Multi-spectral endogenous fluorescence imaging for bacterial differentiation

NASA Astrophysics Data System (ADS)

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

2017-07-01

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

Catheter-based time-gated near-infrared fluorescence/OCT imaging system

NASA Astrophysics Data System (ADS)

Lu, Yuankang; Abran, Maxime; Cloutier, Guy; Lesage, Frédéric

2018-02-01

We developed a new dual-modality intravascular imaging system based on fast time-gated fluorescence intensity imaging and spectral domain optical coherence tomography (SD-OCT) for the purpose of interventional detection of atherosclerosis. A pulsed supercontinuum laser was used for fluorescence and OCT imaging. A double-clad fiber (DCF)- based side-firing catheter was designed and fabricated to have a 23 μm spot size at a 2.2 mm working distance for OCT imaging. Its single-mode core is used for OCT, while its inner cladding transports fluorescence excitation light and collects fluorescent photons. The combination of OCT and fluorescence imaging was achieved by using a DCF coupler. For fluorescence detection, we used a time-gated technique with a novel single-photon avalanche diode (SPAD) working in an ultra-fast gating mode. A custom-made delay chip was integrated in the system to adjust the delay between the excitation laser pulse and the SPAD gate-ON window. This technique allowed to detect fluorescent photons of interest while rejecting most of the background photons, thus leading to a significantly improved signal to noise ratio (SNR). Experiments were carried out in turbid media mimicking tissue with an indocyanine green (ICG) inclusion (1 mM and 100 μM) to compare the time-gated technique and the conventional continuous detection technique. The gating technique increased twofold depth sensitivity, and tenfold SNR at large distances. The dual-modality imaging capacity of our system was also validated with a silicone-based tissue-mimicking phantom.

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.

Reconstruction algorithms based on l1-norm and l2-norm for two imaging models of fluorescence molecular tomography: a comparative study.

PubMed

Yi, Huangjian; Chen, Duofang; Li, Wei; Zhu, Shouping; Wang, Xiaorui; Liang, Jimin; Tian, Jie

2013-05-01

Fluorescence molecular tomography (FMT) is an important imaging technique of optical imaging. The major challenge of the reconstruction method for FMT is the ill-posed and underdetermined nature of the inverse problem. In past years, various regularization methods have been employed for fluorescence target reconstruction. A comparative study between the reconstruction algorithms based on l1-norm and l2-norm for two imaging models of FMT is presented. The first imaging model is adopted by most researchers, where the fluorescent target is of small size to mimic small tissue with fluorescent substance, as demonstrated by the early detection of a tumor. The second model is the reconstruction of distribution of the fluorescent substance in organs, which is essential to drug pharmacokinetics. Apart from numerical experiments, in vivo experiments were conducted on a dual-modality FMT/micro-computed tomography imaging system. The experimental results indicated that l1-norm regularization is more suitable for reconstructing the small fluorescent target, while l2-norm regularization performs better for the reconstruction of the distribution of fluorescent substance.

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.

The Mechanisms and Biomedical Applications of an NIR BODIPY-Based Switchable Fluorescent Probe

PubMed Central

Cheng, Bingbing; Bandi, Venugopal; Yu, Shuai; D’Souza, Francis; Nguyen, Kytai T.; Hong, Yi; Tang, Liping; Yuan, Baohong

2017-01-01

Highly environment-sensitive fluorophores have been desired for many biomedical applications. Because of the noninvasive operation, high sensitivity, and high specificity to the microenvironment change, they can be used as excellent probes for fluorescence sensing/imaging, cell tracking/imaging, molecular imaging for cancer, and so on (i.e., polarity, viscosity, temperature, or pH measurement). In this work, investigations of the switching mechanism of a recently reported near-infrared environment-sensitive fluorophore, ADP(CA)2, were conducted. Besides, multiple potential biomedical applications of this switchable fluorescent probe have been demonstrated, including wash-free live-cell fluorescence imaging, in vivo tissue fluorescence imaging, temperature sensing, and ultrasound-switchable fluorescence (USF) imaging. The fluorescence of the ADP(CA)2 is extremely sensitive to the microenvironment, especially polarity and viscosity. Our investigations showed that the fluorescence of ADP(CA)2 can be switched on by low polarity, high viscosity, or the presence of protein and surfactants. In wash-free live-cell imaging, the fluorescence of ADP(CA)2 inside cells was found much brighter than the dye-containing medium and was retained for at least two days. In all of the fluorescence imaging applications conducted in this study, high target-to-noise (>5-fold) was achieved. In addition, a high temperature sensitivity (73-fold per Celsius degree) of ADP(CA)2-based temperature probes was found in temperature sensing. PMID:28208666

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.

Multimodal optoacoustic and multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sela, Gali; Razansky, Daniel; Shoham, Shy

2013-03-01

Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

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

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells.

PubMed

Nygate, Yoav N; Singh, Gyanendra; Barnea, Itay; Shaked, Natan T

2018-06-01

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

Localization-based super-resolution imaging of cellular structures.

PubMed

Kanchanawong, Pakorn; Waterman, Clare M

2013-01-01

Fluorescence microscopy allows direct visualization of fluorescently tagged proteins within cells. However, the spatial resolution of conventional fluorescence microscopes is limited by diffraction to ~250 nm, prompting the development of super-resolution microscopy which offers resolution approaching the scale of single proteins, i.e., ~20 nm. Here, we describe protocols for single molecule localization-based super-resolution imaging, using focal adhesion proteins as an example and employing either photoswitchable fluorophores or photoactivatable fluorescent proteins. These protocols should also be easily adaptable to imaging a broad array of macromolecular assemblies in cells whose components can be fluorescently tagged and assemble into high density structures.

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

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

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.

