sexual assaults, as well as in identifying a suspect, the detection of human sperm is important. Recently, a kit for fluorescent staining of human spermatozoa, SPERM HY-LITER™, has become available. This kit allows for microscopic observation of the heads of human sperm using an antibody tagged with a fluorescent dye. This kit is specific to human sperm and provides easy detection by luminescence. However, criteria need to be established to objectively evaluate the fluorescent signals and to evaluate the staining efficiency of this kit. These criteria will be indispensable for investigation of forensic samples. In the present study, the SPERM HY-LITER™ Express kit, which is an improved version of SPERM HY-LITER™, was evaluated using an image analysis procedure using Laplacian and Gaussian methods. This method could be used to automatically select important regions of fluorescence produced by sperm. The fluorescence staining performance was evaluated and compared under various experimental conditions, such as for aged traces and in combination with other chemical staining methods. The morphological characteristics of human sperm were incorporated into the criteria for objective identification of sperm, based on quantified features of the fluorescent spots. Using the criteria, non-specific or insignificant fluorescent spots were excluded, and the specificity of the kit for human sperm was confirmed. The image analysis method and criteria established in this study are universal and could be applied under any experimental conditions. These criteria will increase the reliability of operator judgment in the analysis of human sperm samples in forensics.

Accurate and fiducial-marker-free correction for three-dimensional chromatic shift in biological fluorescence microscopy.

PubMed

Matsuda, Atsushi; Schermelleh, Lothar; Hirano, Yasuhiro; Haraguchi, Tokuko; Hiraoka, Yasushi

2018-05-15

Correction of chromatic shift is necessary for precise registration of multicolor fluorescence images of biological specimens. New emerging technologies in fluorescence microscopy with increasing spatial resolution and penetration depth have prompted the need for more accurate methods to correct chromatic aberration. However, the amount of chromatic shift of the region of interest in biological samples often deviates from the theoretical prediction because of unknown dispersion in the biological samples. To measure and correct chromatic shift in biological samples, we developed a quadrisection phase correlation approach to computationally calculate translation, rotation, and magnification from reference images. Furthermore, to account for local chromatic shifts, images are split into smaller elements, for which the phase correlation between channels is measured individually and corrected accordingly. We implemented this method in an easy-to-use open-source software package, called Chromagnon, that is able to correct shifts with a 3D accuracy of approximately 15 nm. Applying this software, we quantified the level of uncertainty in chromatic shift correction, depending on the imaging modality used, and for different existing calibration methods, along with the proposed one. Finally, we provide guidelines to choose the optimal chromatic shift registration method for any given situation.

Imaging a Large Sample with Selective Plane Illumination Microscopy Based on Multiple Fluorescent Microsphere Tracking

NASA Astrophysics Data System (ADS)

Ryu, Inkeon; Kim, Daekeun

2018-04-01

A typical selective plane illumination microscopy (SPIM) image size is basically limited by the field of view, which is a characteristic of the objective lens. If an image larger than the imaging area of the sample is to be obtained, image stitching, which combines step-scanned images into a single panoramic image, is required. However, accurately registering the step-scanned images is very difficult because the SPIM system uses a customized sample mount where uncertainties for the translational and the rotational motions exist. In this paper, an image registration technique based on multiple fluorescent microsphere tracking is proposed in the view of quantifying the constellations and measuring the distances between at least two fluorescent microspheres embedded in the sample. Image stitching results are demonstrated for optically cleared large tissue with various staining methods. Compensation for the effect of the sample rotation that occurs during the translational motion in the sample mount is also discussed.

In vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment.

PubMed

Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

2014-07-01

Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in vivo fluorescence lifetime imaging with HER2-targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pretreatment size. Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (∼0.13 ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment), the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03 ns. The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. ©2014 American Association for Cancer Research.

In-vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment

PubMed Central

Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

2015-01-01

Purpose Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in-vivo fluorescence lifetime imaging with HER2 targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. Experimental Design HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice, bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pre-treatment size. Results Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (~0.13ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment) the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03ns. Conclusions The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in-vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. PMID:24671949

Ultra-small dye-doped silica nanoparticles via modified sol-gel technique

NASA Astrophysics Data System (ADS)

Riccò, R.; Nizzero, S.; Penna, E.; Meneghello, A.; Cretaio, E.; Enrichi, F.

2018-05-01

In modern biosensing and imaging, fluorescence-based methods constitute the most diffused approach to achieve optimal detection of analytes, both in solution and on the single-particle level. Despite the huge progresses made in recent decades in the development of plasmonic biosensors and label-free sensing techniques, fluorescent molecules remain the most commonly used contrast agents to date for commercial imaging and detection methods. However, they exhibit low stability, can be difficult to functionalise, and often result in a low signal-to-noise ratio. Thus, embedding fluorescent probes into robust and bio-compatible materials, such as silica nanoparticles, can substantially enhance the detection limit and dramatically increase the sensitivity. In this work, ultra-small fluorescent silica nanoparticles (NPs) for optical biosensing applications were doped with a fluorescent dye, using simple water-based sol-gel approaches based on the classical Stöber procedure. By systematically modulating reaction parameters, controllable size tuning of particle diameters as low as 10 nm was achieved. Particles morphology and optical response were evaluated showing a possible single-molecule behaviour, without employing microemulsion methods to achieve similar results. [Figure not available: see fulltext.

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

Bazalova, M; Ahmad, M; Fahrig, R

Purpose: To evaluate x-ray fluorescence computed tomography induced with proton beams (pXFCT) for imaging of gold contrast agent. Methods: Proton-induced x-ray fluorescence was studied by means of Monte Carlo (MC) simulations using TOPAS, a MC code based on GEANT4. First, proton-induced K-shell and L-shell fluorescence was studied as a function of proton beam energy and 1) depth in water and 2) size of contrast object. Second, pXFCT images of a 2-cm diameter cylindrical phantom with four 5- mm diameter contrast vials and of a 20-cm diameter phantom with 1-cm diameter vials were simulated. Contrast vials were filled with water andmore » water solutions with 1-5% gold per weight. Proton beam energies were varied from 70-250MeV. pXFCT sinograms were generated based on the net number of gold K-shell or L-shell x-rays determined by interpolations from the neighboring 0.5keV energy bins of spectra collected with an idealized 4π detector. pXFCT images were reconstructed with filtered-back projection, and no attenuation correction was applied. Results: Proton induced x-ray fluorescence spectra showed very low background compared to x-ray induced fluorescence. Proton induced L-shell fluorescence had a higher cross-section compared to K-shell fluorescence. Excitation of L-shell fluorescence was most efficient for low-energy protons, i.e. at the Bragg peak. K-shell fluorescence increased with increasing proton beam energy and object size. The 2% and 5% gold contrast vials were accurately reconstructed in K-shell pXFCT images of both the 2-cm and 20-cm diameter phantoms. Small phantom L-shell pXFCT image required attenuation correction and had a higher sensitivity for 70MeV protons compared to 250MeV protons. With attenuation correction, L-shell pXFCT might be a feasible option for imaging of small size (∼2cm) objects. Imaging doses for all simulations were 5-30cGy. Conclusion: Proton induced x-ray fluorescence CT promises to be an alternative quantitative imaging technique to the commonly considered XFCT imaging with x-ray beams.« less

A simple and sensitive high-throughput GFP screening in woody and herbaceous plants.

PubMed

Hily, Jean-Michel; Liu, Zongrang

2009-03-01

Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T(0) seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.

A practical implementation of multi-frequency widefield frequency-domain FLIM

PubMed Central

Chen, Hongtao

2013-01-01

Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime, especially in kinetic studies in biomedical researches. However, the small range of modulation frequencies available in commercial instruments makes this technique limited in its applications. Here we describe a practical implementation of multi-frequency widefield FD-FLIM using a pulsed supercontinuum laser and a direct digital synthesizer. In this instrument we use a pulse to modulate the image intensifier rather than the more conventional sine wave modulation. This allows parallel multi-frequency FLIM measurement using the Fast Fourier Transform and the cross-correlation technique, which permits precise and simultaneous isolation of individual frequencies. In addition, the pulse modulation at the cathode of image intensifier restored the loss of optical resolution caused by the defocusing effect when the voltage at the cathode is sinusoidally modulated. Furthermore, in our implementation of this technique, data can be graphically analyzed by the phasor method while data are acquired, which allows easy fit-free lifetime analysis of FLIM images. Here our measurements of standard fluorescent samples and a Föster resonance energy transfer pair demonstrate that the widefield multi-frequency FLIM system is a valuable and simple tool in fluorescence imaging studies. PMID:23296945

Using simulated fluorescence cell micrographs for the evaluation of cell image segmentation algorithms.

PubMed

Wiesmann, Veit; Bergler, Matthias; Palmisano, Ralf; Prinzen, Martin; Franz, Daniela; Wittenberg, Thomas

2017-03-18

Manual assessment and evaluation of fluorescent micrograph cell experiments is time-consuming and tedious. Automated segmentation pipelines can ensure efficient and reproducible evaluation and analysis with constant high quality for all images of an experiment. Such cell segmentation approaches are usually validated and rated in comparison to manually annotated micrographs. Nevertheless, manual annotations are prone to errors and display inter- and intra-observer variability which influence the validation results of automated cell segmentation pipelines. We present a new approach to simulate fluorescent cell micrographs that provides an objective ground truth for the validation of cell segmentation methods. The cell simulation was evaluated twofold: (1) An expert observer study shows that the proposed approach generates realistic fluorescent cell micrograph simulations. (2) An automated segmentation pipeline on the simulated fluorescent cell micrographs reproduces segmentation performances of that pipeline on real fluorescent cell micrographs. The proposed simulation approach produces realistic fluorescent cell micrographs with corresponding ground truth. The simulated data is suited to evaluate image segmentation pipelines more efficiently and reproducibly than it is possible on manually annotated real micrographs.

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.

PubMed

Lajevardipour, Alireza; Chon, James W M; Chattopadhyay, Amitabha; Clayton, Andrew H A

2016-11-22

Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C 6 -NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

A Review of Indocyanine Green Fluorescent Imaging in Surgery

PubMed Central

Alander, Jarmo T.; Kaartinen, Ilkka; Laakso, Aki; Pätilä, Tommi; Spillmann, Thomas; Tuchin, Valery V.; Venermo, Maarit; Välisuo, Petri

2012-01-01

The purpose of this paper is to give an overview of the recent surgical intraoperational applications of indocyanine green fluorescence imaging methods, the basics of the technology, and instrumentation used. Well over 200 papers describing this technique in clinical setting are reviewed. In addition to the surgical applications, other recent medical applications of ICG are briefly examined. PMID:22577366

Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation

PubMed Central

Dupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D.C.

2013-01-01

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments. PMID:23746509

Efficient processing of fluorescence images using directional multiscale representations.

PubMed

Labate, D; Laezza, F; Negi, P; Ozcan, B; Papadakis, M

2014-01-01

Recent advances in high-resolution fluorescence microscopy have enabled the systematic study of morphological changes in large populations of cells induced by chemical and genetic perturbations, facilitating the discovery of signaling pathways underlying diseases and the development of new pharmacological treatments. In these studies, though, due to the complexity of the data, quantification and analysis of morphological features are for the vast majority handled manually, slowing significantly data processing and limiting often the information gained to a descriptive level. Thus, there is an urgent need for developing highly efficient automated analysis and processing tools for fluorescent images. In this paper, we present the application of a method based on the shearlet representation for confocal image analysis of neurons. The shearlet representation is a newly emerged method designed to combine multiscale data analysis with superior directional sensitivity, making this approach particularly effective for the representation of objects defined over a wide range of scales and with highly anisotropic features. Here, we apply the shearlet representation to problems of soma detection of neurons in culture and extraction of geometrical features of neuronal processes in brain tissue, and propose it as a new framework for large-scale fluorescent image analysis of biomedical data.

Efficient processing of fluorescence images using directional multiscale representations

PubMed Central

Labate, D.; Laezza, F.; Negi, P.; Ozcan, B.; Papadakis, M.

2017-01-01

Recent advances in high-resolution fluorescence microscopy have enabled the systematic study of morphological changes in large populations of cells induced by chemical and genetic perturbations, facilitating the discovery of signaling pathways underlying diseases and the development of new pharmacological treatments. In these studies, though, due to the complexity of the data, quantification and analysis of morphological features are for the vast majority handled manually, slowing significantly data processing and limiting often the information gained to a descriptive level. Thus, there is an urgent need for developing highly efficient automated analysis and processing tools for fluorescent images. In this paper, we present the application of a method based on the shearlet representation for confocal image analysis of neurons. The shearlet representation is a newly emerged method designed to combine multiscale data analysis with superior directional sensitivity, making this approach particularly effective for the representation of objects defined over a wide range of scales and with highly anisotropic features. Here, we apply the shearlet representation to problems of soma detection of neurons in culture and extraction of geometrical features of neuronal processes in brain tissue, and propose it as a new framework for large-scale fluorescent image analysis of biomedical data. PMID:28804225

Facile synthesis of water-soluble and biocompatible fluorescent nitrogen-doped carbon dots for cell imaging.

PubMed

Wang, Weiping; Lu, Ya-Chun; Huang, Hong; Feng, Jiu-Ju; Chen, Jian-Rong; Wang, Ai-Jun

2014-04-07

A simple, facile and green hydrothermal method was developed in the synthesis of water-soluble nitrogen-doped carbon dots (N-CDs) from streptomycin. The as-prepared N-CDs displayed bright blue fluorescence under the irradiation of UV light, together with a high quantum yield of 7.6% and good biocompatibility as demonstrated by the cell viability assay. Thus, the N-CDs can be used as fluorescent probes for cell imaging, which have potential applications in bioimaging and related fields. This strategy opens a new way for the preparation of fluorescent carbon nanomaterials using small molecules as carbon sources.

Investigating the Functional Role of Prostate-Specific Membrane Antigen and its Enzymatic Activity in Prostate Cancer Metastasis

DTIC Science & Technology

2008-02-01

fluorescent probes for live cell imaging . PSMA distribution of cells grown on different extracellular matrices will be characterized to provide guidance...PCa migration, using in vitro cell model systems and live - cell imaging methods, we characterized the role of PSMA in cell motility and adhesion. Using...Generated fluorescently conjugated anti-PSMA antibodies for live cell imaging . 2. Optimized the siRNA-PSMA transfection and achieved an approximately

Autofluorescence imaging to optimize 5-ALA-induced fluorescence endoscopy of bladder carcinoma.

PubMed

Frimberger, D; Zaak, D; Stepp, H; Knüchel, R; Baumgartner, R; Schneede, P; Schmeller, N; Hofstetter, A

2001-09-01

To design an optical system for detecting autofluorescence (AF) of bladder tumors and to determine the success of reducing the false-positive rate of 5-aminolevulinic acid-induced fluorescence endoscopy (AFE). AFE provides significantly higher sensitivity in detecting and localizing bladder carcinoma compared with white light endoscopy. The specificity of AFE is equivalent to white light endoscopy, mostly because of the false-positive fluorescence of chronic cystitis lesions. Laser-induced spectral autofluorescence detection is also an efficient method in the diagnosis of bladder carcinoma. Bladder tissue was excited to AF using the D-Light (375 to 440 nm) after regular AFE with detection of fluorescence-positive areas. The optical image was produced using a special RGB camera. Biopsies were taken from AFE-positive areas, the peritumoral edges, and normal bladder mucosa. The AF images of the suspicious areas were compared with the AFE images and the histologic results. A total of 43 biopsies were histologically examined (24 benign and 19 neoplastic). AF imaging showed contrast differences between papillary tumors, flat lesions, and normal mucosa. The combination of AFE with AF raised the specificity of AFE alone from 67% to 88%. AF imaging is possible. The value of the method in reducing the false-positive rate of the highly sensitive AFE needs to be validated with higher numbers. The combination of AF with AFE had a 20% higher specificity than AFE alone in our study.

Parallel detecting super-resolution microscopy using correlation based image restoration

NASA Astrophysics Data System (ADS)

Yu, Zhongzhi; Liu, Shaocong; Zhu, Dazhao; Kuang, Cuifang; Liu, Xu

2017-12-01

A novel approach to achieve the image restoration is proposed in which each detector's relative position in the detector array is no longer a necessity. We can identify each detector's relative location by extracting a certain area from one of the detector's image and scanning it on other detectors' images. According to this location, we can generate the point spread functions (PSF) for each detector and perform deconvolution for image restoration. Equipped with this method, the microscope with discretionally designed detector array can be easily constructed without the concern of exact relative locations of detectors. The simulated results and experimental results show the total improvement in resolution with a factor of 1.7 compared to conventional confocal fluorescence microscopy. With the significant enhancement in resolution and easiness for application of this method, this novel method should have potential for a wide range of application in fluorescence microscopy based on parallel detecting.

Characterization of the Distance Relationship Between Localized Serotonin Receptors and Glia Cells on Fluorescence Microscopy Images of Brain Tissue.

PubMed

Jacak, Jaroslaw; Schaller, Susanne; Borgmann, Daniela; Winkler, Stephan M

2015-08-01

We here present two new methods for the characterization of fluorescent localization microscopy images obtained from immunostained brain tissue sections. Direct stochastic optical reconstruction microscopy images of 5-HT1A serotonin receptors and glial fibrillary acidic proteins in healthy cryopreserved brain tissues are analyzed. In detail, we here present two image processing methods for characterizing differences in receptor distribution on glial cells and their distribution on neural cells: One variant relies on skeleton extraction and adaptive thresholding, the other on k-means based discrete layer segmentation. Experimental results show that both methods can be applied for distinguishing classes of images with respect to serotonin receptor distribution. Quantification of nanoscopic changes in relative protein expression on particular cell types can be used to analyze degeneration in tissues caused by diseases or medical treatment.

White light-informed optical properties improve ultrasound-guided fluorescence tomography of photoactive protoporphyrin IX

NASA Astrophysics Data System (ADS)

Flynn, Brendan P.; DSouza, Alisha V.; Kanick, Stephen C.; Davis, Scott C.; Pogue, Brian W.

2013-04-01

Subsurface fluorescence imaging is desirable for medical applications, including protoporphyrin-IX (PpIX)-based skin tumor diagnosis, surgical guidance, and dosimetry in photodynamic therapy. While tissue optical properties and heterogeneities make true subsurface fluorescence mapping an ill-posed problem, ultrasound-guided fluorescence-tomography (USFT) provides regional fluorescence mapping. Here USFT is implemented with spectroscopic decoupling of fluorescence signals (auto-fluorescence, PpIX, photoproducts), and white light spectroscopy-determined bulk optical properties. Segmented US images provide a priori spatial information for fluorescence reconstruction using region-based, diffuse FT. The method was tested in simulations, tissue homogeneous and inclusion phantoms, and an injected-inclusion animal model. Reconstructed fluorescence yield was linear with PpIX concentration, including the lowest concentration used, 0.025 μg/ml. White light spectroscopy informed optical properties, which improved fluorescence reconstruction accuracy compared to the use of fixed, literature-based optical properties, reduced reconstruction error and reconstructed fluorescence standard deviation by factors of 8.9 and 2.0, respectively. Recovered contrast-to-background error was 25% and 74% for inclusion phantoms without and with a 2-mm skin-like layer, respectively. Preliminary mouse-model imaging demonstrated system feasibility for subsurface fluorescence measurement in vivo. These data suggest that this implementation of USFT is capable of regional PpIX mapping in human skin tumors during photodynamic therapy, to be used in dosimetric evaluations.

Imaging bio-distribution of a topically applied dermatological cream on minipig skin using fluorescence lifetime imaging microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Alex, Aneesh; Chaney, Eric J.; Criley, Jennifer M.; Spillman, Darold R.; Hutchison, Phaedra B.; Li, Joanne; Marjanovic, Marina; Frey, Steve; Cook, Steven; Boppart, Stephen A.; Arp, Zane A.

2017-02-01

Currently there is a lack of in vivo techniques to evaluate the spatial bio-distribution of dermal drugs over time without the need to take multiple serial biopsies. To address this gap, we investigated the use of multi-photon optical imaging methods to non-invasively track drug distribution on miniature pig (Species: Sus scrofa, Strain: Göttingen) skin in vivo. Minipig skin is the standard comparative research model to human skin, and is anatomically and functionally similar. We employed fluorescence lifetime imaging microscopy (FLIM) to visualize the spatial distribution and residency time of a topically applied experimental dermatological cream. This was made possible by the endogenous fluorescent optical properties of the experimental drug (fluorescence lifetime > 3000 ps). Two different drug formulations were applied on 2 minipigs for 7 consecutive days, with the control creams applied on the contralateral side, followed by 7 days of post-application monitoring using a multi-modal optical imaging system (MPTflex-CARS, JenLab, Germany). FLIM images were obtained from the treated regions 24 hr post-application from day 1 to day 14 that allowed visualization of cellular and sub-cellular features associated with different dermal layers non-invasively to a depth of 200 µm. Five punch biopsies per animal were obtained from the corresponding treated regions between days 8 and 14 for bioanalytical analysis and comparison with results obtained using FLIM. In conclusion, utilization of non-invasive optical biopsy methods for dermal drug evaluation can provide true longitudinal monitoring of drug spatial distribution, remove sampling limitations, and be more time-efficient compared to traditional methods.

An improved method for differentiating cell-bound from internalized particles by imaging flow cytometry.

PubMed

Smirnov, Asya; Solga, Michael D; Lannigan, Joanne; Criss, Alison K

2015-08-01

Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type. Copyright © 2015 Elsevier B.V. All rights reserved.

Dynamic Assessment of the Endothelialization of Tissue-Engineered Blood Vessels Using an Optical Coherence Tomography Catheter-Based Fluorescence Imaging System.

PubMed

Gurjarpadhye, Abhijit Achyut; DeWitt, Matthew R; Xu, Yong; Wang, Ge; Rylander, Marissa Nichole; Rylander, Christopher G

2015-07-01

Lumen endothelialization of bioengineered vascular scaffolds is essential to maintain small-diameter graft patency and prevent thrombosis postimplantation. Unfortunately, nondestructive imaging methods to visualize this dynamic process are lacking, thus slowing development and clinical translation of these potential tissue-engineering approaches. To meet this need, a fluorescence imaging system utilizing a commercial optical coherence tomography (OCT) catheter was designed to visualize graft endothelialization. C7 DragonFly™ intravascular OCT catheter was used as a channel for delivery and collection of excitation and emission spectra. Poly-dl-lactide (PDLLA) electrospun scaffolds were seeded with endothelial cells (ECs). Seeded cells were exposed to Calcein AM before imaging, causing the living cells to emit green fluorescence in response to blue laser. By positioning the catheter tip precisely over a specimen using high-fidelity electromechanical components, small regions of the specimen were excited selectively. The resulting fluorescence intensities were mapped on a two-dimensional digital grid to generate spatial distribution of fluorophores at single-cell-level resolution. Fluorescence imaging of endothelialization on glass and PDLLA scaffolds was performed using the OCT catheter-based imaging system as well as with a commercial fluorescence microscope. Cell coverage area was calculated for both image sets for quantitative comparison of imaging techniques. Tubular PDLLA scaffolds were maintained in a bioreactor on seeding with ECs, and endothelialization was monitored over 5 days using the OCT catheter-based imaging system. No significant difference was observed in images obtained using our imaging system to those acquired with the fluorescence microscope. Cell area coverage calculated using the images yielded similar values. Nondestructive imaging of endothelialization on tubular scaffolds showed cell proliferation with cell coverage area increasing from 15 ± 4% to 89 ± 6% over 5 days. In this study, we showed the capability of an OCT catheter-based imaging system to obtain single-cell resolution and to quantify endothelialization in tubular electrospun scaffolds. We also compared the resulting images with traditional microscopy, showing high fidelity in image capability. This imaging system, used in conjunction with OCT, could potentially be a powerful tool for in vitro optimization of scaffold cellularization, ensuring long-term graft patency postimplantation.

Normalized Polarization Ratios for the Analysis of Cell Polarity

PubMed Central

Shimoni, Raz; Pham, Kim; Yassin, Mohammed; Ludford-Menting, Mandy J.; Gu, Min; Russell, Sarah M.

2014-01-01

The quantification and analysis of molecular localization in living cells is increasingly important for elucidating biological pathways, and new methods are rapidly emerging. The quantification of cell polarity has generated much interest recently, and ratiometric analysis of fluorescence microscopy images provides one means to quantify cell polarity. However, detection of fluorescence, and the ratiometric measurement, is likely to be sensitive to acquisition settings and image processing parameters. Using imaging of EGFP-expressing cells and computer simulations of variations in fluorescence ratios, we characterized the dependence of ratiometric measurements on processing parameters. This analysis showed that image settings alter polarization measurements; and that clustered localization is more susceptible to artifacts than homogeneous localization. To correct for such inconsistencies, we developed and validated a method for choosing the most appropriate analysis settings, and for incorporating internal controls to ensure fidelity of polarity measurements. This approach is applicable to testing polarity in all cells where the axis of polarity is known. PMID:24963926

Superresolution fluorescence imaging by pump-probe setup using repetitive stimulated transition process

NASA Astrophysics Data System (ADS)

Dake, Fumihiro; Fukutake, Naoki; Hayashi, Seri; Taki, Yusuke

2018-02-01

We proposed superresolution nonlinear fluorescence microscopy with pump-probe setup that utilizes repetitive stimulated absorption and stimulated emission caused by two-color laser beams. The resulting nonlinear fluorescence that undergoes such a repetitive stimulated transition is detectable as a signal via the lock-in technique. As the nonlinear fluorescence signal is produced by the multi-ply combination of incident beams, the optical resolution can be improved. A theoretical model of the nonlinear optical process is provided using rate equations, which offers phenomenological interpretation of nonlinear fluorescence and estimation of the signal properties. The proposed method is demonstrated as having the scalability of optical resolution. Theoretical resolution and bead image are also estimated to validate the experimental result.

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.

Preclinical Whole-body Fluorescence Imaging: Review of Instruments, Methods and Applications

PubMed Central

Leblond, Frederic; Davis, Scott C.; Valdés, Pablo A.; Pogue, Brain W.

2013-01-01

Fluorescence sampling of cellular function is widely used in all aspects of biology, allowing the visualization of cellular and sub-cellular biological processes with spatial resolutions in the range from nanometers up to centimeters. Imaging of fluorescence in vivo has become the most commonly used radiological tool in all pre-clinical work. In the last decade, full-body pre-clinical imaging systems have emerged with a wide range of utilities and niche application areas. The range of fluorescent probes that can be excited in the visible to near-infrared part of the electromagnetic spectrum continues to expand, with the most value for in vivo use being beyond the 630 nm wavelength, because the absorption of light sharply decreases. Whole-body in vivo fluorescence imaging has not yet reached a state of maturity that allows its routine use in the scope of large-scale pre-clinical studies. This is in part due to an incomplete understanding of what the actual fundamental capabilities and limitations of this imaging modality are. However, progress is continuously being made in research laboratories pushing the limits of the approach to consistently improve its performance in terms of spatial resolution, sensitivity and quantification. This paper reviews this imaging technology with a particular emphasis on its potential uses and limitations, the required instrumentation, and the possible imaging geometries and applications. A detailed account of the main commercially available systems is provided as well as some perspective relating to the future of the technology development. Although the vast majority of applications of in vivo small animal imaging are based on epi-illumination planar imaging, the future success of the method relies heavily on the design of novel imaging systems based on state-of-the-art optical technology used in conjunction with high spatial resolution structural modalities such as MRI, CT or ultra-sound. PMID:20031443

Correlative cryogenic tomography of cells using light and soft x-rays.

PubMed

Smith, Elizabeth A; Cinquin, Bertrand P; Do, Myan; McDermott, Gerry; Le Gros, Mark A; Larabell, Carolyn A

2014-08-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryopreserved. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited for correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. © 2013 Elsevier B.V. All rights reserved.

Macroscopic fluorescence imaging: a novel technique to monitor retention and distribution of injected microspheres in an experimental model of ischemic heart failure.

PubMed

Martens, Andreas; Rojas, Sebastian V; Baraki, Hassina; Rathert, Christian; Schecker, Natalie; Hernandez, Sara Rojas; Schwanke, Kristin; Zweigerdt, Robert; Martin, Ulrich; Saito, Shunsuke; Haverich, Axel; Kutschka, Ingo

2014-01-01

The limited effectiveness of cardiac cell therapy has generated concern regarding its clinical relevance. Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly. Surgical aspects and mechanical loss are suspected to be the main culprits behind this phenomenon. As current techniques of monitoring intramyocardial injections are complex and time-consuming, the aim of the study was to develop a fast and simple model to study cardiac retention and distribution following intramyocardial injections. For this purpose, our main hypothesis was that macroscopic fluorescence imaging could adequately serve as a detection method for intramyocardial injections. A total of 20 mice underwent ligation of the left anterior descending artery (LAD) for myocardial infarction. Fluorescent microspheres with cellular dimensions were used as cell surrogates. Particles (5 × 10(5)) were injected into the infarcted area of explanted resting hearts (Ex vivo myocardial injetions EVMI, n = 10) and in vivo into beating hearts (In vivo myocardial injections IVMI, n = 10). Microsphere quantification was performed by fluorescence imaging of explanted organs. Measurements were repeated after a reduction to homogenate dilutions. Cardiac microsphere retention was 2.78 × 10(5) ± 0.31 × 10(5) in the EVMI group. In the IVMI group, cardiac retention of microspheres was significantly lower (0.74 × 10(5) ± 0.18 × 10(5); p<0.05). Direct fluorescence imaging revealed venous drainage through the coronary sinus, resulting in a microsphere accumulation in the left (0.90 × 10(5) ± 0.20 × 10(5)) and the right (1.07 × 10(5) ± 0.17 × 10(5)) lung. Processing to homogenates involved further particle loss (p<0.05) in both groups. We developed a fast and simple direct fluorescence imaging method for biodistribution analysis which enabled the quantification of fluorescent microspheres after intramyocardial delivery using macroscopic fluorescence imaging. This new technique showed massive early particle loss and venous drainage into the right atrium leading to substantial accumulation of graft particles in both lungs.

[Place of indocyanine green coupled with fluorescence imaging in research of breast cancer sentinel node].

PubMed

Vermersch, Charlotte; Raia Barjat, Tiphaine; Perrot, Marianne; Lima, Suzanne; Chauleur, Céline

2016-04-01

The sentinel node has a fundamental role in the management of early breast cancer. Currently, the double detection of blue and radioisotope is recommended. But in common practice, many centers use a single method. However, with a single detection, the risk of false negatives and the identification failure rate increase to a significant extent and the number of sentinel lymph node detected and removed is not enough. Furthermore, the tracers used until now show inconveniences. The purpose of this work is to present a new method of detection, using the green of indocyanine coupled with fluorescence imaging, and to compare it with the already existing methods. The method combined by fluorescence and isotopic is reliable, sure, of fast learning and could constitute a good strategy of detection. The major interest is to obtain a satisfactory number of sentinel nodes. The profit could be even more important for overweight patients. The fluorescence used alone is at the moment not possible. Wide ranging studies are necessary. The FLUOTECH, randomized study of 100 patients, comparing the isotopic method of double isotope technique and fluorescence, is underway to confirm these data. Copyright © 2016 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Mapping absolute tissue endogenous fluorophore concentrations with chemometric wide-field fluorescence microscopy

NASA Astrophysics Data System (ADS)

Xu, Zhang; Reilley, Michael; Li, Run; Xu, Min

2017-06-01

We report chemometric wide-field fluorescence microscopy for imaging the spatial distribution and concentration of endogenous fluorophores in thin tissue sections. Nonnegative factorization aided by spatial diversity is used to learn both the spectral signature and the spatial distribution of endogenous fluorophores from microscopic fluorescence color images obtained under broadband excitation and detection. The absolute concentration map of individual fluorophores is derived by comparing the fluorescence from "pure" fluorophores under the identical imaging condition following the identification of the fluorescence species by its spectral signature. This method is then demonstrated by characterizing the concentration map of endogenous fluorophores (including tryptophan, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide) for lung tissue specimens. The absolute concentrations of these fluorophores are all found to decrease significantly from normal, perilesional, to cancerous (squamous cell carcinoma) tissue. Discriminating tissue types using the absolute fluorophore concentration is found to be significantly more accurate than that achievable with the relative fluorescence strength. Quantification of fluorophores in terms of the absolute concentration map is also advantageous in eliminating the uncertainties due to system responses or measurement details, yielding more biologically relevant data, and simplifying the assessment of competing imaging approaches.

In vivo tomographic imaging of deep seated cancer using fluorescence lifetime contrast

PubMed Central

Rice, William L.; Shcherbakova, Daria M; Verkusha, Vladislav V.; Kumar, Anand T.N.

2015-01-01

Preclinical cancer research would benefit from non-invasive imaging methods that allow tracking and visualization of early stage metastasis in vivo. While fluorescent proteins revolutionized intravital microscopy, two major challenges which still remain are tissue autofluorescence and hemoglobin absorption, which act to limit intravital optical techniques to large or subcutaneous tumors. Here we employ time-domain technology for the effective separation of tissue autofluorescence from extrinsic fluorophores, based on their distinct fluorescence lifetimes. Additionally, we employ cancer cells labelled with near infra-red fluorescent proteins (iRFP) to allow deep-tissue imaging. Our results demonstrate that time-domain imaging allows the detection of metastasis in deep-seated organs of living mice with a more than 20-fold increase in sensitivity compared to conventional continuous wave techniques. Furthermore, the distinct fluorescence lifetimes of each iRFP enables lifetime multiplexing of three different tumors, each expressing unique iRFP labels in the same animal. Fluorescence tomographic reconstructions reveal 3D distributions of iRFP720-expressing cancer cells in lungs and brain of live mice, allowing ready longitudinal monitoring of cancer cell fate with greater sensitivity than otherwise currently possible. PMID:25670171

A low-cost method for visible fluorescence imaging.

PubMed

Tarver, Crissy L; Pusey, Marc

2017-12-01

A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using β-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

Laser-induced dental caries and plaque diagnosis on patients by sensitive autofluorescence spectroscopy and time-gated video imaging: preliminary studies

NASA Astrophysics Data System (ADS)

Koenig, Karsten; Schneckenburger, Herbert

1994-09-01

The laser-induced in vivo autofluorescence of human teeth was investigated by means of time- resolved/time-gated fluorescence techniques. The aim of these studies was non-contact caries and plaque detection. Carious lesions and dental plaque fluoresce in the red spectral region. This autofluorescence seems to be based on porphyrin-producing bacteria. We report on preliminary studies on patients using a novel method of autofluorescence imaging. A special device was constructed for time-gated video imaging. Nanosecond laser pulses for fluorescence excitation were provided by a frequency-doubled, Q-switched Nd:YAG laser. Autofluorescence was detected in an appropriate nanosecond time window using a video camera with a time-gated image intensifier (minimal time gate: 5 ns). Laser-induced autofluorescence based on porphyrin-producing bacteria seems to be an appropriate tool for detecting dental lesions and for creating `caries-images' and `dental plaque' images.