Evaluation of slide based cytometry (SBC) for concentration measurements of fluorescent dyes in solution

NASA Astrophysics Data System (ADS)

Pierzchalski, Arkadiusz; Marecka, Monika; Müller, Hans-Willy; Bocsi, József; Tárnok, Attila

2009-02-01

Flow cytometers (FCM) are built for particle measurements. In principle, concentration measurement of a homogeneous solution is not possible with FCM due to the lack of a trigger signal. In contrast to FCM slide based cytometry systems could act as tools for the measurement of concentrations using volume defined cell counting chambers. These chambers enable to analyze a well defined volume. Sensovation AG (Stockach, Germany) introduced an automated imaging system that combines imaging with cytometric features analysis. Aim of this study was to apply this imaging system to quantify the fluorescent molecule concentrations. The Lumisens (Sensovation AG) slide-based technology based on fluorescence digital imaging microscopy was used. The instrument is equipped with an inverted microscope, blue and red LEDs, double band-pass filters and a high-resolution cooled 16-bit digital camera. The instrument was focussed on the bottom of 400μm deep 6 chamber slides (IBIDI GmbH, Martinsried, Germany) or flat bottom 96 well plates (Greiner Bio One GmbH, Frickenhausen, Germany). Fluorescent solutions were imaged under 90% pixel saturation in a broad concentration range (FITC: 0.0002-250 μg/ml, methylene blue (MethB): 0.0002-250 μg/ml). Exposition times were recorded. Images were analysed by the iCys (CompuCyte Corp., Cambridge, MA, USA) image analysis software with the phantom contour function. Relative fluorescence intensities were calculated from mean fluorescence intensities per phantom contours divided by the exposition time. Solution concentrations could be distinguished over a broad dynamic range of 3.5 to 5.5 decades log (range FITC: 0.0002-31.25μg/ml, MethB: 0.0076-31.25μg/ml) with a good linear relationship between dye concentration and relative fluorescence intensity. The minimal number of fluorescent molecules per pixel as determined by the mean fluorescence intensity and the molecular weight of the fluorochrome were about 800 molecules FITC and ~2.000 MethB. The novel slide-based imaging system is suitable for detection of fluorescence differences over a broad range of concentrations. This approach may lead to novel assays for measuring concentration differences in cell free solutions and cell cultures e.g. in secretion assays.

Use of image analysis to estimate anthocyanin and UV-excited fluorescent phenolic compound levels in strawberry fruit

PubMed Central

Yoshioka, Yosuke; Nakayama, Masayoshi; Noguchi, Yuji; Horie, Hideki

2013-01-01

Strawberry is rich in anthocyanins, which are responsible for the red color, and contains several colorless phenolic compounds. Among the colorless phenolic compounds, some, such as hydroxycinammic acid derivatives, emit blue-green fluorescence when excited with ultraviolet (UV) light. Here, we investigated the effectiveness of image analyses for estimating the levels of anthocyanins and UV-excited fluorescent phenolic compounds in fruit. The fruit skin and cut surface of 12 cultivars were photographed under visible and UV light conditions; colors were evaluated based on the color components of images. The levels of anthocyanins and UV-excited fluorescent compounds in each fruit were also evaluated by spectrophotometric and high performance liquid chromatography (HPLC) analyses, respectively and relationships between these levels and the image data were investigated. Red depth of the fruits differed greatly among the cultivars and anthocyanin content was well estimated based on the color values of the cut surface images. Strong UV-excited fluorescence was observed on the cut surfaces of several cultivars, and the grayscale values of the UV-excited fluorescence images were markedly correlated with the levels of those fluorescent compounds as evaluated by HPLC analysis. These results indicate that image analyses can select promising genotypes rich in anthocyanins and fluorescent phenolic compounds. PMID:23853516

Dendrimer probes for enhanced photostability and localization in fluorescence imaging.

PubMed

Kim, Younghoon; Kim, Sung Hoon; Tanyeri, Melikhan; Katzenellenbogen, John A; Schroeder, Charles M

2013-04-02

Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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.

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

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

Morpholine Derivative-Functionalized Carbon Dots-Based Fluorescent Probe for Highly Selective Lysosomal Imaging in Living Cells.

PubMed

Wu, Luling; Li, Xiaolin; Ling, Yifei; Huang, Chusen; Jia, Nengqin

2017-08-30

The development of a suitable fluorescent probe for the specific labeling and imaging of lysosomes through the direct visual fluorescent signal is extremely important for understanding the dysfunction of lysosomes, which might induce various pathologies, including neurodegenerative diseases, cancer, and Alzheimer's disease. Herein, a new carbon dot-based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. In this probe, PEI (polyethylenimine) is introduced to improve water solubility and provide abundant amine groups for the as-prepared CDs-PEI, and the morpholine group (ML) serves as a targeting unit for lysosomes. More importantly, passivation with PEI could dramatically increase the fluorescence quantum yield of CDs-PEI-ML as well as their stability in fluorescence emission under different excitation wavelength. Consequently, experimental data demonstrated that the target probe CDs-PEI-ML has low cytotoxicity and excellent photostability. Additionally, further live cell imaging experiment indicated that CDs-PEI-ML is a highly selective fluorescent probe for lysosomes. We speculate the mechanism for selective staining of lysosomes that CDs-PEI-ML was initially taken up by lysosomes through the endocytic pathway and then accumulated in acidic lysosomes. It is notable that there was less diffusion of CDs-PEI-ML into cytoplasm, which could be ascribed to the presence of lysosome target group morpholine on surface of CDs-PEI-ML. The blue emission wavelength combined with the high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML become an alternative fluorescent probe for multicolor labeling and long-term tracking of lysosomes in live cells and the potential application in super-resolution imaging. To best of our knowledge, there are still limited carbon dots-based fluorescent probes that have been studied for specific lysosomal imaging in live cells. The concept of surface functionality of carbon dots will also pave a new avenue for developing carbon dots-based fluorescent probes for subcellular labeling.