An Automatic Segmentation Method Combining an Active Contour Model and a Classification Technique for Detecting Polycomb-group Proteinsin High-Throughput Microscopy Images.

PubMed

Gregoretti, Francesco; Cesarini, Elisa; Lanzuolo, Chiara; Oliva, Gennaro; Antonelli, Laura

2016-01-01

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.

3D quantitative phase imaging of neural networks using WDT

NASA Astrophysics Data System (ADS)

Kim, Taewoo; Liu, S. C.; Iyer, Raj; Gillette, Martha U.; Popescu, Gabriel

2015-03-01

White-light diffraction tomography (WDT) is a recently developed 3D imaging technique based on a quantitative phase imaging system called spatial light interference microscopy (SLIM). The technique has achieved a sub-micron resolution in all three directions with high sensitivity granted by the low-coherence of a white-light source. Demonstrations of the technique on single cell imaging have been presented previously; however, imaging on any larger sample, including a cluster of cells, has not been demonstrated using the technique. Neurons in an animal body form a highly complex and spatially organized 3D structure, which can be characterized by neuronal networks or circuits. Currently, the most common method of studying the 3D structure of neuron networks is by using a confocal fluorescence microscope, which requires fluorescence tagging with either transient membrane dyes or after fixation of the cells. Therefore, studies on neurons are often limited to samples that are chemically treated and/or dead. WDT presents a solution for imaging live neuron networks with a high spatial and temporal resolution, because it is a 3D imaging method that is label-free and non-invasive. Using this method, a mouse or rat hippocampal neuron culture and a mouse dorsal root ganglion (DRG) neuron culture have been imaged in order to see the extension of processes between the cells in 3D. Furthermore, the tomogram is compared with a confocal fluorescence image in order to investigate the 3D structure at synapses.

Robust demarcation of basal cell carcinoma by dependent component analysis-based segmentation of multi-spectral fluorescence images.

PubMed

Kopriva, Ivica; Persin, Antun; Puizina-Ivić, Neira; Mirić, Lina

2010-07-02

This study was designed to demonstrate robust performance of the novel dependent component analysis (DCA)-based approach to demarcation of the basal cell carcinoma (BCC) through unsupervised decomposition of the red-green-blue (RGB) fluorescent image of the BCC. Robustness to intensity fluctuation is due to the scale invariance property of DCA algorithms, which exploit spectral and spatial diversities between the BCC and the surrounding tissue. Used filtering-based DCA approach represents an extension of the independent component analysis (ICA) and is necessary in order to account for statistical dependence that is induced by spectral similarity between the BCC and surrounding tissue. This generates weak edges what represents a challenge for other segmentation methods as well. By comparative performance analysis with state-of-the-art image segmentation methods such as active contours (level set), K-means clustering, non-negative matrix factorization, ICA and ratio imaging we experimentally demonstrate good performance of DCA-based BCC demarcation in two demanding scenarios where intensity of the fluorescent image has been varied almost two orders of magnitude. Copyright 2010 Elsevier B.V. All rights reserved.

Advanced forensic validation for human spermatozoa identification using SPERM HY-LITER™ Express with quantitative image analysis.

PubMed

Takamura, Ayari; Watanabe, Ken; Akutsu, Tomoko

2017-07-01

Identification of human semen is indispensable for the investigation of sexual assaults. Fluorescence staining methods using commercial kits, such as the series of SPERM HY-LITER™ kits, have been useful to detect human sperm via strong fluorescence. These kits have been examined from various forensic aspects. However, because of a lack of evaluation methods, these studies did not provide objective, or quantitative, descriptions of the results nor clear criteria for the decisions reached. In addition, the variety of validations was considerably limited. In this study, we conducted more advanced validations of SPERM HY-LITER™ Express using our established image analysis method. Use of this method enabled objective and specific identification of fluorescent sperm's spots and quantitative comparisons of the sperm detection performance under complex experimental conditions. For body fluid mixtures, we examined interference with the fluorescence staining from other body fluid components. Effects of sample decomposition were simulated in high humidity and high temperature conditions. Semen with quite low sperm concentrations, such as azoospermia and oligospermia samples, represented the most challenging cases in application of the kit. Finally, the tolerance of the kit against various acidic and basic environments was analyzed. The validations herein provide useful information for the practical applications of the SPERM HY-LITER™ Express kit, which were previously unobtainable. Moreover, the versatility of our image analysis method toward various complex cases was demonstrated.

Monte Carlo based method for fluorescence tomographic imaging with lifetime multiplexing using time gates

PubMed Central

Chen, Jin; Venugopal, Vivek; Intes, Xavier

2011-01-01

Time-resolved fluorescence optical tomography allows 3-dimensional localization of multiple fluorophores based on lifetime contrast while providing a unique data set for improved resolution. However, to employ the full fluorescence time measurements, a light propagation model that accurately simulates weakly diffused and multiple scattered photons is required. In this article, we derive a computationally efficient Monte Carlo based method to compute time-gated fluorescence Jacobians for the simultaneous imaging of two fluorophores with lifetime contrast. The Monte Carlo based formulation is validated on a synthetic murine model simulating the uptake in the kidneys of two distinct fluorophores with lifetime contrast. Experimentally, the method is validated using capillaries filled with 2.5nmol of ICG and IRDye™800CW respectively embedded in a diffuse media mimicking the average optical properties of mice. Combining multiple time gates in one inverse problem allows the simultaneous reconstruction of multiple fluorophores with increased resolution and minimal crosstalk using the proposed formulation. PMID:21483610

Wide-field imaging through scattering media by scattered light fluorescence microscopy

NASA Astrophysics Data System (ADS)

Zhou, Yulan; Li, Xun

2017-08-01

To obtain images through scattering media, scattered light fluorescence (SLF) microscopy that utilizes the optical memory effect has been developed. However, the small field of view (FOV) of SLF microscopy limits its application. In this paper, we have introduced a re-modulation method to achieve wide-field imaging through scattering media by SLF microscopy. In the re-modulation method, to raster scan the focus across the object plane, the incident wavefront is re-modulated via a spatial light modulator (SLM) in the updated phase compensation calculated using the optimized iterative algorithm. Compared with the conventional optical memory effect method, the re-modulation method can greatly increase the FOV of a SLF microscope. With the phase compensation theoretically calculated, the process of updating the phase compensation of a high speed SLM is fast. The re-modulation method does not increase the imaging time. The re-modulation method is, therefore, expected to make SLF microscopy have much wider applications in biology, medicine and physiology.

An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy.

PubMed

Warner, Cherish A; Biedrzycki, Meredith L; Jacobs, Samuel S; Wisser, Randall J; Caplan, Jeffrey L; Sherrier, D Janine

2014-12-01

We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process. © 2014 American Society of Plant Biologists. All Rights Reserved.

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

Kang, Hyeonggon; Attota, Ravikiran, E-mail: ravikiran.attota@nist.gov; Tondare, Vipin

We present a method that uses conventional optical microscopes to determine the number of nanoparticles in a cluster, which is typically not possible using traditional image-based optical methods due to the diffraction limit. The method, called through-focus scanning optical microscopy (TSOM), uses a series of optical images taken at varying focus levels to achieve this. The optical images cannot directly resolve the individual nanoparticles, but contain information related to the number of particles. The TSOM method makes use of this information to determine the number of nanoparticles in a cluster. Initial good agreement between the simulations and the measurements ismore » also presented. The TSOM method can be applied to fluorescent and non-fluorescent as well as metallic and non-metallic nano-scale materials, including soft materials, making it attractive for tag-less, high-speed, optical analysis of nanoparticles down to 45 nm diameter.« less

Super-resolution fluorescence microscopy by stepwise optical saturation

PubMed Central

Zhang, Yide; Nallathamby, Prakash D.; Vigil, Genevieve D.; Khan, Aamir A.; Mason, Devon E.; Boerckel, Joel D.; Roeder, Ryan K.; Howard, Scott S.

2018-01-01

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples. PMID:29675306

Univariate and multivariate methods for chemical mapping of cervical cancer cells

NASA Astrophysics Data System (ADS)

Duraipandian, Shiyamala; Zheng, Wei; Huang, Zhiwei

2012-01-01

Visualization of cells and subcellular organelles are currently carried out using available microscopy methods such as cryoelectron microscopy, and fluorescence microscopy. These methods require external labeling using fluorescent dyes and extensive sample preparations to access the subcellular structures. However, Raman micro-spectroscopy provides a non-invasive, label-free method for imaging the cells with chemical specificity at sub-micrometer spatial resolutions. The scope of this paper is to image the biochemical/molecular distributions in cells associated with cancerous changes. Raman map data sets were acquired from the human cervical carcinoma cell lines (HeLa) after fixation under 785 nm excitation wavelength. The individual spectrum was recorded by raster-scanning the laser beam over the sample with 1μm step size and 10s exposure time. Images revealing nucleic acids, lipids and proteins (phenylalanine, amide I) were reconstructed using univariate methods. In near future, the small pixel to pixel variations will also be imaged using different multivariate methods (PCA, clustering (HCA, K-means, FCM)) to determine the main cellular constitutions. The hyper-spectral image of cell was reconstructed utilizing the spectral contrast at different pixels of the cell (due to the variation in the biochemical distribution) without using fluorescent dyes. Normal cervical squamous cells will also be imaged in order to differentiate normal and cancer cells of cervix using the biochemical changes in different grades of cancer. Based on the information obtained from the pseudo-color maps, constructed from the hyper-spectral cubes, the primary cellular constituents of normal and cervical cancer cells were identified.

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.

Point-of-care testing for streptomycin based on aptamer recognizing and digital image colorimetry by smartphone.

PubMed

Lin, Bixia; Yu, Ying; Cao, Yujuan; Guo, Manli; Zhu, Debin; Dai, Jiaxing; Zheng, Minshi

2018-02-15

The rapid detection of antibiotic residual in everyday life is very important for food safety. In order to realize the on-site and visual detection of antibiotic, a POCT method was established by using digital image colorimetry based on smartphone. Streptomycin was taken as the analyte model of antibiotics, streptomycin aptamer preferentially recognized analyte, and the excess aptamer hybridized with the complementary DNA to form the dsDNA. SYBR Green I combined with the dsDNA and then emitted obvious green fluorescence, thus the fluorescence intensity decreased with the increasing of streptomycin concentration. Then a smartphone-based device was constructed as the fluorescence readout. The smartphone camera acquired the images of the fluorescence derived from the samples, and the Touch Color APP installed in smartphone read out the RGB values of the images. There was a linear relationship between the G values and the streptomycin concentrations in the range of 0.1-100µM. The detection limit was 94nM, which was lower than the maximum residue limit defined by World Health Organization. The POCT method was applied for determining streptomycin in chicken and milk samples with recoveries in 94.1-110%. This method had the advantages of good selectivity, simple operation and on-site visualization. Copyright © 2017 Elsevier B.V. All rights reserved.

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

PubMed

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

In vivo magnetic resonance and fluorescence dual imaging of tumor sites by using dye-doped silica-coated iron oxide nanoparticles

NASA Astrophysics Data System (ADS)

Jang, Haeyun; Lee, Chaedong; Nam, Gi-Eun; Quan, Bo; Choi, Hyuck Jae; Yoo, Jung Sun; Piao, Yuanzhe

2016-02-01

The difficulty in delineating tumor is a major obstacle for better outcomes in cancer treatment of patients. The use of single-imaging modality is often limited by inadequate sensitivity and resolution. Here, we present the synthesis and the use of monodisperse iron oxide nanoparticles coated with fluorescent silica nano-shells for fluorescence and magnetic resonance dual imaging of tumor. The as-synthesized core-shell nanoparticles were designed to improve the accuracy of diagnosis via simultaneous tumor imaging with dual imaging modalities by a single injection of contrast agent. The iron oxide nanocrystals ( 11 nm) were coated with Rhodamine B isothiocyanate-doped silica shells via reverse microemulsion method. Then, the core-shell nanoparticles ( 54 nm) were analyzed to confirm their size distribution by transmission electron microscopy and dynamic laser scattering. Photoluminescence spectroscopy was used to characterize the fluorescent property of the dye-doped silica shell-coated nanoparticles. The cellular compatibility of the as-prepared nanoparticles was confirmed by a trypan blue dye exclusion assay and the potential as a dual-imaging contrast agent was verified by in vivo fluorescence and magnetic resonance imaging. The experimental results show that the uniform-sized core-shell nanoparticles are highly water dispersible and the cellular toxicity of the nanoparticles is negligible. In vivo fluorescence imaging demonstrates the capability of the developed nanoparticles to selectively target tumors by the enhanced permeability and retention effects and ex vivo tissue analysis was corroborated this. Through in vitro phantom test, the core/shell nanoparticles showed a T2 relaxation time comparable to Feridex® with smaller size, indicating that the as-made nanoparticles are suitable for imaging tumor. This new dual-modality-nanoparticle approach has promised for enabling more accurate tumor imaging.

In Vivo Near-Infrared Imaging of Fibrin Deposition in Thromboembolic Stroke in Mice

PubMed Central

Zhang, Yi; Fan, Shufeng; Yao, Yuyu; Ding, Jie; Wang, Yu; Zhao, Zhen; Liao, Lei; Li, Peicheng; Zang, Fengchao; Teng, Gao-Jun

2012-01-01

Objectives Thrombus and secondary thrombosis plays a key role in stroke. Recent molecular imaging provides in vivo imaging of activated factor XIII (FXIIIa), an important mediator of thrombosis or fibrinolytic resistance. The present study was to investigate the fibrin deposition in a thromboembolic stroke mice model by FXIIIa–targeted near-infrared fluorescence (NIRF) imaging. Materials and Methods The experimental protocol was approved by our institutional animal use committee. Seventy-six C57B/6J mice were subjected to thromboembolic middle cerebral artery occlusion or sham operation. Mice were either intravenously injected with the FXIIIa-targeted probe or control probe. In vivo and ex vivo NIRF imaging were performed thereafter. Probe distribution was assessed with fluorescence microscopy by spectral imaging and quantification system. MR scans were performed to measure lesion volumes in vivo, which were correlated with histology after animal euthanasia. Results In vivo significant higher fluorescence intensity over the ischemia-affected hemisphere, compared to the contralateral side, was detected in mice that received FXIIIa-targeted probe, but not in the controlled mice. Significantly NIRF signals showed time-dependent processes from 8 to 96 hours after injection of FXIIIa-targeted probes. Ex vivo NIRF image showed an intense fluorescence within the ischemic territory only in mice injected with FXIIIa-targeted probe. The fluorescence microscopy demonstrated distribution of FXIIIa-targeted probe in the ischemic region and nearby micro-vessels, and FXIIIa-targeted probe signals showed good overlap with immune-fluorescent fibrin staining images. There was a significant correlation between total targeted signal from in vivo or ex vivo NIRF images and lesion volume. Conclusion Non-invasive detection of fibrin deposition in ischemic mouse brain using NIRF imaging is feasible and this technique may provide an in vivo experimental tool in studying the role of fibrin in stroke. PMID:22272319

Cell nuclei segmentation in fluorescence microscopy images using inter- and intra-region discriminative information.

PubMed

Song, Yang; Cai, Weidong; Feng, David Dagan; Chen, Mei

2013-01-01

Automated segmentation of cell nuclei in microscopic images is critical to high throughput analysis of the ever increasing amount of data. Although cell nuclei are generally visually distinguishable for human, automated segmentation faces challenges when there is significant intensity inhomogeneity among cell nuclei or in the background. In this paper, we propose an effective method for automated cell nucleus segmentation using a three-step approach. It first obtains an initial segmentation by extracting salient regions in the image, then reduces false positives using inter-region feature discrimination, and finally refines the boundary of the cell nuclei using intra-region contrast information. This method has been evaluated on two publicly available datasets of fluorescence microscopic images with 4009 cells, and has achieved superior performance compared to popular state of the art methods using established metrics.

Near-infrared optical imaging of nucleic acid nanocarriers in vivo

PubMed Central

Rome, Claire; Gravier, Julien; Morille, Marie; Divita, Gilles; Bolcato-Bellemin, Anne-Laure; Josserand, Véronique; Coll, Jean-Luc

2013-01-01

Summary Non-invasive, real time optical imaging methods are particularly well suited for the in vivo follow up of the distribution of nucleic acids nanocarriers, their dissociation and finally the resulting gene expression or inhibition. Indeed, most small animal imaging devices are performing bioluminescence and fluorescence measurements without moving the animal, allowing a simple, rapid and cost effective method of investigation of several parameters at a time, in longitudinal experiments that can last for days or weeks. Here we propose to help the reader choosing adapted near-infrared (NIR) fluorophores or pairs of fluorophores for FRET assays, reporter genes as well as nanocarriers for in vivo gene and siRNA delivery. In addition, we present the labeling methods of these macromolecules, and of their payload and the protocols to detect them using bioluminescence and NIR fluorescence imaging in mice. PMID:23070763

Automated segmentation of retinal pigment epithelium cells in fluorescence adaptive optics images.