Facilitating in vivo tumor localization by principal component analysis based on dynamic fluorescence molecular imaging

NASA Astrophysics Data System (ADS)

Gao, Yang; Chen, Maomao; Wu, Junyu; Zhou, Yuan; Cai, Chuangjian; Wang, Daliang; Luo, Jianwen

2017-09-01

Fluorescence molecular imaging has been used to target tumors in mice with xenograft tumors. However, tumor imaging is largely distorted by the aggregation of fluorescent probes in the liver. A principal component analysis (PCA)-based strategy was applied on the in vivo dynamic fluorescence imaging results of three mice with xenograft tumors to facilitate tumor imaging, with the help of a tumor-specific fluorescent probe. Tumor-relevant features were extracted from the original images by PCA and represented by the principal component (PC) maps. The second principal component (PC2) map represented the tumor-related features, and the first principal component (PC1) map retained the original pharmacokinetic profiles, especially of the liver. The distribution patterns of the PC2 map of the tumor-bearing mice were in good agreement with the actual tumor location. The tumor-to-liver ratio and contrast-to-noise ratio were significantly higher on the PC2 map than on the original images, thus distinguishing the tumor from its nearby fluorescence noise of liver. The results suggest that the PC2 map could serve as a bioimaging marker to facilitate in vivo tumor localization, and dynamic fluorescence molecular imaging with PCA could be a valuable tool for future studies of in vivo tumor metabolism and progression.

Spectroscopic imaging using acousto-optic tunable filters

NASA Astrophysics Data System (ADS)

Bouhifd, Mounir; Whelan, Maurice

2007-07-01

We report on novel hyper-spectral imaging filter-modules based on acousto-optic tuneable filters (AOTF). The AOTF functions as a full-field tuneable bandpass filter which offers fast continuous or random access tuning with high filtering efficiency. Due to the diffractive nature of the device, the unfiltered zero-order and the filtered first-order images are geometrically separated. The modules developed exploit this feature to simultaneously route both the transmitted white-light image and the filtered fluorescence image to two separate cameras. Incorporation of prisms in the optical paths and careful design of the relay optics in the filter module have overcome a number of aberrations inherent to imaging through AOTFs, leading to excellent spatial resolution. A number of practical uses of this technique, both for in vivo auto-fluorescence endoscopy and in vitro fluorescence microscopy were demonstrated. We describe the operational principle and design of recently improved prototype instruments for fluorescence-based diagnostics and demonstrate their performance by presenting challenging hyper-spectral fluorescence imaging applications.

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.

Multifunctional PHPMA-Derived Polymer for Ratiometric pH Sensing, Fluorescence Imaging, and Magnetic Resonance Imaging.

PubMed

Su, Fengyu; Agarwal, Shubhangi; Pan, Tingting; Qiao, Yuan; Zhang, Liqiang; Shi, Zhengwei; Kong, Xiangxing; Day, Kevin; Chen, Meiwan; Meldrum, Deirdre; Kodibagkar, Vikram D; Tian, Yanqing

2018-01-17

In this paper, we report synthesis and characterization of a novel multimodality (MRI/fluorescence) probe for pH sensing and imaging. A multifunctional polymer was derived from poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) and integrated with a naphthalimide-based-ratiometric fluorescence probe and a gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex (Gd-DOTA complex). The polymer was characterized using UV-vis absorption spectrophotometry, fluorescence spectrofluorophotometry, magnetic resonance imaging (MRI), and confocal microscopy for optical and MRI-based pH sensing and cellular imaging. In vitro labeling of macrophage J774 and esophageal CP-A cell lines shows the polymer's ability to be internalized in the cells. The transverse relaxation time (T 2 ) of the polymer was observed to be pH-dependent, whereas the spin-lattice relaxation time (T 1 ) was not. The pH probe in the polymer shows a strong fluorescence-based ratiometric pH response with emission window changes, exhibiting blue emission under acidic conditions and green emission under basic conditions, respectively. This study provides new materials with multimodalities for pH sensing and imaging.

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

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.

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.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice.

PubMed

Feeks, James A; Hunter, Jennifer J

2017-05-01

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina.

Snapshot imaging Fraunhofer line discriminator for detection of plant fluorescence

NASA Astrophysics Data System (ADS)

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

2015-05-01

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

Co-registered photoacoustic and fluorescent imaging of a switchable nanoprobe based on J-aggregates of indocyanine green

NASA Astrophysics Data System (ADS)

Dumani, Diego S.; Brecht, Hans-Peter; Ivanov, Vassili; Deschner, Ryan; Harris, Justin T.; Homan, Kimberly A.; Cook, Jason R.; Emelianov, Stanislav Y.; Ermilov, Sergey A.

2018-02-01

We introduce a preclinical imaging platform - a 3D photoacoustic/fluorescence tomography (PAFT) instrument augmented with an environmentally responsive dual-contrast biocompatible nanoprobe. The PAFT instrument was designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct co-registration of the two imaging modalities. The nanoprobe was based on liposomes loaded with J-aggregates of indocyanine green (PAtrace). Once PAtrace interacts with the environment, a transition from J-aggregate to monomeric ICG is induced. The subsequent recovery of monomeric ICG is characterized by dramatic changes in the optical absorption spectrum and reinstated fluorescence. In the activated state, PAtrace can be simultaneously detected by both imaging modes of the PAFT instrument using 780 nm excitation and fluorescence detection at 810 nm. The fluorescence imaging component is used to boost detection sensitivity by providing lowresolution map of activated nanoprobes, which are then more precisely mapped in 3D by the photoacoustic imaging component. Activated vs non-activated particles can be distinguished based on their different optical absorption peaks, removing the requirements for complex image registration between reference and detection scans. Preliminary phantom and in vivo animal imaging results showed successful activation and visualization of PAtrace with high sensitivity and resolution. The proposed PAFT-PAtrace imaging platform could be used in various functional and molecular imaging applications including multi-point in vivo assessment of early metastasis.

MRI-guided fluorescence tomography of the breast: a phantom study

NASA Astrophysics Data System (ADS)

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

2009-02-01

Tissue phantoms simulating the human breast were used to demonstrate the imaging capabilities of an MRI-coupled fluorescence molecular tomography (FMT) imaging system. Specifically, phantoms with low tumor-to-normal drug contrast and complex internal structure were imaged with the MR-coupled FMT system. Images of indocyanine green (ICG) fluorescence yield were recovered using a diffusion model-based approach capable of estimating the distribution of fluorescence activity in a tissue volume from tissue-boundary measurements of transmitted light. Tissue structural information, which can be determined from standard T1 and T2 MR images, was used to guide the recovery of fluorescence activity. The study revealed that this spatial guidance is critical for recovering images of fluorescence yield in tissue with low tumor-to-normal drug contrast.

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.

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.

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.

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

NASA Astrophysics Data System (ADS)

Wang, Taejun; Jang, Won Hyuk; Lee, Seunghun; Yoon, Calvin J.; Lee, Jun Ho; Kim, Bumju; Hwang, Sekyu; Hong, Chun-Pyo; Yoon, Yeoreum; Lee, Gilgu; Le, Viet-Hoan; Bok, Seoyeon; Ahn, G.-One; Lee, Jaewook; Gho, Yong Song; Chung, Euiheon; Kim, Sungjee; Jang, Myoung Ho; Myung, Seung-Jae; Kim, Myoung Joon; So, Peter T. C.; Kim, Ki Hean

2016-06-01

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

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

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

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

Community detection for fluorescent lifetime microscopy image segmentation

NASA Astrophysics Data System (ADS)

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

2014-03-01

Multiresolution community detection (CD) method has been suggested in a recent work as an efficient method for performing unsupervised segmentation of fluorescence lifetime (FLT) images of live cell images containing fluorescent molecular probes.1 In the current paper, we further explore this method in FLT images of ex vivo tissue slices. The image processing problem is framed as identifying clusters with respective average FLTs against a background or "solvent" in FLT imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the multiresolution CD method from different starting points. In this paper, our method is found to be more efficient than a current state-of-the-art image segmentation method based on mixture of Gaussian distributions. It offers more than 1:25 times diversity based on Shannon index than the latter method, in selecting clusters with distinct average FLTs in NIR FLIM images.

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

PubMed Central

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

2017-01-01

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

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.

Fluorescence tomography characterization for sub-surface imaging with protoporphyrin IX

PubMed Central

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

2009-01-01

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

Frequency division multiplexed multi-color fluorescence microscope system

NASA Astrophysics Data System (ADS)

Le, Vu Nam; Yang, Huai Dong; Zhang, Si Chun; Zhang, Xin Rong; Jin, Guo Fan

2017-10-01

Grayscale camera can only obtain gray scale image of object, while the multicolor imaging technology can obtain the color information to distinguish the sample structures which have the same shapes but in different colors. In fluorescence microscopy, the current method of multicolor imaging are flawed. Problem of these method is affecting the efficiency of fluorescence imaging, reducing the sampling rate of CCD etc. In this paper, we propose a novel multiple color fluorescence microscopy imaging method which based on the Frequency division multiplexing (FDM) technology, by modulating the excitation lights and demodulating the fluorescence signal in frequency domain. This method uses periodic functions with different frequency to modulate amplitude of each excitation lights, and then combine these beams for illumination in a fluorescence microscopy imaging system. The imaging system will detect a multicolor fluorescence image by a grayscale camera. During the data processing, the signal obtained by each pixel of the camera will be processed with discrete Fourier transform, decomposed by color in the frequency domain and then used inverse discrete Fourier transform. After using this process for signals from all of the pixels, monochrome images of each color on the image plane can be obtained and multicolor image is also acquired. Based on this method, this paper has constructed and set up a two-color fluorescence microscope system with two excitation wavelengths of 488 nm and 639 nm. By using this system to observe the linearly movement of two kinds of fluorescent microspheres, after the data processing, we obtain a two-color fluorescence dynamic video which is consistent with the original image. This experiment shows that the dynamic phenomenon of multicolor fluorescent biological samples can be generally observed by this method. Compared with the current methods, this method can obtain the image signals of each color at the same time, and the color video's frame rate is consistent with the frame rate of the camera. The optical system is simpler and does not need extra color separation element. In addition, this method has a good filtering effect on the ambient light or other light signals which are not affected by the modulation process.

Smartphone Cortex Controlled Real-Time Image Processing and Reprocessing for Concentration Independent LED Induced Fluorescence Detection in Capillary Electrophoresis.

PubMed

Szarka, Mate; Guttman, Andras

2017-10-17

We present the application of a smartphone anatomy based technology in the field of liquid phase bioseparations, particularly in capillary electrophoresis. A simple capillary electrophoresis system was built with LED induced fluorescence detection and a credit card sized minicomputer to prove the concept of real time fluorescent imaging (zone adjustable time-lapse fluorescence image processor) and separation controller. The system was evaluated by analyzing under- and overloaded aminopyrenetrisulfonate (APTS)-labeled oligosaccharide samples. The open source software based image processing tool allowed undistorted signal modulation (reprocessing) if the signal was inappropriate for the actual detection system settings (too low or too high). The novel smart detection tool for fluorescently labeled biomolecules greatly expands dynamic range and enables retrospective correction for injections with unsuitable signal levels without the necessity to repeat the analysis.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice

PubMed Central

Feeks, James A.; Hunter, Jennifer J.

2017-01-01

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina. PMID:28663886

Multistage morphological segmentation of bright-field and fluorescent microscopy images

NASA Astrophysics Data System (ADS)

Korzyńska, A.; Iwanowski, M.