PubMed

Rangel-Fonseca, Piero; Gómez-Vieyra, Armando; Malacara-Hernández, Daniel; Wilson, Mario C; Williams, David R; Rossi, Ethan A

2013-12-01

Adaptive optics (AO) imaging methods allow the histological characteristics of retinal cell mosaics, such as photoreceptors and retinal pigment epithelium (RPE) cells, to be studied in vivo. The high-resolution images obtained with ophthalmic AO imaging devices are rich with information that is difficult and/or tedious to quantify using manual methods. Thus, robust, automated analysis tools that can provide reproducible quantitative information about the cellular mosaics under examination are required. Automated algorithms have been developed to detect the position of individual photoreceptor cells; however, most of these methods are not well suited for characterizing the RPE mosaic. We have developed an algorithm for RPE cell segmentation and show its performance here on simulated and real fluorescence AO images of the RPE mosaic. Algorithm performance was compared to manual cell identification and yielded better than 91% correspondence. This method can be used to segment RPE cells for morphometric analysis of the RPE mosaic and speed the analysis of both healthy and diseased RPE mosaics.

Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy

PubMed Central

Brooker, Gary; Siegel, Nisan; Wang, Victor; Rosen, Joseph

2011-01-01

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging. PMID:21445140

In Vivo Time-gated Fluorescence Imaging with Biodegradable Luminescent Porous Silicon Nanoparticles

PubMed Central

Gu, Luo; Hall, David J.; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J.; Howell, Stephen B.; Sailor, Michael J.

2014-01-01

Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (< 10 ns) emission signals from organic chromophores or tissue autofluorescence.Here, using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by > 50-fold in vitro and by > 20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed. PMID:23933660

Reconstruction of fluorescence molecular tomography with a cosinoidal level set method.

PubMed

Zhang, Xuanxuan; Cao, Xu; Zhu, Shouping

2017-06-27

Implicit shape-based reconstruction method in fluorescence molecular tomography (FMT) is capable of achieving higher image clarity than image-based reconstruction method. However, the implicit shape method suffers from a low convergence speed and performs unstably due to the utilization of gradient-based optimization methods. Moreover, the implicit shape method requires priori information about the number of targets. A shape-based reconstruction scheme of FMT with a cosinoidal level set method is proposed in this paper. The Heaviside function in the classical implicit shape method is replaced with a cosine function, and then the reconstruction can be accomplished with the Levenberg-Marquardt method rather than gradient-based methods. As a result, the priori information about the number of targets is not required anymore and the choice of step length is avoided. Numerical simulations and phantom experiments were carried out to validate the proposed method. Results of the proposed method show higher contrast to noise ratios and Pearson correlations than the implicit shape method and image-based reconstruction method. Moreover, the number of iterations required in the proposed method is much less than the implicit shape method. The proposed method performs more stably, provides a faster convergence speed than the implicit shape method, and achieves higher image clarity than the image-based reconstruction method.

Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

NASA Astrophysics Data System (ADS)

Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

2015-07-01

Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ˜16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

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

Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

PubMed Central

Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

2015-01-01

Abstract. Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations. PMID:26160347

Motion estimation of subcellular structures from fluorescence microscopy images.

PubMed

Vallmitjana, A; Civera-Tregon, A; Hoenicka, J; Palau, F; Benitez, R

2017-07-01

We present an automatic image processing framework to study moving intracellular structures from live cell fluorescence microscopy. The system includes the identification of static and dynamic structures from time-lapse images using data clustering as well as the identification of the trajectory of moving objects with a probabilistic tracking algorithm. The method has been successfully applied to study mitochondrial movement in neurons. The approach provides excellent performance under different experimental conditions and is robust to common sources of noise including experimental, molecular and biological fluctuations.

Enumerating viruses by using fluorescence and the nature of the nonviral background fraction.

PubMed

Pollard, Peter C

2012-09-01

Bulk fluorescence measurements could be a faster and cheaper way of enumerating viruses than epifluorescence microscopy, flow cytometry, or transmission electron microscopy (TEM). However, since viruses are not imaged, the background fluorescence compromises the signal, and we know little about its nature. In this paper the size ranges of nucleotides that fluoresce in the presence of SYBR gold were determined for wastewater and a range of freshwater samples using a differential filtration method. Fluorescence excitation-emission matrices (FEEMs) showed that >70% of the SYBR fluorescence was in the <10-nm size fraction (background) and was not associated with intact viruses. This was confirmed using TEM. The use of FEEMs to develop a fluorescence-based method for counting viruses is an approach that is fundamentally different from the epifluorescence microscopy technique used for enumerating viruses. This high fluorescence background is currently overlooked, yet it has had a most pervasive influence on the development of a simple fluorescence-based method for quantifying viral abundance in water.

A ratiometric threshold for determining presence of cancer during fluorescence-guided surgery.

PubMed

Warram, Jason M; de Boer, Esther; Moore, Lindsay S; Schmalbach, Cecelia E; Withrow, Kirk P; Carroll, William R; Richman, Joshua S; Morlandt, Anthony B; Brandwein-Gensler, Margaret; Rosenthal, Eben L

2015-07-01

Fluorescence-guided imaging to assist in identification of malignant margins has the potential to dramatically improve oncologic surgery. However, a standardized method for quantitative assessment of disease-specific fluorescence has not been investigated. Introduced here is a ratiometric threshold derived from mean fluorescent tissue intensity that can be used to semi-quantitatively delineate tumor from normal tissue. Open-field and a closed-field imaging devices were used to quantify fluorescence in punch biopsy tissues sampled from primary tumors collected during a phase 1 trial evaluating the safety of cetuximab-IRDye800 in patients (n = 11) undergoing surgical intervention for head and neck cancer. Fluorescence ratios were calculated using mean fluorescence intensity (MFI) from punch biopsy normalized by MFI of patient-matched tissues. Ratios were compared to pathological assessment and a ratiometric threshold was established to predict presence of cancer. During open-field imaging using an intraoperative device, the threshold for muscle normalized tumor fluorescence was found to be 2.7, which produced a sensitivity of 90.5% and specificity of 78.6% for delineating disease tissue. The skin-normalized threshold generated greater sensitivity (92.9%) and specificity (81.0%). Successful implementation of a semi-quantitative threshold can provide a scientific methodology for delineating disease from normal tissue during fluorescence-guided resection of cancer. © 2015 Wiley Periodicals, Inc.

System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents

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

Levenson, Richard; Demos, Stavros

A method is disclosed for analyzing a thin tissue sample and adapted to be supported on a slide. The tissue sample may be placed on a slide and exposed to one or more different exogenous fluorophores excitable in a range of about 300 nm-200 nm, and having a useful emission band from about 350 nm-900 nm, and including one or more fluorescent dyes or fluorescently labeled molecular probes that accumulate in tissue or cellular components. The fluorophores may be excited with a first wavelength of UV light between about 200 nm-290 nm. An optical system collects emissions from the fluorophoresmore » at a second wavelength, different from the first wavelength, which are generated in response to the first wavelength of UV light, to produce an image for analysis.« less

Wide-field fluorescence diffuse optical tomography with epi-illumination of sinusoidal pattern

NASA Astrophysics Data System (ADS)

Li, Tongxin; Gao, Feng; Chen, Weiting; Qi, Caixia; Yan, Panpan; Zhao, Huijuan

2017-02-01

We present a wide-field fluorescence tomography with epi-illumination of sinusoidal pattern. In this scheme, a DMD projector is employed as a spatial light modulator to generate independently wide-field sinusoidal illumination patterns at varying spatial frequencies on a sample, and then the emitted photons at the sample surface were captured with a EM-CCD camera. This method results in a significantly reduced number of the optical field measurements as compared to the point-source-scanning ones and thereby achieves a fast data acquisition that is desired for a dynamic imaging application. Fluorescence yield images are reconstructed using the normalized-Born formulated inversion of the diffusion model. Experimental reconstructions are presented on a phantom embedding the fluorescent targets and compared for a combination of the multiply frequencies. The results validate the ability of the method to determine the target relative depth and quantification with an increasing accuracy.

High-Throughput Live-Cell Microscopy Analysis of Association Between Chromosome Domains and the Nucleolus in S. cerevisiae.

PubMed

Wang, Renjie; Normand, Christophe; Gadal, Olivier

2016-01-01

Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell's nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.

In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds.

PubMed

Tseng, Jen-Chieh; Kung, Andrew L

2015-06-24

Here we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as β-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry. Efficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular β-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity. In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

Non-invasive dual fluorescence in vivo imaging for detection of macrophage infiltration and matrix metalloproteinase (MMP) activity in inflammatory arthritic joints

PubMed Central

Cho, Hongsik; Bhatti, Fazal-Ur-Rehman; Yoon, Tae Won; Hasty, Karen A.; Stuart, John M.; Yi, Ae-Kyung

2016-01-01

Detection and intervention at an early stage is a critical factor to impede arthritis progress. Here we present a non-invasive method to detect inflammatory changes in joints of arthritic mice. Inflammation was monitored by dual fluorescence optical imaging for near-infrared fluorescent (750F) matrix-metalloproteinase activatable agent and allophycocyanin-conjugated anti-mouse CD11b. Increased intensity of allophycocyanin (indication of macrophage accumulation) and 750F (indication of matrix-metalloproteinase activity) showed a biological relationship with the arthritis severity score and the histopathology score of arthritic joints. Our results demonstrate that this method can be used to detect early stages of arthritis with minimum intervention in small animal models. PMID:27231625

A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation.

PubMed

Pelet, S; Previte, M J R; Laiho, L H; So, P T C

2004-10-01

Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained analysis. Convergence speed can be greatly accelerated by providing appropriate initial guesses. Realizing that the image morphology often correlates with fluorophore distribution, a global fitting algorithm has been developed to assign initial guesses throughout an image based on a segmentation analysis. This algorithm was tested on both simulated data sets and time-domain lifetime measurements. We have successfully measured fluorophore distribution in fibroblasts stained with Hoechst and calcein. This method further allows second harmonic generation from collagen and elastin autofluorescence to be differentiated in fluorescence lifetime imaging microscopy images of ex vivo human skin. On our experimental measurement, this algorithm increased convergence speed by over two orders of magnitude and achieved significantly better fits. Copyright 2004 Biophysical Society

Highly sensitive fluorescence detection of metastatic lymph nodes of gastric cancer with photo-oxidation of protoporphyrin IX.

PubMed

Koizumi, N; Harada, Y; Beika, M; Minamikawa, T; Yamaoka, Y; Dai, P; Murayama, Y; Yanagisawa, A; Otsuji, E; Tanaka, H; Takamatsu, T

2016-08-01

The establishment of a precise and rapid method to detect metastatic lymph nodes (LNs) is essential to perform less invasive surgery with reduced gastrectomy along with reduced lymph node dissection. We herein describe a novel imaging strategy to detect 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence in excised LNs specifically with reduced effects of tissue autofluorescence based on photo-oxidation of PpIX. We applied the method in a clinical setting, and evaluated its feasibility. To reduce the unfavorable effect of autofluorescence, we focused on photo-oxidation of PpIX: Following light irradiation, PpIX changes into another substance, photo-protoporphyrin, via an oxidative process, which has a different spectral peak, at 675 nm, whereas PpIX has its spectral peak at 635 nm. Based on the unique spectral alteration, fluorescence spectral imaging before and after light irradiation and subsequent originally-developed image processing was performed. Following in vitro study, we applied this method to a total of 662 excised LNs obtained from 30 gastric cancer patients administered 5-ALA preoperatively. Specific visualization of PpIX was achieved in in vitro study. The method allowed highly sensitive detection of metastatic LNs, with sensitivity of 91.9% and specificity of 90.8% in the in vivo clinical trial. Receiver operating characteristic analysis indicated high diagnostic accuracy, with the area under the curve of 0.926. We established a highly sensitive and specific 5-ALA-induced fluorescence imaging method applicable in clinical settings. The novel method has a potential to become a useful tool for intraoperative rapid diagnosis of LN metastasis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

PubMed Central

Maynard, Carrie; Plaks, Vicki

2016-01-01

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment. PMID:26862704

Indocyanine Green Loaded Reduced Graphene Oxide for In Vivo Photoacoustic/Fluorescence Dual-Modality Tumor Imaging

NASA Astrophysics Data System (ADS)

Chen, Jingqin; Liu, Chengbo; Zeng, Guang; You, Yujia; Wang, Huina; Gong, Xiaojing; Zheng, Rongqin; Kim, Jeesu; Kim, Chulhong; Song, Liang

2016-02-01

Multimodality imaging based on multifunctional nanocomposites holds great promise to fundamentally augment the capability of biomedical imaging. Specifically, photoacoustic and fluorescence dual-modality imaging is gaining much interest because of their non-invasiveness and the complementary nature of the two modalities in terms of imaging resolution, depth, sensitivity, and speed. Herein, using a green and facile method, we synthesize indocyanine green (ICG) loaded, polyethylene glycol (PEG)ylated, reduced nano-graphene oxide nanocomposite (rNGO-PEG/ICG) as a new type of fluorescence and photoacoustic dual-modality imaging contrast. The nanocomposite is shown to have minimal toxicity and excellent photoacoustic/fluorescence signals both in vitro and in vivo. Compared with free ICG, the nanocomposite is demonstrated to possess greater stability, longer blood circulation time, and superior passive tumor targeting capability. In vivo study shows that the circulation time of rNGO-PEG/ICG in the mouse body can sustain up to 6 h upon intravenous injection; while after 1 day, no obvious accumulation of rNGO-PEG/ICG is found in any major organs except the tumor regions. The demonstrated high fluorescence/photoacoustic dual contrasts, together with its low toxicity and excellent circulation life time, suggest that the synthesized rNGO-PEG/ICG can be a promising candidate for further translational studies on both the early diagnosis and image-guided therapy/surgery of cancer.

Analysis of doxorubicin distribution in MCF-7 cells treated with drug-loaded nanoparticles by combination of two fluorescence-based techniques, confocal spectral imaging and capillary electrophoresis.

PubMed

Gautier, Juliette; Munnier, Emilie; Soucé, Martin; Chourpa, Igor; Douziech Eyrolles, Laurence

2015-05-01

The intracellular distribution of the antiancer drug doxorubicin (DOX) was followed qualitatively by fluorescence confocal spectral imaging (FCSI) and quantitatively by capillary electrophoresis (CE). FCSI permits the localization of the major fluorescent species in cell compartments, with spectral shifts indicating the polarity of the respective environment. However, distinction between drug and metabolites by FCSI is difficult due to their similar fluorochromes, and direct quantification of their fluorescence is complicated by quantum yield variation between different subcellular environments. On the other hand, capillary electrophoresis with fluorescence detection (CE-LIF) is a quantitative method capable of separating doxorubicin and its metabolites. In this paper, we propose a method for determining drug and metabolite concentration in enriched nuclear and cytosolic fractions of cancer cells by CE-LIF, and we compare these data with those of FCSI. Significant differences in the subcellular distribution of DOX are observed between the drug administered as a molecular solution or as a suspension of drug-loaded iron oxide nanoparticles coated with polyethylene glycol. Comparative analysis of the CE-LIF vs FCSI data may lead to a tentative calibration of this latter method in terms of DOX fluorescence quantum yields in the nucleus and more or less polar regions of the cytosol.

Preclinical Evaluation of Robotic-Assisted Sentinel Lymph Node Fluorescence Imaging

PubMed Central

Liss, Michael A.; Farshchi-Heydari, Salman; Qin, Zhengtao; Hickey, Sean A.; Hall, David J.; Kane, Christopher J.; Vera, David R.

2015-01-01

An ideal substance to provide convenient and accurate targeting for sentinel lymph node (SLN) mapping during robotic-assisted surgery has yet to be found. We used an animal model to determine the ability of the FireFly camera system to detect fluorescent SLNs after administration of a dual-labeled molecular imaging agent. Methods We injected the footpads of New Zealand White rabbits with 1.7 or 8.4 nmol of tilmanocept labeled with 99mTc and a near-infrared fluorophore, IRDye800CW. One and 36 h after injection, popliteal lymph nodes, representing the SLNs, were dissected with the assistance of the FireFly camera system, a fluorescence-capable endoscopic imaging system. After excision of the paraaortic lymph nodes, which represented non-SLNs, we assayed all lymph nodes for radioactivity and fluorescence intensity. Results Fluorescence within all popliteal lymph nodes was easily detected by the FireFly camera system. Fluorescence within the lymph channel could be imaged during the 1-h studies. When compared with the paraaortic lymph nodes, the popliteal lymph nodes retain greater than 95% of the radioactivity at both 1 and 36 h after injection. At both doses (1.7 and 8.4 nmol), the popliteal nodes had higher (P < 0.050) optical fluorescence intensity than the paraaortic nodes at the 1- and 36-h time points. Conclusion The FireFly camera system can easily detect tilmanocept labeled with a near-infrared fluorophore at least 36 h after administration. This ability will permit image acquisition and subsequent verification of fluorescence-labeled SLNs during robotic-assisted surgery. PMID:25024425

Imaging of Neuronal Activity in Awake Mice by Measurements of Flavoprotein Autofluorescence Corrected for Cerebral Blood Flow.