2012-06-01

This paper describes the multistage morphological segmentation method (MSMA) for microscopic cell images. The proposed method enables us to study the cell behaviour by using a sequence of two types of microscopic images: bright field images and/or fluorescent images. The proposed method is based on two types of information: the cell texture coming from the bright field images and intensity of light emission, done by fluorescent markers. The method is dedicated to the image sequences segmentation and it is based on mathematical morphology methods supported by other image processing techniques. The method allows for detecting cells in image independently from a degree of their flattening and from presenting structures which produce the texture. It makes use of some synergic information from the fluorescent light emission image as the support information. The MSMA method has been applied to images acquired during the experiments on neural stem cells as well as to artificial images. In order to validate the method, two types of errors have been considered: the error of cell area detection and the error of cell position using artificial images as the "gold standard".

Navigation of a robot-integrated fluorescence laparoscope in preoperative SPECT/CT and intraoperative freehand SPECT imaging data: a phantom study

NASA Astrophysics Data System (ADS)

van Oosterom, Matthias Nathanaël; Engelen, Myrthe Adriana; van den Berg, Nynke Sjoerdtje; KleinJan, Gijs Hendrik; van der Poel, Henk Gerrit; Wendler, Thomas; van de Velde, Cornelis Jan Hadde; Navab, Nassir; van Leeuwen, Fijs Willem Bernhard

2016-08-01

Robot-assisted laparoscopic surgery is becoming an established technique for prostatectomy and is increasingly being explored for other types of cancer. Linking intraoperative imaging techniques, such as fluorescence guidance, with the three-dimensional insights provided by preoperative imaging remains a challenge. Navigation technologies may provide a solution, especially when directly linked to both the robotic setup and the fluorescence laparoscope. We evaluated the feasibility of such a setup. Preoperative single-photon emission computed tomography/X-ray computed tomography (SPECT/CT) or intraoperative freehand SPECT (fhSPECT) scans were used to navigate an optically tracked robot-integrated fluorescence laparoscope via an augmented reality overlay in the laparoscopic video feed. The navigation accuracy was evaluated in soft tissue phantoms, followed by studies in a human-like torso phantom. Navigation accuracies found for SPECT/CT-based navigation were 2.25 mm (coronal) and 2.08 mm (sagittal). For fhSPECT-based navigation, these were 1.92 mm (coronal) and 2.83 mm (sagittal). All errors remained below the <1-cm detection limit for fluorescence imaging, allowing refinement of the navigation process using fluorescence findings. The phantom experiments performed suggest that SPECT-based navigation of the robot-integrated fluorescence laparoscope is feasible and may aid fluorescence-guided surgery procedures.

Boronic acids for fluorescence imaging of carbohydrates.

PubMed

Sun, Xiaolong; Zhai, Wenlei; Fossey, John S; James, Tony D

2016-02-28

"Fluorescence imaging" is a particularly exciting and rapidly developing area of research; the annual number of publications in the area has increased ten-fold over the last decade. The rapid increase of interest in fluorescence imaging will necessitate the development of an increasing number of molecular receptors and binding agents in order to meet the demand in this rapidly expanding area. Carbohydrate biomarkers are particularly important targets for fluorescence imaging given their pivotal role in numerous important biological events, including the development and progression of many diseases. Therefore, the development of new fluorescent receptors and binding agents for carbohydrates is and will be increasing in demand. This review highlights the development of fluorescence imaging agents based on boronic acids a particularly promising class of receptors given their strong and selective binding with carbohydrates in aqueous media.

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.

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Statistical image segmentation for the detection of skin lesion borders in UV fluorescence excitation

NASA Astrophysics Data System (ADS)

Ortega-Martinez, Antonio; Padilla-Martinez, Juan Pablo; Franco, Walfre

2016-04-01

The skin contains several fluorescent molecules or fluorophores that serve as markers of structure, function and composition. UV fluorescence excitation photography is a simple and effective way to image specific intrinsic fluorophores, such as the one ascribed to tryptophan which emits at a wavelength of 345 nm upon excitation at 295 nm, and is a marker of cellular proliferation. Earlier, we built a clinical UV photography system to image cellular proliferation. In some samples, the naturally low intensity of the fluorescence can make it difficult to separate the fluorescence of cells in higher proliferation states from background fluorescence and other imaging artifacts -- like electronic noise. In this work, we describe a statistical image segmentation method to separate the fluorescence of interest. Statistical image segmentation is based on image averaging, background subtraction and pixel statistics. This method allows to better quantify the intensity and surface distributions of fluorescence, which in turn simplify the detection of borders. Using this method we delineated the borders of highly-proliferative skin conditions and diseases, in particular, allergic contact dermatitis, psoriatic lesions and basal cell carcinoma. Segmented images clearly define lesion borders. UV fluorescence excitation photography along with statistical image segmentation may serve as a quick and simple diagnostic tool for clinicians.

A series of fluorene-based two-photon absorbing molecules: synthesis, linear and nonlinear characterization, and bioimaging

PubMed Central

Andrade, Carolina D.; Yanez, Ciceron O.; Rodriguez, Luis; Belfield, Kevin D.

2010-01-01

The synthesis, structural, and photophysical characterization of a series of new fluorescent donor–acceptor and acceptor-acceptor molecules, based on the fluorenyl ring system, with two-photon absorbing properties is described. These new compounds exhibited large Stokes shifts, high fluorescent quantum yields, and, significantly, high two-photon absorption cross sections, making them well suited for two-photon fluorescence microscopy (2PFM) imaging. Confocal and two-photon fluorescence microscopy imaging of COS-7 and HCT 116 cells incubated with probe I showed endosomal selectivity, demonstrating the potential of this class of fluorescent probes in multiphoton fluorescence microscopy. PMID:20481596

[Development of a near-infrared fluorescence imaging system based on fluorescence properties of methylene blue].