PubMed

Takahashi, Manami; Urushihata, Takuya; Takuwa, Hiroyuki; Sakata, Kazumi; Takado, Yuhei; Shimizu, Eiji; Suhara, Tetsuya; Higuchi, Makoto; Ito, Hiroshi

2017-01-01

Green fluorescence imaging (e.g., flavoprotein autofluorescence imaging, FAI) can be used to measure neuronal activity and oxygen metabolism in living brains without expressing fluorescence proteins. It is useful for understanding the mechanism of various brain functions and their abnormalities in age-related brain diseases. However, hemoglobin in cerebral blood vessels absorbs green fluorescence, hampering accurate assessments of brain function in animal models with cerebral blood vessel dysfunctions and subsequent cerebral blood flow (CBF) alterations. In the present study, we developed a new method to correct FAI signals for hemoglobin-dependent green fluorescence reductions by simultaneous measurements of green fluorescence and intrinsic optical signals. Intrinsic optical imaging enabled evaluations of light absorption and scatters by hemoglobin, which could then be applied to corrections of green fluorescence intensities. Using this method, enhanced flavoprotein autofluorescence by sensory stimuli was successfully detected in the brains of awake mice, despite increases of CBF, and hemoglobin interference. Moreover, flavoprotein autofluorescence could be properly quantified in a resting state and during sensory stimulation by a CO 2 inhalation challenge, which modified vascular responses without overtly affecting neuronal activities. The flavoprotein autofluorescence signal data obtained here were in good agreement with the previous findings from a condition with drug-induced blockade of cerebral vasodilation, justifying the current assaying methodology. Application of this technology to studies on animal models of brain diseases with possible changes of CBF, including age-related neurological disorders, would provide better understanding of the mechanisms of neurovascular coupling in pathological circumstances.

Fluorescently labeled therapeutic antibodies for detection of microscopic melanoma

PubMed Central

Day, Kristine E.; Beck, Lauren N.; Deep, Nicholas L.; Kovar, Joy; Zinn, Kurt R; Rosenthal, Eben L.

2013-01-01

Objective Detection of microscopic disease during surgical resection of melanoma remains a significant challenge. To assess real-time optical imaging for visualization of microscopic cancer, we evaluated three FDA-approved therapeutic monoclonal antibodies. Study Design Prospective, basic science Methods Melanoma cell lines (A375 and SKMEL5) were xenografted into the ears of immunodeficient mice. Bevacizumab, panitumumab, tocilizumab, or a non-specific IgG were covalently linked to a near-infrared (NIR) fluorescent probe (IRDye800CW) and systemically injected. Primary tumors were imaged and then resected under fluorescent guidance using the SPY, an NIR imaging system used in plastic and reconstructive surgeries to evaluate perfusion. Mice were also imaged with the Pearl Impulse small animal imager, an NIR imaging system designed for use with IRDye800CW. Post-resection, small tissue fragments were fluorescently imaged and presence of tumor subsequently confirmed by correlation with histology. Results All fluorescently-labeled therapeutic monoclonal antibodies could adequately delineate tumor from normal tissue based on tumor-to-background ratios (TBR) compared to IgG-IRDye800CW. On serial imaging, panitumumab achieved the highest TBRs with both SPY and Pearl (3.8 and 6.6). When used to guide resections, the antibody-dye conjugates generated TBRs in the range of 1.3-2.2 (average=1.6) using the SPY and 1.9-6.3 (average=2.7) using the Pearl. There was no significant difference amongst the antibodies with either imaging modality or cell line (one-way ANOVA). Conclusion Our data suggests that FDA approved antibodies may be suitable targeting agents for the intraoperative fluorescent detection of melanoma. Level of Evidence N/A PMID:23616260

Visualization of oxygen transfer across the air-water interface using a fluorescence oxygen visualization method.

PubMed

Lee, Minhee

2002-04-01

Oxygen concentration fields in a water body were visualized by the fluorescence oxygen visualization (FOV) method. Pyrenebutyric acid (PBA) was used as a fluorescent indicator of oxygen, and an intensive charge coupled-device (ICCD) camera as an image detector. Sequential images (over 2000 images) of the oxygen concentration field around the surface water of the tank (1 x 1 x 0.75 m3) were produced during the 3 h experiment. From image processing, the accurate pathway of oxygen-rich, cold water at the water surface was also visualized. The amount of oxygen transferred through the air-water interface during the experiment was measured and the oxygen transfer coefficient (K(L)) was determined as 0.22 m/d, which was much higher than that is expected in molecular diffusion. Results suggest that vertical penetration of cold water was the main pathway of oxygen in the water body in the tank. The average velocity of cold water penetrating downward in water body was also measured from consecutive images and the value was 0.3-0.6 mm/s. The FOV method used in this research should have wide application in experimental fluid mechanics and can also provide a phenomenological description of oxygen transfer under physically realizable natural conditions in lakes and reservoirs.

Portable hyperspectral imager with continuous wave green laser for identification and detection of untreated latent fingerprints on walls.

PubMed

Nakamura, Atsushi; Okuda, Hidekazu; Nagaoka, Takashi; Akiba, Norimitsu; Kurosawa, Kenji; Kuroki, Kenro; Ichikawa, Fumihiko; Torao, Akira; Sota, Takayuki

2015-09-01

Untreated latent fingerprints are known to exhibit fluorescence under UV laser excitation. Previously, the hyperspectral imager (HSI) has been primarily evaluated in terms of its potential to enhance the sensitivity of latent fingerprint detection following treatment by conventional chemical methods in the forensic science field. In this study however, the potential usability of the HSI for the visualization and detection of untreated latent fingerprints by measuring their inherent fluorescence under continuous wave (CW) visible laser excitation was examined. Its potential to undertake spectral separation of overlapped fingerprints was also evaluated. The excitation wavelength dependence of fluorescent images was examined using an untreated palm print on a steel based wall, and it was found that green laser excitation is superior to blue and yellow lasers' excitation for the production of high contrast fluorescence images. In addition, a spectral separation method for overlapped fingerprints/palm prints on a plaster wall was proposed using new images converted by the division and subtraction of two single wavelength images constructed based on measured hyperspectral data (HSD). In practical tests, the relative isolation of two overlapped fingerprints/palm prints was successful in twelve out of seventeen cases. Only one fingerprint/palm print was extracted for an additional three cases. These results revealed that the feasibility of overlapped fingerprint/palm print spectral separation depends on the difference in the temporal degeneration of each fluorescence spectrum. The present results demonstrate that a combination of a portable HSI and CW green laser has considerable potential for the identification and detection of untreated latent fingerprints/palm prints on the walls under study, while the use of HSD makes it practically possible for doubly overlapped fingerprints/palm prints to be separated spectrally. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Emerging biomedical applications of time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Lakowicz, Joseph R.; Szmacinski, Henryk; Koen, Peter A.

1994-07-01

Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics, and chemical physics. Advances in laser technology, the development of long-wavelength probes, and the use of lifetime-based methods are resulting in the rapid migration of time-resolved fluorescence to the clinical chemistry lab, to the patient's bedside, to flow cytometers, to the doctor's office, and even to home health care. Additionally, time-resolved imaging is now a reality in fluorescence microscopy, and will provide chemical imaging of a variety of intracellular analytes and/or cellular phenomena. In this overview paper we attempt to describe some of the opportunities available using chemical sensing based on fluorescence lifetimes, and to predict those applications of lifetime-based sensing which are most likely in the near future.

Multispectral guided fluorescence diffuse optical tomography using upconverting nanoparticles

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

Svenmarker, Pontus, E-mail: pontus.svenmarker@physics.umu.se; Department of Physics, Umeå University, SE-901 87 Umeå; Centre for Microbial Research

2014-02-17

We report on improved image detectability for fluorescence diffuse optical tomography using upconverting nanoparticles doped with rare-earth elements. Core-shell NaYF{sub 4}:Yb{sup 3+}/Er{sup 3+}@NaYF{sub 4} upconverting nanoparticles were synthesized through a stoichiometric method. The Yb{sup 3+}/Er{sup 3+} sensitizer-activator pair yielded two anti-Stokes shifted fluorescence emission bands at 540 nm and 660 nm, here used to a priori estimate the fluorescence source depth with sub-millimeter precision. A spatially varying regularization incorporated the a priori fluorescence source depth estimation into the tomography reconstruction scheme. Tissue phantom experiments showed both an improved resolution and contrast in the reconstructed images as compared to not using any amore » priori information.« less

In vivo quantitative imaging of point-like bioluminescent and fluorescent sources: Validation studies in phantoms and small animals post mortem

NASA Astrophysics Data System (ADS)

Comsa, Daria Craita

2008-10-01

There is a real need for improved small animal imaging techniques to enhance the development of therapies in which animal models of disease are used. Optical methods for imaging have been extensively studied in recent years, due to their high sensitivity and specificity. Methods like bioluminescence and fluorescence tomography report promising results for 3D reconstructions of source distributions in vivo. However, no standard methodology exists for optical tomography, and various groups are pursuing different approaches. In a number of studies on small animals, the bioluminescent or fluorescent sources can be reasonably approximated as point or line sources. Examples include images of bone metastases confined to the bone marrow. Starting with this premise, we propose a simpler, faster, and inexpensive technique to quantify optical images of point-like sources. The technique avoids the computational burden of a tomographic method by using planar images and a mathematical model based on diffusion theory. The model employs in situ optical properties estimated from video reflectometry measurements. Modeled and measured images are compared iteratively using a Levenberg-Marquardt algorithm to improve estimates of the depth and strength of the bioluminescent or fluorescent inclusion. The performance of the technique to quantify bioluminescence images was first evaluated on Monte Carlo simulated data. Simulated data also facilitated a methodical investigation of the effect of errors in tissue optical properties on the retrieved source depth and strength. It was found that, for example, an error of 4 % in the effective attenuation coefficient led to 4 % error in the retrieved depth for source depths of up to 12mm, while the error in the retrieved source strength increased from 5.5 % at 2mm depth, to 18 % at 12mm depth. Experiments conducted on images from homogeneous tissue-simulating phantoms showed that depths up to 10mm could be estimated within 8 %, and the relative source strength within 20 %. For sources 14mm deep, the inaccuracy in determining the relative source strength increased to 30 %. Measurements on small animals post mortem showed that the use of measured in situ optical properties to characterize heterogeneous tissue resulted in a superior estimation of the source strength and depth compared to when literature optical properties for organs or tissues were used. Moreover, it was found that regardless of the heterogeneity of the implant location or depth, our algorithm consistently showed an advantage over the simple assessment of the source strength based on the signal strength in the emission image. Our bioluminescence algorithm was generally able to predict the source strength within a factor of 2 of the true strength, but the performance varied with the implant location and depth. In fluorescence imaging a more complex technique is required, including knowledge of tissue optical properties at both the excitation and emission wavelengths. A theoretical study using simulated fluorescence data showed that, for example, for a source 5 mm deep in tissue, errors of up to 15 % in the optical properties would give rise to errors of +/-0.7 mm in the retrieved depth and the source strength would be over- or under-estimated by a factor ranging from 1.25 to 2. Fluorescent sources implanted in rats post mortem at the same depth were localized with an error just slightly higher than predicted theoretically: a root-mean-square value of 0.8 mm was obtained for all implants 5 mm deep. However, for this source depth, the source strength was assessed within a factor ranging from 1.3 to 4.2 from the value estimated in a controlled medium. Nonetheless, similarly to the bioluminescence study, the fluorescence quantification algorithm consistently showed an advantage over the simple assessment of the source strength based on the signal strength in the fluorescence image. Few studies have been reported in the literature that reconstruct known sources of bioluminescence or fluorescence in vivo or in heterogeneous phantoms. The few reported results show that the 3D tomographic methods have not yet reached their full potential. In this context, the simplicity of our technique emerges as a strong advantage.

Fluorescence life-time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles.

PubMed

Schwartz, Shmulik; Fixler, Dror; Popovtzer, Rachela; Shefi, Orit

2015-11-01

Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications. Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY-GNP (Middle) enable the differentiation between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Fluorescent signal detection of chromatographic chip by algorithms of pyramid connection and Gaussian mixture model].

PubMed

Hu, Beibei; Zhang, Xueqing; Chen, Haopeng; Cui, Daxiang

2011-03-01

We proposed a new algorithm for automatic identification of fluorescent signal. Based on the features of chromatographic chips, mathematic morphology in RGB color space was used to filter and enhance the images, pyramid connection was used to segment the areas of fluorescent signal, and then the method of Gaussian Mixture Model was used to detect the fluorescent signal. Finally we calculated the average fluorescent intensity in obtained fluorescent areas. Our results show that the algorithm has a good efficacy to segment the fluorescent areas, can detect the fluorescent signal quickly and accurately, and finally realize the quantitative detection of fluorescent signal in chromatographic chip.

Anatomical image-guided fluorescence molecular tomography reconstruction using kernel method

NASA Astrophysics Data System (ADS)

Baikejiang, Reheman; Zhao, Yue; Fite, Brett Z.; Ferrara, Katherine W.; Li, Changqing

2017-05-01

Fluorescence molecular tomography (FMT) is an important in vivo imaging modality to visualize physiological and pathological processes in small animals. However, FMT reconstruction is ill-posed and ill-conditioned due to strong optical scattering in deep tissues, which results in poor spatial resolution. It is well known that FMT image quality can be improved substantially by applying the structural guidance in the FMT reconstruction. An approach to introducing anatomical information into the FMT reconstruction is presented using the kernel method. In contrast to conventional methods that incorporate anatomical information with a Laplacian-type regularization matrix, the proposed method introduces the anatomical guidance into the projection model of FMT. The primary advantage of the proposed method is that it does not require segmentation of targets in the anatomical images. Numerical simulations and phantom experiments have been performed to demonstrate the proposed approach's feasibility. Numerical simulation results indicate that the proposed kernel method can separate two FMT targets with an edge-to-edge distance of 1 mm and is robust to false-positive guidance and inhomogeneity in the anatomical image. For the phantom experiments with two FMT targets, the kernel method has reconstructed both targets successfully, which further validates the proposed kernel method.

Caries Detection around Restorations Using ICDAS and Optical Devices.

PubMed

Diniz, Michele Baffi; Eckert, George Joseph; González-Cabezas, Carlos; Cordeiro, Rita de Cássia Loiola; Ferreira-Zandona, Andrea Gonçalves

2016-01-01

Secondary caries is the major reason for replacement of restorations in operative dentistry. New detection methods and technology have the potential to improve the accuracy for diagnosis of secondary carious lesions. This in vitro study evaluated the performance of the ICDAS (International Caries Detection and Assessment System) visual criteria and optical devices for detecting secondary caries around amalgam and composite resin restorations in permanent teeth. A total of 180 extracted teeth with Class I amalgam (N = 90) and resin composite (N = 90) restorations were selected. Two examiners analyzed the teeth twice using the visual criteria (ICDAS), laser fluorescence (LF), light-emitting diode device (MID), quantitative light-induced fluorescence system (QLF), and a prototype system based on the Fluorescence Enamel Imaging technique (Professional Caries Detection System, PCDS). The gold standard was determined by means of confocal laser scanning microscopy. High-reproducibility values were shown for all methods, except for MID in the amalgam group. For both groups the QLF and PCDS were the most sensitive methods, whereas the other methods presented better specificity (p < 0.05). All methods, except the MID device appeared to be potential methods for detecting secondary caries only around resin composite restorations, whereas around amalgam restorations all methods seemed to be questionable. Using Internal Caries Detection and Assessment System (ICDAS), an LF device, quantitative light-induced fluorescence and a novel method based on Fluorescence Enamel Imaging technique may be effective for evaluating secondary caries around composite resin restorations. © 2016 Wiley Periodicals, Inc.

Measuring mRNA copy-number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization (smFISH)

PubMed Central

Skinner, Samuel O.; Sepúlveda, Leonardo A.; Xu, Heng; Golding, Ido

2014-01-01

We present a method for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently-labeled oligonucleotide probes are hybridized to the target mRNA, so that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The number of target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete “spots” as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy-number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 days, with another 2-3 days typically required for image and data analysis. PMID:23680982

Combination of fluorescence imaging and local spectrophotometry in fluorescence diagnostics of early cancer of larynx and bronchi

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

Sokolov, Vladimir V; Filonenko, E V; Telegina, L V

2002-11-30

The results of comparative studies of autofluorescence and 5-ALA-induced fluorescence of protoporphyrin IX, used in the diagnostics of early cancer of larynx and bronchi, are presented. The autofluorescence and 5-ALA-induced fluorescence images of larynx and bronchial tissues are analysed during the endoscopic study. The method of local spectrophotometry is used to verify findings obtained from fluorescence images. It is shown that such a combined approach can be efficiently used to improve the diagnostics of precancer and early cancer, to detect a primary multiple tumours, as well as for the diagnostics of a residual tumour or an early recurrence after themore » endoscopic, surgery or X-ray treatment. The developed approach allows one to minimise the number of false-positive results and to reduce the number of biopsies, which are commonly used in the white-light bronchoscopy search for occult cancerous loci. (laser biology and medicine)« less

Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture

NASA Astrophysics Data System (ADS)

Horilova, Julia; Cunderlikova, Beata; Marcek Chorvatova, Alzbeta

2015-05-01

Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

Using Cell-ID 1.4 with R for Microscope-Based Cytometry

PubMed Central

Bush, Alan; Chernomoretz, Ariel; Yu, Richard; Gordon, Andrew

2012-01-01

This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposefully defocused transmission image (sometimes referred to as bright-field or BF) is acquired to segment the image and locate each cell. Fluorescent images (one for each of the color channels to be analyzed) are then acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses the image processing capabilities of Cell-ID (Gordon et al., 2007, as updated here) and data analysis by the statistical programming framework R (R-Development-Team, 2008), which we have supplemented with a package of routines for analyzing Cell-ID output. Both Cell-ID and the analysis package are open-source. PMID:23026908

Nucleocytoplasmic shuttling: the ins and outs of quantitative imaging.