PubMed

Huang, Lu-Mao; DU, Pei-Yan; Chen, Lan; Zhang, Sa; Zhou, Di-Fu; Chen, Chun-Lin; Xin, Xue-Gang

2018-04-20

To develop a near-infrared fluorescence imaging system based on the fluorescence properties of methylene blue. According to the optical properties of methylene blue, we used a custom-made specific LED light source and an interference filter, a CCD camera and other relevant components to construct the near-infrared fluorescence imaging system. We tested the signal-to-background ratio (SBR) of this imaging system for detecting methylene blue under different experimental conditions and analyzed the SBR in urine samples collected from 15 Wistar rats with intravenous injection of methylene blue at the doses of 0, 1.4, 1.6, 1.8, or 2.0 0 mg/kg methylene blue. The SBR of this imaging system for detecting methylene blue was affected by the concentration of methylene blue and the distance from the sample (P<0.05). In the urine samples from Wistar rats, the SBR varied with the the injection dose, and the rats injected with 1.6 mg/kg methylene blue showed the highest SBR (8.71∓0.20) in the urine (P<0.05). This near-infrared fluorescence imaging system is useful for fluorescence detection of methylene blue and can be used for real-time recognition of ureters during abdominal surgery.

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

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.

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.

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.

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

Preliminary study of diagnostic spectroscopic imaging for nasopharyngeal carcinoma

NASA Astrophysics Data System (ADS)

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

2003-12-01

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

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

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.

In Vivo Deep Tissue Fluorescence and Magnetic Imaging Employing Hybrid Nanostructures.

PubMed

Ortgies, Dirk H; de la Cueva, Leonor; Del Rosal, Blanca; Sanz-Rodríguez, Francisco; Fernández, Nuria; Iglesias-de la Cruz, M Carmen; Salas, Gorka; Cabrera, David; Teran, Francisco J; Jaque, Daniel; Martín Rodríguez, Emma

2016-01-20

Breakthroughs in nanotechnology have made it possible to integrate different nanoparticles in one single hybrid nanostructure (HNS), constituting multifunctional nanosized sensors, carriers, and probes with great potential in the life sciences. In addition, such nanostructures could also offer therapeutic capabilities to achieve a wider variety of multifunctionalities. In this work, the encapsulation of both magnetic and infrared emitting nanoparticles into a polymeric matrix leads to a magnetic-fluorescent HNS with multimodal magnetic-fluorescent imaging abilities. The magnetic-fluorescent HNS are capable of simultaneous magnetic resonance imaging and deep tissue infrared fluorescence imaging, overcoming the tissue penetration limits of classical visible-light based optical imaging as reported here in living mice. Additionally, their applicability for magnetic heating in potential hyperthermia treatments is assessed.

A mitochondria-selective near-infrared-emitting fluorescent dye for cellular imaging studies.

PubMed

Choi, Peter; Noguchi, Katsuya; Ishiyama, Munetaka; Denny, William A; Jose, Jiney

2018-05-03

This communication details the synthesis, evaluation of photophysical properties, and cellular imaging studies of cyanine chromophore based fluorescent dye 1 as a selective imaging agent for mitochondria. Copyright © 2018 Elsevier Ltd. All rights reserved.

Application of fluorescence spectroscopy and imaging in the detection of a photosensitizer in photodynamic therapy

NASA Astrophysics Data System (ADS)

Zang, Lixin; Zhao, Huimin; Zhang, Zhiguo; Cao, Wenwu

2017-02-01

Photodynamic therapy (PDT) is currently an advanced optical technology in medical applications. However, the application of PDT is limited by the detection of photosensitizers. This work focuses on the application of fluorescence spectroscopy and imaging in the detection of an effective photosenzitizer, hematoporphyrin monomethyl ether (HMME). Optical properties of HMME were measured and analyzed based on its absorption and fluorescence spectra. The production mechanism of its fluorescence emission was analyzed. The detection device for HMME based on fluorescence spectroscopy was designed. Ratiometric method was applied to eliminate the influence of intensity change of excitation sources, fluctuates of excitation sources and photo detectors, and background emissions. The detection limit of this device is 6 μg/L, and it was successfully applied to the diagnosis of the metabolism of HMME in the esophageal cancer cells. To overcome the limitation of the point measurement using fluorescence spectroscopy, a two-dimensional (2D) fluorescence imaging system was established. The algorithm of the 2D fluorescence imaging system is deduced according to the fluorescence ratiometric method using bandpass filters. The method of multiple pixel point addition (MPPA) was used to eliminate fluctuates of signals. Using the method of MPPA, SNR was improved by about 30 times. The detection limit of this imaging system is 1.9 μg/L. Our systems can be used in the detection of porphyrins to improve the PDT effect.

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells.

PubMed

Wang, Sheng; Chen, Xuanze; Chang, Lei; Ding, Miao; Xue, Ruiying; Duan, Haifeng; Sun, Yujie

2018-06-05

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

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.

Patch-based anisotropic diffusion scheme for fluorescence diffuse optical tomography--part 2: image reconstruction.

PubMed

Correia, Teresa; Koch, Maximilian; Ale, Angelique; Ntziachristos, Vasilis; Arridge, Simon

2016-02-21

Fluorescence diffuse optical tomography (fDOT) provides 3D images of fluorescence distributions in biological tissue, which represent molecular and cellular processes. The image reconstruction problem is highly ill-posed and requires regularisation techniques to stabilise and find meaningful solutions. Quadratic regularisation tends to either oversmooth or generate very noisy reconstructions, depending on the regularisation strength. Edge preserving methods, such as anisotropic diffusion regularisation (AD), can preserve important features in the fluorescence image and smooth out noise. However, AD has limited ability to distinguish an edge from noise. We propose a patch-based anisotropic diffusion regularisation (PAD), where regularisation strength is determined by a weighted average according to the similarity between patches around voxels within a search window, instead of a simple local neighbourhood strategy. However, this method has higher computational complexity and, hence, we wavelet compress the patches (PAD-WT) to speed it up, while simultaneously taking advantage of the denoising properties of wavelet thresholding. Furthermore, structural information can be incorporated into the image reconstruction with PAD-WT to improve image quality and resolution. In this case, the weights used to average voxels in the image are calculated using the structural image, instead of the fluorescence image. The regularisation strength depends on both structural and fluorescence images, which guarantees that the method can preserve fluorescence information even when it is not structurally visible in the anatomical images. In part 1, we tested the method using a denoising problem. Here, we use simulated and in vivo mouse fDOT data to assess the algorithm performance. Our results show that the proposed PAD-WT method provides high quality and noise free images, superior to those obtained using AD.