PubMed

Molenaar, Chris; Weeks, Kate L

2018-05-17

Nucleocytoplasmic protein shuttling is integral to the transmission of signals between the nucleus and the cytoplasm. The nuclear/cytoplasmic distribution of proteins of interest can be determined via fluorescence microscopy, following labelling of the target protein with fluorophore-conjugated antibodies (immunofluorescence) or by tagging the target protein with an autofluorescent protein, such as green fluorescent protein (GFP). The latter enables live cell imaging, a powerful approach that precludes many of the artefacts associated with indirect immunofluorescence in fixed cells. In this review, we discuss important considerations for the design and implementation of fluorescence microscopy experiments to quantify the nuclear/cytoplasmic distribution of a protein of interest. We summarise the pros and cons of detecting endogenous proteins in fixed cells by immunofluorescence and ectopically-expressed fluorescent fusion proteins in living cells. We discuss the suitability of widefield fluorescence microscopy and of 2D, 3D and 4D imaging by confocal microscopy for different applications, and describe two different methods for quantifying the nuclear/cytoplasmic distribution of a protein of interest from the fluorescent signal. Finally, we discuss the importance of eliminating sources of bias and subjectivity during image acquisition and post-imaging analyses. This is critical for the accurate and reliable quantification of nucleocytoplasmic shuttling. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Tumor-stem cells interactions by fluorescence imaging

NASA Astrophysics Data System (ADS)

Meleshina, Aleksandra V.; Cherkasova, Elena I.; Sergeeva, Ekaterina; Turchin, Ilya V.; Kiseleva, Ekaterina V.; Dashinimaev, Erdem B.; Shirmanova, Marina V.; Zagaynova, Elena V.

2013-02-01

Recently, great deal of interest is investigation the function of the stem cells (SC) in tumors. In this study, we studied «recipient-tumor- fluorescent stem cells » system using the methods of in vivo imaging and laser scanning microscopy (LSM). We used adipose-derived adult stem (ADAS) cells of human lentiviral transfected with the gene of fluorescent protein Turbo FP635. ADAS cells were administrated into nude mice with transplanted tumor HeLa Kyoto (human cervical carcinoma) at different stages of tumor growth (0-8 days) intravenously or into tumor. In vivo imaging was performed on the experimental setup for epi - luminescence bioimaging (IAP RAS, Nizhny Novgorod). The results of the imaging showed localization of fluorophore tagged stem cells in the spleen on day 5-9 after injection. The sensitivity of the technique may be improved by spectral separation autofluorescence and fluorescence of stem cells. We compared the results of in vivo imaging and confocal laser scanning microscopy (LSM 510 META, Carl Zeiss, Germany). Internal organs of the animals and tumor tissue were investigated. It was shown that with i.v. injection of ADAS, bright fluorescent structures with spectral characteristics corresponding to TurboFP635 protein are locally accumulated in the marrow, lungs and tumors of animals. These findings indicate that ADAS cells integrate in the animal body with transplanted tumor and can be identified by fluorescence bioimaging techniques in vivo and ex vivo.

An automated segmentation methodology for quantifying immunoreactive puncta number and fluorescence intensity in tissue sections.

PubMed

Fish, Kenneth N; Sweet, Robert A; Deo, Anthony J; Lewis, David A

2008-11-13

A number of human brain diseases have been associated with disturbances in the structure and function of cortical synapses. Answering fundamental questions about the synaptic machinery in these disease states requires the ability to image and quantify small synaptic structures in tissue sections and to evaluate protein levels at these major sites of function. We developed a new automated segmentation imaging method specifically to answer such fundamental questions. The method takes advantage of advances in spinning disk confocal microscopy, and combines information from multiple iterations of a fluorescence intensity/morphological segmentation protocol to construct three-dimensional object masks of immunoreactive (IR) puncta. This new methodology is unique in that high- and low-fluorescing IR puncta are equally masked, allowing for quantification of the number of fluorescently-labeled puncta in tissue sections. In addition, the shape of the final object masks highly represents their corresponding original data. Thus, the object masks can be used to extract information about the IR puncta (e.g., average fluorescence intensity of proteins of interest). Importantly, the segmentation method presented can be easily adapted for use with most existing microscopy analysis packages.

Evaluation of methods for detection of fluorescence labeled subcellular objects in microscope images.

PubMed

Ruusuvuori, Pekka; Aijö, Tarmo; Chowdhury, Sharif; Garmendia-Torres, Cecilia; Selinummi, Jyrki; Birbaumer, Mirko; Dudley, Aimée M; Pelkmans, Lucas; Yli-Harja, Olli

2010-05-13

Several algorithms have been proposed for detecting fluorescently labeled subcellular objects in microscope images. Many of these algorithms have been designed for specific tasks and validated with limited image data. But despite the potential of using extensive comparisons between algorithms to provide useful information to guide method selection and thus more accurate results, relatively few studies have been performed. To better understand algorithm performance under different conditions, we have carried out a comparative study including eleven spot detection or segmentation algorithms from various application fields. We used microscope images from well plate experiments with a human osteosarcoma cell line and frames from image stacks of yeast cells in different focal planes. These experimentally derived images permit a comparison of method performance in realistic situations where the number of objects varies within image set. We also used simulated microscope images in order to compare the methods and validate them against a ground truth reference result. Our study finds major differences in the performance of different algorithms, in terms of both object counts and segmentation accuracies. These results suggest that the selection of detection algorithms for image based screens should be done carefully and take into account different conditions, such as the possibility of acquiring empty images or images with very few spots. Our inclusion of methods that have not been used before in this context broadens the set of available detection methods and compares them against the current state-of-the-art methods for subcellular particle detection.

Peering into Cells One Molecule at a Time: Single-molecule and plasmon-enhanced fluorescence super-resolution imaging

NASA Astrophysics Data System (ADS)

Biteen, Julie

2013-03-01

Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations of live bacterial cells, and have used these techniques to investigate thee important prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each system presents unique challenges, and we will discuss the important methods developed for each system. Furthermore, we use the plasmon modes of bio-compatible metal nanoparticles to enhance the emissivity of single-molecule fluorophores. The resolution of single-molecule imaging in cells is generally limited to 20-40 nm, far worse than the 1.5-nm localization accuracies which have been attained in vitro. We use plasmonics to improve the brightness and stability of single-molecule probes, and in particular fluorescent proteins, which are widely used for bio-imaging. We find that gold-coupled fluorophores demonstrate brighter, longer-lived emission, yielding an overall enhancement in total photons detected. Ultimately, this results in increased localization accuracy for single-molecule imaging. Furthermore, since fluorescence intensity is proportional to local electromagnetic field intensity, these changes in decay intensity and rate serve as a nm-scale read-out of the field intensity. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

High sensitive fundus autofluorescence imaging combined with speckle-free optical coherence tomography

NASA Astrophysics Data System (ADS)

Stremplewski, Patrycjusz; Komar, Katarzyna; Szkulmowski, Maciej; Motoczyńska, Marta; Wojtkowski, Maciej

2013-03-01

Scattering and fluorescence images provide complementary information about the health condition of the human eye, so getting them in a single measurement, using a single device may significantly improve a quality of diagnosis as it has been already demonstrated in Spectralis (Heidelberg Eng.) OCT instrument. There is still challenge to improve quality of fundus autofluorescence (FAF) images. The biggest obstacle in obtaining in vivo images of sufficient quality is very low fluorescence signal. For eye safety reasons, and because of patient comfort, using highpower fluorescence excitation is not an adequate solution to the low signal problem. In this contribution we show a new detection method in the retinal autofluorescence imaging, which may improve the sensitivity. We used a fast modulated (up to 500 MHz) diode laser of wavelength 473 nm and detected fluorescence in the spectral range 500-680 nm by photomultiplier and lock-in amplifier. Average power of the collimated blue beam on the cornea used for FAF measurements was set to 50 μW, 10 μW, and even 4.5 μW.

Multifunctional gold nanorods for image-guided surgery and photothermal therapy

NASA Astrophysics Data System (ADS)

Barriere, Clement; Qi, Ji; Garcia-Allende, P. Beatriz; Newton, Richard; Elson, Daniel S.

2012-03-01

Nanoparticles are viewed as a promising tool for numerous medical applications, for instance imaging and photothermal therapy (PTT) has been proposed using gold nanorods. We are developing multi-functional gold nanorods (m-GNRs) which have potential for image guided endoscopic surgery of tumour tissue with a modified laparoscope system. A new synthesis method potentially allows any useful acid functionalised molecules to be bonded at the surface. We have created fluorescent m-GNRs which can be used for therapy as they absorb light in the infrared, which may penetrate deep into the tissue and produce localised heating. We have performed a tissue based experiment to demonstrate the feasibility of fluorescence guided PTT using m- GNRs. Ex vivo tests were performed using sheep heart. This measurement, correlated with the fluorescence signal of the m-GNRs measured by the laparoscope allows the clear discrimination of the artery system containing m-GNRs. A laser diode was used to heat the m-GNRs and a thermal camera was able to record the heat distribution. These images were compared to the fluorescence images for validation.

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

Application autofluorescence diagnosis method in endoscopy for investigation mucosal structure in gastrointestinal tract

NASA Astrophysics Data System (ADS)

Varlamova, Larisa; Abramov, Dmitrii; Golovin, Arsenii; Seledkina, Ekaterina

2017-05-01

One of the promising methods for early diagnosis of malignant diseases of the respiratory organs and the gastrointestinal tract (GIT) is now considered a fluorescence method. Application autofluorescence phenomenon in endoscopy allows to obtain a fluorescent image of the mucosa, which shows the difference in the intensity of the autofluorescence of healthy and the affected tissue in the green and red regions of the spectrum. The result of the work is to determine on the basis of scientific research and prototyping capabilities of creating fluorescence video endoscope and the development of fluorescent light (illuminator FLU) for videoendoscopy complex. The solution of this problem is based on the method of studying biological objects in lifetime condition.

Using multimodal information for the segmentation of fluorescent micrographs with application to virology and microbiology.

PubMed

Held, Christian; Wenzel, Jens; Webel, Rike; Marschall, Manfred; Lang, Roland; Palmisano, Ralf; Wittenberg, Thomas

2011-01-01

In order to improve reproducibility and objectivity of fluorescence microscopy based experiments and to enable the evaluation of large datasets, flexible segmentation methods are required which are able to adapt to different stainings and cell types. This adaption is usually achieved by the manual adjustment of the segmentation methods parameters, which is time consuming and challenging for biologists with no knowledge on image processing. To avoid this, parameters of the presented methods automatically adapt to user generated ground truth to determine the best method and the optimal parameter setup. These settings can then be used for segmentation of the remaining images. As robust segmentation methods form the core of such a system, the currently used watershed transform based segmentation routine is replaced by a fast marching level set based segmentation routine which incorporates knowledge on the cell nuclei. Our evaluations reveal that incorporation of multimodal information improves segmentation quality for the presented fluorescent datasets.

Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

PubMed

Mari, João Fernando; Saito, José Hiroki; Neves, Amanda Ferreira; Lotufo, Celina Monteiro da Cruz; Destro-Filho, João-Batista; Nicoletti, Maria do Carmo

2015-12-01

Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

Chlorophyll fluorescence emission as a reporter on cold tolerance in Arabidopsis thaliana accessions

PubMed Central

Mishra, Anamika; Höermiller, Imke I; Heyer, Arnd G; Nedbal, Ladislav

2011-01-01

Non-invasive, high-throughput screening methods are valuable tools in breeding for abiotic stress tolerance in plants. Optical signals such as chlorophyll fluorescence emission can be instrumental in developing new screening techniques. In order to examine the potential of chlorophyll fluorescence to reveal plant tolerance to low temperatures, we used a collection of nine Arabidopsis thaliana accessions and compared their fluorescence features with cold tolerance quantified by the well established electrolyte leakage method on detached leaves. We found that, during progressive cooling, the minimal chlorophyll fluorescence emission rose strongly and that this rise was highly dependent on the cold tolerance of the accessions. Maximum quantum yield of PSII photochemistry and steady state fluorescence normalized to minimal fluorescence were also highly correlated to the cold tolerance measured by the electrolyte leakage method. In order to further increase the capacity of the fluorescence detection to reveal the low temperature tolerance, we applied combinatorial imaging that employs plant classification based on multiple fluorescence features. We found that this method, by including the resolving power of several fluorescence features, can be well employed to detect cold tolerance already at mild sub-zero temperatures. Therefore, there is no need to freeze the screened plants to the largely damaging temperatures of around −15°C. This, together with the method's easy applicability, represents a major advantage of the fluorescence technique over the conventional electrolyte leakage method. PMID:21427532

Detecting thermal phase transitions in corneal stroma by fluorescence micro-imaging analysis

NASA Astrophysics Data System (ADS)

Matteini, P.; Rossi, F.; Ratto, F.; Bruno, I.; Nesi, P.; Pini, R.

2008-02-01

Thermal modifications induced in corneal stroma were investigated by the use of fluorescence microscopy. Freshly extracted porcine corneas were immersed for 5 minutes in a water bath at temperatures in the 35-90°C range and stored in formalin. The samples were then sliced in 200-μm-thick transversal sections and analyzed under a stereomicroscope to assess corneal shrinkage. Fluorescence images of the thermally treated corneal samples were acquired using a slow-scan cooled CCD camera, after staining the slices with Indocyanine Green (ICG) fluorescent dye which allowed to detect fluorescence signal from the whole tissue. All measurements were performed using an inverted epifluorescence microscope equipped with a mercury lamp. The thermally-induced modifications to the corneal specimens were evaluated by studying the grey level distribution in the fluorescence images. For each acquired image, Discrete Fourier Transform (DFT) and entropy analyses were performed. The spatial distribution of DFT absolute value indicated the spatial orientation of the lamellar planes, while entropy was used to study the image texture, correlated to the stromal structural transitions. As a result, it was possible to indicate a temperature threshold value (62°C) for high thermal damage, resulting in a disorganization of the lamellar planes and in full agreement with the measured temperature for corneal shrinkage onset. Analysis of the image entropy evidenced five strong modifications in stromal architecture at temperatures of ~45°C, 53°C, 57°C, 66°C, 75°C. The proposed procedure proved to be an effective micro-imaging method capable of detecting subtle changes in corneal tissue subjected to thermal treatment.

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

PubMed Central

Volpe, Alessia; Man, Francis; Lim, Lindsay; Khoshnevisan, Alex; Blower, Julia; Blower, Philip J.; Fruhwirth, Gilbert O.

2018-01-01

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest. PMID:29608157

The border-to-border distribution method for analysis of cytoplasmic particles and organelles.

PubMed

Yacovone, Shalane K; Ornelles, David A; Lyles, Douglas S

2016-02-01

Comparing the distribution of cytoplasmic particles and organelles between different experimental conditions can be challenging due to the heterogeneous nature of cell morphologies. The border-to-border distribution method was created to enable the quantitative analysis of fluorescently labeled cytoplasmic particles and organelles of multiple cells from images obtained by confocal microscopy. The method consists of four steps: (1) imaging of fluorescently labeled cells, (2) division of the image of the cytoplasm into radial segments, (3) selection of segments of interest, and (4) population analysis of fluorescence intensities at the pixel level either as a function of distance along the selected radial segments or as a function of angle around an annulus. The method was validated using the well-characterized effect of brefeldin A (BFA) on the distribution of the vesicular stomatitis virus G protein, in which intensely labeled Golgi membranes are redistributed within the cytoplasm. Surprisingly, in untreated cells, the distribution of fluorescence in Golgi membrane-containing radial segments was similar to the distribution of fluorescence in other G protein-containing segments, indicating that the presence of Golgi membranes did not shift the distribution of G protein towards the nucleus compared to the distribution of G protein in other regions of the cell. Treatment with BFA caused only a slight shift in the distribution of the brightest G protein-containing segments which had a distribution similar to that in untreated cells. Instead, the major effect of BFA was to alter the annular distribution of G protein in the perinuclear region.