Self-Assembly of Electron Donor-Acceptor-Based Carbazole Derivatives: Novel Fluorescent Organic Nanoprobes for Both One- and Two-Photon Cellular Imaging.

PubMed

Zhang, Jinfeng; Chen, Wencheng; Kalytchuk, Sergii; Li, King Fai; Chen, Rui; Adachi, Chihaya; Chen, Zhan; Rogach, Andrey L; Zhu, Guangyu; Yu, Peter K N; Zhang, Wenjun; Cheah, Kok Wai; Zhang, Xiaohong; Lee, Chun-Sing

2016-05-11

In this study, we report fluorescent organic nanoprobes with intense blue, green, and orange-red emissions prepared by self-assembling three carbazole derivatives into nanorods/nanoparticles. The three compounds consist of two or four electron-donating carbazole groups linked to a central dicyanobenzene electron acceptor. Steric hindrance from the carbazole groups leads to noncoplanar 3D molecular structures favorable to fluorescence in the solid state, while the donor-acceptor structures endow the molecules with good two-photon excited emission properties. The fluorescent organic nanoprobes exhibit good water dispersibility, low cytotoxicity, superior resistance against photodegradation and photobleaching. Both one- and two-photon fluorescent imaging were shown in the A549 cell line. Two-photon fluorescence imaging with the fluorescent probes was demonstrated to be more effective in visualizing and distinguishing cellular details compared to conventional one-photon fluorescence imaging.

An off-on fluorescence probe targeting mitochondria based on oxidation-reduction response for tumor cell and tissue imaging

NASA Astrophysics Data System (ADS)

Yao, Hanchun; Cao, Li; Zhao, Weiwei; Zhang, Suge; Zeng, Man; Du, Bin

2017-10-01

In this study, a tumor-targeting poly( d, l-lactic-co-glycolic acid) (PLGA) loaded "off-on" fluorescent probe nanoparticle (PFN) delivery system was developed to evaluate the region of tumor by off-on fluorescence. The biodegradability of the nanosize PFN delivery system readily released the probe under tumor acidic conditions. The probe with good biocompatibility was used to monitor the intracellular glutathione (GSH) of cancer cells and selectively localize to mitochondria for tumor imaging. The incorporated tumor-targeting probe was based on the molecular photoinduced electron transfer (PET) mechanism preventing fluorescence ("off" state) and could be easily released under tumor acidic conditions. However, the released tumor-targeting fluorescence probe molecule was selective towards GSH with high selectivity and an ultra-sensitivity for the mitochondria of cancer cells and tissues significantly increasing the probe molecule fluorescence signal ("on" state). The tumor-targeting fluorescence probe showed sensitivity to GSH avoiding interference from cysteine and homocysteine. The PFNs could enable fluorescence-guided cancer imaging during cancer therapy. This work may expand the biological applications of PFNs as a diagnostic reagent, which will be beneficial for fundamental research in tumor imaging. [Figure not available: see fulltext.

Facile synthesis of Curcuma longa tuber powder engineered metal nanoparticles for bioimaging applications

NASA Astrophysics Data System (ADS)

Sankar, Renu; Rahman, Pattanathu K. S. M.; Varunkumar, Krishnamoorthy; Anusha, Chidambaram; Kalaiarasi, Arunachalam; Shivashangari, Kanchi Subramanian; Ravikumar, Vilwanathan

2017-02-01

Nanomaterials based fluorescent agents are rapidly becoming significant and promising transformative tools for improving medical diagnostics for extensive in vivo imaging modalities. Compared with conventional fluorescent agents, nano-fluorescence has capabilities to improve the in vivo detection and enriched targeting efficiencies. In our laboratory we synthesized fluorescent metal nanoparticles of silver, copper and iron using Curcuma longa tuber powder by simple reduction. The physicochemical properties of the synthesized metal nanoparticles were attained using UV-visible spectrophotometry, scanning electron microscopy with EDAX spectroscopy, dynamic light scattering, Fourier-transform infrared spectroscopy and X-ray diffraction. The Curcuma longa tuber powder has one of the bioactive compound Curcumin might act as a capping agent during the synthesis of nanoparticles. The synthesized metal nanoparticles fluorescence property was confirmed by spectrofluorometry. When compared with copper and iron nanoparticles the silver nanoparticles showed high fluorescence intensity under spectrofluorometry. Moreover, in vitro cell images of the silver nanoparticles in A549 cell lines also correlated with the results of spectrofluorometry. These silver nanoparticles show inspiring cell-imaging applications. They enter into cells without any further modifications, and the fluorescence property can be utilized for fluorescence-based cell imaging applications.