Multimodal fluorescence molecular imaging for in vivo characterization of skin cancer using endogenous and exogenous fluorophores

NASA Astrophysics Data System (ADS)

Miller, Jessica P.; Habimana-Griffin, LeMoyne; Edwards, Tracy S.; Achilefu, Samuel

2017-06-01

Similarity of skin cancer with many benign skin pathologies requires reliable methods to detect and differentiate the different types of these lesions. Previous studies have explored the use of disparate optical techniques to identify and estimate the invasive nature of melanoma and basal cell carcinoma with varying outcomes. Here, we used a concerted approach that provides complementary information for rapid screening and characterization of tumors, focusing on squamous cell carcinoma (SCC) of the skin. Assessment of in vivo autofluorescence lifetime (FLT) imaging of endogenous fluorophores that are excitable at longer wavelengths (480 nm) than conventional NADH and FAD revealed a decrease in the short FLT component for SCC compared to normal skin, with mean values of 0.57±0.026 ns and 0.61±0.021 ns, respectively (p=0.004). Subsequent systemic administration of a near-infrared fluorescent molecular probe in SCC bearing mice, followed by the implementation of image processing methods on data acquired from two-dimensional and three-dimensional fluorescence molecular imaging, allowed us to estimate the tumor volume and depth, as well as quantify the fluorescent probe in the tumor. The result suggests the involvement of lipofuscin-like lipopigments and riboflavin in SCC metabolism and serves as a model for staging SCC.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

PubMed Central

Valades Cruz, Cesar Augusto; Shaban, Haitham Ahmed; Kress, Alla; Bertaux, Nicolas; Monneret, Serge; Mavrakis, Manos; Savatier, Julien; Brasselet, Sophie

2016-01-01

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells. PMID:26831082

Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP

PubMed Central

Guo, Wenyan; Liu, Xiuli; Liu, Yurong; Gang, Yadong; He, Xiaobin; Jia, Yao; Yin, Fangfang; Li, Pei; Huang, Fei; Zhou, Hongfu; Wang, Xiaojun; Gong, Hui; Luo, Qingming; Xu, Fuqiang; Zeng, Shaoqun

2017-01-01

The pH-sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EGFP or EYFP is good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is an urgent need. Here a pH-sensitive red fluorescent protein, pHuji, is selected and verified to remain pH-sensitive in HM20 resin. We observe 183% fluorescence intensity of pHuji in resin-embeded mouse brain and 29.08-fold fluorescence intensity of reactivated pHuji compared to the quenched state. pHuji and EGFP can be quenched and chemically reactivated simultaneously in resin, thus enabling simultaneous two-color micro-optical sectioning tomography of resin-embedded mouse brain. This method may greatly facilitate the visualization of neuronal morphology and neural circuits to promote understanding of the structure and function of the brain. PMID:28717566

Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP.

PubMed

Guo, Wenyan; Liu, Xiuli; Liu, Yurong; Gang, Yadong; He, Xiaobin; Jia, Yao; Yin, Fangfang; Li, Pei; Huang, Fei; Zhou, Hongfu; Wang, Xiaojun; Gong, Hui; Luo, Qingming; Xu, Fuqiang; Zeng, Shaoqun

2017-07-01

The pH-sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EGFP or EYFP is good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is an urgent need. Here a pH-sensitive red fluorescent protein, pHuji, is selected and verified to remain pH-sensitive in HM20 resin. We observe 183% fluorescence intensity of pHuji in resin-embeded mouse brain and 29.08-fold fluorescence intensity of reactivated pHuji compared to the quenched state. pHuji and EGFP can be quenched and chemically reactivated simultaneously in resin, thus enabling simultaneous two-color micro-optical sectioning tomography of resin-embedded mouse brain. This method may greatly facilitate the visualization of neuronal morphology and neural circuits to promote understanding of the structure and function of the brain.

Live imaging of dense-core vesicles in primary cultured hippocampal neurons.

PubMed

Kwinter, David M; Silverman, Michael A; Kwinter, David; Michael, Silverman

2009-05-29

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Segmentation of Vasculature from Fluorescently Labeled Endothelial Cells in Multi-Photon Microscopy Images.

PubMed

Bates, Russell; Irving, Benjamin; Markelc, Bostjan; Kaeppler, Jakob; Brown, Graham; Muschel, Ruth J; Brady, Sir Michael; Grau, Vicente; Schnabel, Julia A

2017-08-09

Vasculature is known to be of key biological significance, especially in the study of tumors. As such, considerable effort has been focused on the automated segmentation of vasculature in medical and pre-clinical images. The majority of vascular segmentation methods focus on bloodpool labeling methods, however, particularly in the study of tumors it is of particular interest to be able to visualize both perfused and non-perfused vasculature. Imaging vasculature by highlighting the endothelium provides a way to separate the morphology of vasculature from the potentially confounding factor of perfusion. Here we present a method for the segmentation of tumor vasculature in 3D fluorescence microscopy images using signals from the endothelial and surrounding cells. We show that our method can provide complete and semantically meaningful segmentations of complex vasculature using a supervoxel-Markov Random Field approach. We show that in terms of extracting meaningful segmentations of the vasculature, our method out-performs both a state-ofthe- art method, specific to these data, as well as more classical vasculature segmentation methods.

A portable fluorescence microscopic imaging system for cholecystectomy

NASA Astrophysics Data System (ADS)

Ye, Jian; Yang, Chaoyu; Gan, Qi; Ma, Rong; Zhang, Zeshu; Chang, Shufang; Shao, Pengfei; Zhang, Shiwu; Liu, Chenhai; Xu, Ronald

2016-03-01

In this paper we proposed a portable fluorescence microscopic imaging system to prevent iatrogenic biliary injuries from occurring during cholecystectomy due to misidentification of the cystic structures. The system consisted of a light source module, a CMOS camera, a Raspberry Pi computer and a 5 inch HDMI LCD. Specifically, the light source module was composed of 690 nm and 850 nm LEDs, allowing the CMOS camera to simultaneously acquire both fluorescence and background images. The system was controlled by Raspberry Pi using Python programming with the OpenCV library under Linux. We chose Indocyanine green(ICG) as a fluorescent contrast agent and then tested fluorescence intensities of the ICG aqueous solution at different concentration levels by our fluorescence microscopic system compared with the commercial Xenogen IVIS system. The spatial resolution of the proposed fluorescence microscopic imaging system was measured by a 1951 USAF resolution target and the dynamic response was evaluated quantitatively with an automatic displacement platform. Finally, we verified the technical feasibility of the proposed system in mouse models of bile duct, performing both correct and incorrect gallbladder resection. Our experiments showed that the proposed system can provide clear visualization of the confluence between the cystic duct and common bile duct or common hepatic duct, suggesting that this is a potential method for guiding cholecystectomy. The proposed portable system only cost a total of $300, potentially promoting its use in resource-limited settings.

In vivo stepwise multi-photon activation fluorescence imaging of melanin in human skin

NASA Astrophysics Data System (ADS)

Lai, Zhenhua; Gu, Zetong; Abbas, Saleh; Lowe, Jared; Sierra, Heidy; Rajadhyaksha, Milind; DiMarzio, Charles

2014-03-01

The stepwise multi-photon activated fluorescence (SMPAF) of melanin is a low cost and reliable method of detecting melanin because the activation and excitation can be a continuous-wave (CW) mode near infrared (NIR) laser. Our previous work has demonstrated the melanin SMPAF images in sepia melanin, mouse hair, and mouse skin. In this study, we show the feasibility of using SMPAF to detect melanin in vivo. in vivo melanin SMPAF images of normal skin and benign nevus are demonstrated. SMPAF images add specificity for melanin detection than MPFM images and CRM images. Melanin SMPAF is a promising technology to enable early detection of melanoma for dermatologists.

Optically trapped atomic resonant devices for narrow linewidth spectral imaging

NASA Astrophysics Data System (ADS)

Qian, Lipeng

This thesis focuses on the development of atomic resonant devices for spectroscopic applications. The primary emphasis is on the imaging properties of optically thick atomic resonant fluorescent filters and their applications. In addition, this thesis presents a new concept for producing very narrow linewidth light as from an atomic vapor lamp pumped by a nanosecond pulse system. This research was motivated by application for missile warning system, and presents an innovative approach to a wide angle, ultra narrow linewidth imaging filter using a potassium vapor cell. The approach is to image onto and collect the fluorescent photons emitted from the surface of an optically thick potassium vapor cell, generating a 2 GHz pass-band imaging filter. This linewidth is narrow enough to fall within a Fraunhefer dark zone in the solar spectrum, thus make the detection solar blind. Experiments are conducted to measure the absorption line shape of the potassium resonant filter, the quantum efficiency of the fluorescent behavior, and the resolution of the fluorescent image. Fluorescent images with different spatial frequency components are analyzed by using a discrete Fourier transform, and the imaging capability of the fluorescent filter is described by its Modulation Transfer Function. For the detection of radiation that is spectrally broader than the linewidth of the potassium imaging filter, the fluorescent image is seen to be blurred by diffuse fluorescence from the slightly off resonant photons. To correct this, an ultra-thin potassium imaging filter is developed and characterized. The imaging property of the ultra-thin potassium imaging cell is tested with a potassium seeded flame, yielding a resolution image of ˜ 20 lines per mm. The physics behind the atomic resonant fluorescent filter is radiation trapping. The diffusion process of the resonant photons trapped in the atomic vapor is theoretically described in this thesis. A Monte Carlo method is used to simulate the absorption and fluorescence. The optimum resolution of the fluorescent image is predicted by simulation. Radiation trapping is also shown to be useful for the generation of ultra-narrow linewidth light from an atomic vapor flash lamp. A 2 nanosecond, high voltage pulse is used to excite low pressure mercury vapor mixed with noble gases, producing high intensity emission at the mercury resonant line at 253.7 nm. With a nanosecond pumping time and high electrical current, the radiation intensity of the mercury discharge is increased significantly compared to a normal glow discharge lamp, while simultaneously suppressing the formation of an arc discharge. By avoiding the arc discharge, discrete spectral lines of mercury were kept at narrow bandwidth. Due to radiation trapping, the emission linewidth from the nanosecond mercury lamp decreases with time and produces ultra-narrow linewidth emission 100 ns after of the excitation, this linewidth is verified by absorption measurements through low pressure mercury absorption filter. The lamp is used along with mercury absorption filters for spectroscopic applications, including Filtered Rayleigh Scattering with different CO2 pressures and Raman scattering from methanol.

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

PubMed

Rich, Ryan M; Stankowska, Dorota L; Maliwal, Badri P; Sørensen, Thomas Just; Laursen, Bo W; Krishnamoorthy, Raghu R; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Fudala, Rafal

2013-02-01

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.

Effects of Photobleaching on Microplastics

NASA Astrophysics Data System (ADS)

Ferrone, Salvatore; Sullivan, Kelley

The presence of microplastics in our oceans and lakes is a contemporary environmental issue. A popular method for studying microplastics is fluorescence microscopy. We are studying the effects of fluorescence photo-bleaching on the imaging of microplastics. Our goal is to find out to what extent microplastics photo-bleach and if the photo-bleaching is recoverable. Photo-bleaching may entirely destroy the plastics' ability to fluoresce, hamper it for a short time, or have a minuscule effects. For this project, we consider the seven recyclable plastics. For each plastic type, we record a video of the micro-plastics for several hours under 405 nm light, then analyze and plot the image intensity as a function of time to see if the outputted light from the plastic decays over time. We then take single images at different time intervals to check if the intensity recovers. Our results will help set conditions under which fluorescence techniques can be used on microplastics. Undergraduate Student.

The total antioxidant capacity and fluorescence imaging of selected plant leaves commonly consumed in Brunei Darussalam

NASA Astrophysics Data System (ADS)

Watu, Aswani; Metussin, Nurzaidah; Yasin, Hartini M.; Usman, Anwar

2018-02-01

We investigated the total antioxidant capacity and fluorescence imaging of several selected plants, namely Centella asiatica, Aidia borneensis and Anacardium occidentale, which are grown and traditionally consumed in Brunei Darussalam. The total antioxidant capacities of aqueous-methanolic infusions of their leaves were measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, and microscopic fluorescence images were measured to identify the fluorescent substances bound in the leaves. We found that the total antioxidant capacity of their infusions is estimated to be 150, 25, 15 folds, respectively, lower compared with that of the standard gallic acid. Accordingly, we demonstrated that the relative antioxidant activity of young and matured leaves agrees with the intensity of red light emission of their fresh leaves upon UV excitation. Thus, this non-invasive spectroscopic method can be potentially utilized to indicate the antioxidants in plant leaves qualitatively.

Brain vascular image enhancement based on gradient adjust with split Bregman

NASA Astrophysics Data System (ADS)

Liang, Xiao; Dong, Di; Hui, Hui; Zhang, Liwen; Fang, Mengjie; Tian, Jie

2016-04-01

Light Sheet Microscopy is a high-resolution fluorescence microscopic technique which enables to observe the mouse brain vascular network clearly with immunostaining. However, micro-vessels are stained with few fluorescence antibodies and their signals are much weaker than large vessels, which make micro-vessels unclear in LSM images. In this work, we developed a vascular image enhancement method to enhance micro-vessel details which should be useful for vessel statistics analysis. Since gradient describes the edge information of the vessel, the main idea of our method is to increase the gradient values of the enhanced image to improve the micro-vessels contrast. Our method contained two steps: 1) calculate the gradient image of LSM image, and then amplify high gradient values of the original image to enhance the vessel edge and suppress low gradient values to remove noises. Then we formulated a new L1-norm regularization optimization problem to find an image with the expected gradient while keeping the main structure information of the original image. 2) The split Bregman iteration method was used to deal with the L1-norm regularization problem and generate the final enhanced image. The main advantage of the split Bregman method is that it has both fast convergence and low memory cost. In order to verify the effectiveness of our method, we applied our method to a series of mouse brain vascular images acquired from a commercial LSM system in our lab. The experimental results showed that our method could greatly enhance micro-vessel edges which were unclear in the original images.

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.

Technical Review: Microscopy and Image Processing Tools to Analyze Plant Chromatin: Practical Considerations.

PubMed

Baroux, Célia; Schubert, Veit

2018-01-01

In situ nucleus and chromatin analyses rely on microscopy imaging that benefits from versatile, efficient fluorescent probes and proteins for static or live imaging. Yet the broad choice in imaging instruments offered to the user poses orientation problems. Which imaging instrument should be used for which purpose? What are the main caveats and what are the considerations to best exploit each instrument's ability to obtain informative and high-quality images? How to infer quantitative information on chromatin or nuclear organization from microscopy images? In this review, we present an overview of common, fluorescence-based microscopy systems and discuss recently developed super-resolution microscopy systems, which are able to bridge the resolution gap between common fluorescence microscopy and electron microscopy. We briefly present their basic principles and discuss their possible applications in the field, while providing experience-based recommendations to guide the user toward best-possible imaging. In addition to raw data acquisition methods, we discuss commercial and noncommercial processing tools required for optimal image presentation and signal evaluation in two and three dimensions.

Non-invasive assessment of the liver using imaging

NASA Astrophysics Data System (ADS)

Thorling Thompson, Camilla; Wang, Haolu; Liu, Xin; Liang, Xiaowen; Crawford, Darrell H.; Roberts, Michael S.

2016-12-01

Chronic liver disease causes 2,000 deaths in Australia per year and early diagnosis is crucial to avoid progression to cirrhosis and end stage liver disease. There is no ideal method to evaluate liver function. Blood tests and liver biopsies provide spot examinations and are unable to track changes in function quickly. Therefore better techniques are needed. Non-invasive imaging has the potential to extract increased information over a large sampling area, continuously tracking dynamic changes in liver function. This project aimed to study the ability of three imaging techniques, multiphoton and fluorescence lifetime imaging microscopy, infrared thermography and photoacoustic imaging, in measuring liver function. Collagen deposition was obvious in multiphoton and fluorescence lifetime imaging in fibrosis and cirrhosis and comparable to conventional histology. Infrared thermography revealed a significantly increased liver temperature in hepatocellular carcinoma. In addition, multiphoton and fluorescence lifetime imaging and photoacoustic imaging could both track uptake and excretion of indocyanine green in rat liver. These results prove that non-invasive imaging can extract crucial information about the liver continuously over time and has the potential to be translated into clinic in the assessment of liver disease.

Fluorescence diffuse tomography for detection of RFP-expressed tumors in small animals

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Meerovich, Irina G.; Arslanbaeva, Lyaisan R.; Jerdeva, Viktoria V.; Orlova, Anna G.; Kleshnin, Mikhail S.; Shirmanova, Marina V.; Fiks, Ilya I.

2007-02-01

Conventional optical imaging is restricted with tumor size due to high tissue scattering. Labeling of tumors by fluorescent markers improves sensitivity of tumor detection thus increasing the value of optical imaging dramatically. Creation of tumor cell lines transfected with fluorescent proteins gives the possibility not only to detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and TurboRFP genes. Emission of RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. In vivo experiments were conducted immediately after the subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug responce.

Automatic tracking of cells for video microscopy in patch clamp experiments.

PubMed

Peixoto, Helton M; Munguba, Hermany; Cruz, Rossana M S; Guerreiro, Ana M G; Leao, Richardson N

2014-06-20

Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices.

Quantitative fluorescence microscopy and image deconvolution.

PubMed

Swedlow, Jason R

2013-01-01

Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used to remove blurred signal from an image. There are two major types of deconvolution approaches, deblurring and restoration algorithms. Deblurring algorithms remove blur, but treat a series of optical sections as individual two-dimensional entities, and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed. Copyright © 1998 Elsevier Inc. All rights reserved.

Multi-Modal Imaging in a Mouse Model of Orthotopic Lung Cancer.

PubMed

Patel, Priya; Kato, Tatsuya; Ujiie, Hideki; Wada, Hironobu; Lee, Daiyoon; Hu, Hsin-Pei; Hirohashi, Kentaro; Ahn, Jin Young; Zheng, Jinzi; Yasufuku, Kazuhiro

2016-01-01

Investigation of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine green (ICG) and iohexol, for real-time near infrared (NIR) fluorescence and computed tomography (CT) image-guided surgery in an orthotopic lung cancer model in nude mice. CF800 was intravenously administered into 13 mice bearing the H460 orthotopic human lung cancer. At 48 h post-injection (peak imaging agent accumulation time point), ex vivo NIR and CT imaging was performed. A clinical NIR imaging system (SPY®, Novadaq) was used to measure fluorescence intensity of tumor and lung. Tumor-to-background-ratios (TBR) were calculated in inflated and deflated states. The mean Hounsfield unit (HU) of lung tumor was quantified using the CT data set and a semi-automated threshold-based method. Histological evaluation using H&E, the macrophage marker F4/80 and the endothelial cell marker CD31, was performed, and compared to the liposomal fluorescence signal obtained from adjacent tissue sections. The fluorescence TBR measured when the lung is in the inflated state (2.0 ± 0.58) was significantly greater than in the deflated state (1.42 ± 0.380 (n = 7, p<0.003). Mean fluorescent signal in tumor was highly variable across samples, (49.0 ± 18.8 AU). CT image analysis revealed greater contrast enhancement in lung tumors (a mean increase of 110 ± 57 HU) when CF800 is administered compared to the no contrast enhanced tumors (p = 0.0002). Preliminary data suggests that the high fluorescence TBR and CT tumor contrast enhancement provided by CF800 may have clinical utility in localization of lung cancer during CT and NIR image-guided surgery.

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.

Automated image analysis for quantitative fluorescence in situ hybridization with environmental samples.

PubMed

Zhou, Zhi; Pons, Marie Noëlle; Raskin, Lutgarde; Zilles, Julie L

2007-05-01

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An automated image analysis program that detects cells from 4',6'-diamidino-2-phenylindole (DAPI) micrographs and extracts the maximum and mean fluorescence intensities for each cell from corresponding FISH images was developed with the software Visilog. Intensity thresholds were not consistent even for duplicate analyses, so alternative ways of classifying signals were investigated. In the resulting method, the intensity data were divided into clusters using fuzzy c-means clustering, and the resulting clusters were classified as target (positive) or nontarget (negative). A manual quality control confirmed this classification. With this method, 50.4, 72.1, and 64.9% of the cells in two swine manure samples and one soil sample, respectively, were positive as determined with a 16S rRNA-targeted bacterial probe (S-D-Bact-0338-a-A-18). Manual counting resulted in corresponding values of 52.3, 70.6, and 61.5%, respectively. In two swine manure samples and one soil sample 21.6, 12.3, and 2.5% of the cells were positive with an archaeal probe (S-D-Arch-0915-a-A-20), respectively. Manual counting resulted in corresponding values of 22.4, 14.0, and 2.9%, respectively. This automated method should facilitate quantitative analysis of FISH images for a variety of complex environmental samples.

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

PubMed

Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

2018-04-19

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

A near-infrared BSA coated DNA-AgNCs for cellular imaging.

PubMed

Mu, Wei-Yu; Yang, Rui; Robertson, Akrofi; Chen, Qiu-Yun

2018-02-01

Near-infrared silver nanoclusters, have potential applications in the field of biosensing and biological imaging. However, less stability of most DNA-AgNCs limits their application. To obtain stable near-infrared fluorescence DNA-AgNCs for biological imaging, a new kind of near-infrared fluorescent DNA-Ag nanoclusters was constructed using the C3A rich aptamer as a synthesis template, GAG as the enhancer. In particular, a new DNA-AgNCs-Trp@BSA was obtained based on the self-assembly of bovine serum albumin (BSA) and tryptophan loaded DNA-AgNCs by hydrophobic interaction. This self-assembly method can be used to stabilize DNAn-Ag (n = 1-3) nanoclusters. Hence, the near-infrared fluorescence DNA-AgNCs-Trp@BSA was applied in cellular imaging of HepG-2 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Imaging of pharmacokinetic rates of indocyanine green in mouse liver with a hybrid fluorescence molecular tomography/x-ray computed tomography system.

PubMed

Zhang, Guanglei; Liu, Fei; Zhang, Bin; He, Yun; Luo, Jianwen; Bai, Jing

2013-04-01

Pharmacokinetic rates have the potential to provide quantitative physiological and pathological information for biological studies and drug development. Fluorescence molecular tomography (FMT) is an attractive imaging tool for three-dimensionally resolving fluorophore distribution in small animals. In this letter, pharmacokinetic rates of indocyanine green (ICG) in mouse liver are imaged with a hybrid FMT and x-ray computed tomography (XCT) system. A recently developed FMT method using structural priors from an XCT system is adopted to improve the quality of FMT reconstruction. In the in vivo experiments, images of uptake and excretion rates of ICG in mouse liver are obtained, which can be used to quantitatively evaluate liver function. The accuracy of the results is validated by a fiber-based fluorescence measurement system.

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.

Rapid Raman spectroscopy of musculoskeletal tissue using a visible laser and an electron-multiplying CCD (EMCCD) detector

NASA Astrophysics Data System (ADS)

Golcuk, Kurtulus; Mandair, Gurjit S.; Callender, Andrew F.; Finney, William F.; Sahar, Nadder; Kohn, David H.; Morris, Michael D.

2006-02-01

Background fluorescence can often complicate the use of Raman microspectroscopy in the study of musculoskeletal tissues. Such fluorescence interferences are undesirable as the Raman spectra of matrix and mineral phases can be used to differentiate between normal and pathological or microdamaged bone. Photobleaching with the excitation laser provides a non-invasive method for reducing background fluorescence, enabling 532 nm Raman hyperspectral imaging of bone tissue. The signal acquisition time for a 400 point Raman line image is reduced to 1-4 seconds using electronmultiplying CCD (EMCCD) detector, enabling acquisition of Raman images in less than 10 minutes. Rapid photobleaching depends upon multiple scattering effects in the tissue specimen and is applicable to some, but not all experimental situations.

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.

Semi-automated segmentation of neuroblastoma nuclei using the gradient energy tensor: a user driven approach

NASA Astrophysics Data System (ADS)

Kromp, Florian; Taschner-Mandl, Sabine; Schwarz, Magdalena; Blaha, Johanna; Weiss, Tamara; Ambros, Peter F.; Reiter, Michael

2015-02-01

We propose a user-driven method for the segmentation of neuroblastoma nuclei in microscopic fluorescence images involving the gradient energy tensor. Multispectral fluorescence images contain intensity and spatial information about antigene expression, fluorescence in situ hybridization (FISH) signals and nucleus morphology. The latter serves as basis for the detection of single cells and the calculation of shape features, which are used to validate the segmentation and to reject false detections. Accurate segmentation is difficult due to varying staining intensities and aggregated cells. It requires several (meta-) parameters, which have a strong influence on the segmentation results and have to be selected carefully for each sample (or group of similar samples) by user interactions. Because our method is designed for clinicians and biologists, who may have only limited image processing background, an interactive parameter selection step allows the implicit tuning of parameter values. With this simple but intuitive method, segmentation results with high precision for a large number of cells can be achieved by minimal user interaction. The strategy was validated on handsegmented datasets of three neuroblastoma cell lines.

Simulation-based evaluation of the resolution and quantitative accuracy of temperature-modulated fluorescence tomography

PubMed Central

Lin, Yuting; Nouizi, Farouk; Kwong, Tiffany C.; Gulsen, Gultekin

2016-01-01

Conventional fluorescence tomography (FT) can recover the distribution of fluorescent agents within a highly scattering medium. However, poor spatial resolution remains its foremost limitation. Previously, we introduced a new fluorescence imaging technique termed “temperature-modulated fluorescence tomography” (TM-FT), which provides high-resolution images of fluorophore distribution. TM-FT is a multimodality technique that combines fluorescence imaging with focused ultrasound to locate thermo-sensitive fluorescence probes using a priori spatial information to drastically improve the resolution of conventional FT. In this paper, we present an extensive simulation study to evaluate the performance of the TM-FT technique on complex phantoms with multiple fluorescent targets of various sizes located at different depths. In addition, the performance of the TM-FT is tested in the presence of background fluorescence. The results obtained using our new method are systematically compared with those obtained with the conventional FT. Overall, TM-FT provides higher resolution and superior quantitative accuracy, making it an ideal candidate for in vivo preclinical and clinical imaging. For example, a 4 mm diameter inclusion positioned in the middle of a synthetic slab geometry phantom (D:40 mm × W :100 mm) is recovered as an elongated object in the conventional FT (x = 4.5 mm; y = 10.4 mm), while TM-FT recovers it successfully in both directions (x = 3.8 mm; y = 4.6 mm). As a result, the quantitative accuracy of the TM-FT is superior because it recovers the concentration of the agent with a 22% error, which is in contrast with the 83% error of the conventional FT. PMID:26368884

Clinical application of photodynamic medicine technology using light-emitting fluorescence imaging based on a specialized luminous source.

PubMed

Namikawa, Tsutomu; Fujisawa, Kazune; Munekage, Eri; Iwabu, Jun; Uemura, Sunao; Tsujii, Shigehiro; Maeda, Hiromichi; Kitagawa, Hiroyuki; Fukuhara, Hideo; Inoue, Keiji; Sato, Takayuki; Kobayashi, Michiya; Hanazaki, Kazuhiro

2018-04-04

The natural amino acid 5-aminolevulinic acid (ALA) is a protoporphyrin IX (PpIX) precursor and a new-generation photosensitive substance that accumulates specifically in cancer cells. When indocyanine green (ICG) is irradiated with near-infrared (NIR) light, it shifts to a higher energy state and emits infrared light with a longer wavelength than the irradiated NIR light. Photodynamic diagnosis (PDD) using ALA and ICG-based NIR fluorescence imaging has emerged as a new diagnostic technique. Specifically, in laparoscopic examinations for serosa-invading advanced gastric cancer, peritoneal metastases could be detected by ALA-PDD, but not by conventional visible-light imaging. The HyperEye Medical System (HEMS) can visualize ICG fluorescence as color images simultaneously projected with visible light in real time. This ICG fluorescence method is widely applicable, including for intraoperative identification of sentinel lymph nodes, visualization of blood vessels in organ resection, and blood flow evaluation during surgery. Fluorescence navigation by ALA-PDD and NIR using ICG imaging provides good visualization and detection of the target lesions that is not possible with the naked eye. We propose that this technique should be used in fundamental research on the relationship among cellular dynamics, metabolic enzymes, and tumor tissues, and to evaluate clinical efficacy and safety in multicenter cooperative clinical trials.

Oxygen-doped carbon nanotubes for near-infrared fluorescent labels and imaging probes.

PubMed

Iizumi, Yoko; Yudasaka, Masako; Kim, Jaeho; Sakakita, Hajime; Takeuchi, Tsukasa; Okazaki, Toshiya

2018-04-19

Chemical modification of carbon nanotube surface can controllably modulate their optical properties. Here we report a simple and effective synthesis method of oxygen-doped single-walled carbon nanotubes (o-SWCNTs), in which a thin film of SWCNTs is just irradiated under the UV light for a few minutes in air. By using this method, the epoxide-type oxygen-adducts (ep-SWCNTs) were produced in addition to the ether-type oxygen-adducts (eth-SWCNTs). The Treated (6, 5) ep-SWCNTs show a red-shifted luminescence at ~1280 nm, which corresponds to the most transparent regions for bio-materials. Immunoassay, fluorescence vascular angiography and observation of the intestinal contractile activity of mice were demonstrated by using the produced o-SWCNTs as infrared fluorescent labels and imaging agents.

A fast reconstruction algorithm for fluorescence optical diffusion tomography based on preiteration.

PubMed

Song, Xiaolei; Xiong, Xiaoyun; Bai, Jing

2007-01-01

Fluorescence optical diffusion tomography in the near-infrared (NIR) bandwidth is considered to be one of the most promising ways for noninvasive molecular-based imaging. Many reconstructive approaches to it utilize iterative methods for data inversion. However, they are time-consuming and they are far from meeting the real-time imaging demands. In this work, a fast preiteration algorithm based on the generalized inverse matrix is proposed. This method needs only one step of matrix-vector multiplication online, by pushing the iteration process to be executed offline. In the preiteration process, the second-order iterative format is employed to exponentially accelerate the convergence. Simulations based on an analytical diffusion model show that the distribution of fluorescent yield can be well estimated by this algorithm and the reconstructed speed is remarkably increased.

Thin-film tunable filters for hyperspectral fluorescence microscopy

PubMed Central

Favreau, Peter; Hernandez, Clarissa; Lindsey, Ashley Stringfellow; Alvarez, Diego F.; Rich, Thomas; Prabhat, Prashant

2013-01-01

Abstract. Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging—the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes. PMID:24077519

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography

PubMed Central

Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

2016-01-01

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT®). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors. PMID:27809256

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography.

PubMed

Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

2016-10-31

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT ® ). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors.

Stereoscopic Imaging in Hypersonics Boundary Layers using Planar Laser-Induced Fluorescence

NASA Technical Reports Server (NTRS)

Danehy, Paul M.; Bathel, Brett; Inman, Jennifer A.; Alderfer, David W.; Jones, Stephen B.

2008-01-01

Stereoscopic time-resolved visualization of three-dimensional structures in a hypersonic flow has been performed for the first time. Nitric Oxide (NO) was seeded into hypersonic boundary layer flows that were designed to transition from laminar to turbulent. A thick laser sheet illuminated and excited the NO, causing spatially-varying fluorescence. Two cameras in a stereoscopic configuration were used to image the fluorescence. The images were processed in a computer visualization environment to provide stereoscopic image pairs. Two methods were used to display these image pairs: a cross-eyed viewing method which can be viewed by naked eyes, and red/blue anaglyphs, which require viewing through red/blue glasses. The images visualized three-dimensional information that would be lost if conventional planar laser-induced fluorescence imaging had been used. Two model configurations were studied in NASA Langley Research Center's 31-Inch Mach 10 Air Wind tunnel. One model was a 10 degree half-angle wedge containing a small protuberance to force the flow to transition. The other model was a 1/3-scale, truncated Hyper-X forebody model with blowing through a series of holes to force the boundary layer flow to transition to turbulence. In the former case, low flowrates of pure NO seeded and marked the boundary layer fluid. In the latter, a trace concentration of NO was seeded into the injected N2 gas. The three-dimensional visualizations have an effective time resolution of about 500 ns, which is fast enough to freeze this hypersonic flow. The 512x512 resolution of the resulting images is much higher than high-speed laser-sheet scanning systems with similar time response, which typically measure 10-20 planes.

Quantifying cancer cell receptors with paired-agent fluorescent imaging: a novel method to account for tissue optical property effects

NASA Astrophysics Data System (ADS)

Sadeghipour, Negar; Davis, Scott C.; Tichauer, Kenneth M.

2018-02-01

Dynamic fluorescence imaging approaches can be used to estimate the concentration of cell surface receptors in vivo. Kinetic models are used to generate the final estimation by taking the targeted imaging agent concentration as a function of time. However, tissue absorption and scattering properties cause the final readout signal to be on a different scale than the real fluorescent agent concentration. In paired-agent imaging approaches, simultaneous injection of a suitable control imaging agent with a targeted one can account for non-specific uptake and retention of the targeted agent. Additionally, the signal from the control agent can be a normalizing factor to correct for tissue optical property differences. In this study, the kinetic model used for paired-agent imaging analysis (i.e., simplified reference tissue model) is modified and tested in simulation and experimental data in a way that accounts for the scaling correction within the kinetic model fit to the data to ultimately extract an estimate of the targeted biomarker concentration.

Demonstration of plant fluorescence by imaging technique and Intelligent FluoroSensor

NASA Astrophysics Data System (ADS)

Lenk, Sándor; Gádoros, Patrik; Kocsányi, László; Barócsi, Attila

2015-10-01

Photosynthesis is a process that converts carbon-dioxide into organic compounds, especially into sugars, using the energy of sunlight. The absorbed light energy is used mainly for photosynthesis initiated at the reaction centers of chlorophyll-protein complexes, but part of it is lost as heat and chlorophyll fluorescence. Therefore, the measurement of the latter can be used to estimate the photosynthetic activity. The basic method, when illuminating intact leaves with strong light after a dark adaptation of at least 20 minutes resulting in a transient change of fluorescence emission of the fluorophore chlorophyll-a called `Kautsky effect', is demonstrated by an imaging setup. The experimental kit includes a high radiant blue LED and a CCD camera (or a human eye) equipped with a red transmittance filter to detect the changing fluorescence radiation. However, for the measurement of several fluorescence parameters, describing the plant physiological processes in detail, the variation of several excitation light sources and an adequate detection method are needed. Several fluorescence induction protocols (e.g. traditional Kautsky, pulse amplitude modulated and excitation kinetic), are realized in the Intelligent FluoroSensor instrument. Using it, students are able to measure different plant fluorescence induction curves, quantitatively determine characteristic parameters and qualitatively interpret the measured signals.

NIR fluorescence lifetime sensing through a multimode fiber for intravascular molecular probing

NASA Astrophysics Data System (ADS)

Ingelberts, H.; Hernot, S.; Debie, P.; Lahoutte, T.; Kuijk, M.

2016-04-01

Coronary artery disease (CAD) contributes to millions of deaths each year. The identification of vulnerable plaques is essential to the diagnosis of CAD but is challenging. Molecular probes can improve the detection of these plaques using intravascular imaging methods. Fluorescence lifetime sensing is a safe and robust method to image these molecular probes. We present two variations of an optical system for intravascular near-infrared (NIR) fluorescence lifetime sensing through a multimode fiber. Both systems are built around a recently developed fast and efficient CMOS detector, the current-assisted photonic sampler (CAPS) that is optimized for sub-nanosecond NIR fluorescence lifetime sensing. One system mimics the optical setup of an epifluorescence microscope while the other uses a practical fiber optic coupler to separate fluorescence excitation and emission. We test both systems by measuring the lifetime of several NIR dyes in DMSO solutions and we show that these systems are capable of detecting lifetimes of solutions with concentrations down to 370 nM and this with short acquisition times. These results are compared with time-correlated single photon counting (TCSPC) measurements for reference.