Compact whole-body fluorescent imaging of nude mice bearing EGFP expressing tumor

NASA Astrophysics Data System (ADS)

Chen, Yanping; Xiong, Tao; Chu, Jun; Yu, Li; Zeng, Shaoqun; Luo, Qingming

2005-01-01

Issue of tumor has been a hotspot of current medicine. It is important for tumor research to detect tumors bearing in animal models easily, fast, repetitively and noninvasivly. Many researchers have paid their increasing interests on the detecting. Some contrast agents, such as green fluorescent protein (GFP) and Discosoma red fluorescent protein (Dsred) were applied to enhance image quality. Three main kinds of imaging scheme were adopted to visualize fluorescent protein expressing tumors in vivo. These schemes based on fluorescence stereo microscope, cooled charge-coupled-device (CCD) or camera as imaging set, and laser or mercury lamp as excitation light source. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes. The authors set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a Kodak digital camera (model DC290), fluorescence filters(B and G2;HB Optical, Shenyang, Liaoning, P.R. China) and a mercury 50-W lamp power supply (U-LH50HG;Olympus Optical, Japan) as excitation light source. The EGFP was excited directly by mercury lamp with D455/70 nm band-pass filter and fluorescence was recorded by digital camera with 520nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For half a year our experiments suggested the imaging scheme was feasible. Whole-body fluorescence optical imaging for fluorescent expressing tumors in nude mouse is an ideal tool for antitumor, antimetastatic, and antiangiogenesis drug screening.

Fluorescence lifetime imaging ophthalmoscopy.

PubMed

Dysli, Chantal; Wolf, Sebastian; Berezin, Mikhail Y; Sauer, Lydia; Hammer, Martin; Zinkernagel, Martin S

2017-09-01

Imaging techniques based on retinal autofluorescence have found broad applications in ophthalmology because they are extremely sensitive and noninvasive. Conventional fundus autofluorescence imaging measures fluorescence intensity of endogenous retinal fluorophores. It mainly derives its signal from lipofuscin at the level of the retinal pigment epithelium. Fundus autofluorescence, however, can not only be characterized by the spatial distribution of the fluorescence intensity or emission spectrum, but also by a characteristic fluorescence lifetime function. The fluorescence lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo measurement of lifetimes of endogenous retinal fluorophores. Recent reports in this field have contributed to our understanding of the pathophysiology of various macular and retinal diseases. Within this review, the basic concept of fluorescence lifetime imaging is provided. It includes technical background information and correlation with in vitro measurements of individual retinal metabolites. In a second part, clinical applications of fluorescence lifetime imaging and fluorescence lifetime features of selected retinal diseases such as Stargardt disease, age-related macular degeneration, choroideremia, central serous chorioretinopathy, macular holes, diabetic retinopathy, and retinal artery occlusion are discussed. Potential areas of use for fluorescence lifetime imaging ophthalmoscopy will be outlined at the end of this review. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Near-Infrared Squaraine Dye Encapsulated Micelles for in Vivo Fluorescence and Photoacoustic Bimodal Imaging.

PubMed

Sreejith, Sivaramapanicker; Joseph, James; Lin, Manjing; Menon, Nishanth Venugopal; Borah, Parijat; Ng, Hao Jun; Loong, Yun Xian; Kang, Yuejun; Yu, Sidney Wing-Kwong; Zhao, Yanli

2015-06-23

Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1(micelle)) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1(micelle) exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1(micelle) in the NIR window of 800-900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1(micelle), as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1(micelle) proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging.

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.

Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions

NASA Astrophysics Data System (ADS)

Sharifzadeh, Mohsen; Bernstein, Paul S.; Gellermann, Werner

2006-10-01

We have developed a CCD-camera-based nonmydriatic instrument that detects fluorescence from retinal lipofuscin chromophores ("autofluorescence") as a means to indirectly quantify and spatially image the distribution of macular pigment (MP). The lipofuscin fluorescence intensity is reduced at all retinal locations containing MP, since MP has a competing absorption in the blue-green wavelength region. Projecting a large diameter, 488 nm excitation spot onto the retina, centered on the fovea, but extending into the macular periphery, and comparing lipofuscin fluorescence intensities outside and inside the foveal area, it is possible to spatially map out the distribution of MP. Spectrally selective detection of the lipofuscin fluorescence reveals an important wavelength dependence of the obtainable image contrast and deduced MP optical density levels, showing that it is important to block out interfering fluorescence contributions in the detection setup originating from ocular media such as the lens. Measuring 70 healthy human volunteer subjects with no ocular pathologies, we find widely varying spatial extent of MP, distinctly differing distribution patterns of MP, and strongly differing absolute MP levels among individuals. Our population study suggests that MP imaging based on lipofuscin fluorescence is useful as a relatively simple, objective, and quantitative noninvasive optical technique suitable to rapidly screen MP levels and distributions in healthy humans with undilated pupils.

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

Shcheslavskiy, V. I.; Institute of Biomedical Technologies, Nizhny Novgorod State Medical Academy, Minin and Pozharsky Square, 10/1, Nizhny Novgorod 603005; Neubauer, A.

We present a lifetime imaging technique that simultaneously records the fluorescence and phosphorescence lifetime images in confocal laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional time-correlated single photon counting board. We demonstrate our technique on the recording of the fluorescence/phosphorescence lifetime images of human embryonic kidney cells at different environmental conditions.

Photocontrollable Fluorescent Proteins for Superresolution Imaging

PubMed Central

Shcherbakova, Daria M.; Sengupta, Prabuddha; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V.

2014-01-01

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization–based and nonlinear ensemble–based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine. PMID:24895855

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).

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

PubMed

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

2017-04-01

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

Non-invasive Photoacoustic and Fluorescence Sentinel Lymph Node Identification using Dye-loaded Perfluorocarbon Nanoparticles

PubMed Central

Akers, Walter J.; Kim, Chulhong; Berezin, Mikhail; Guo, Kevin; Fuhrhop, Ralph; Lanza, Gregory M.; Fischer, Georg M.; Daltrozzo, Ewald; Zumbusch, Andreas; Cai, Xin; Wang, Lihong V.; Achilefu, Samuel

2010-01-01

The contrast mechanisms used for photoacoustic tomography (PAT) and fluorescence imaging differ in subtle but significant ways. Design of contrast agents for each or both modalities requires an understanding of the spectral characteristics as well as intra- and intermolecular interactions that occur during formulation. We found that fluorescence quenching that occurs in the formulation of near infrared (NIR) fluorescent dyes in nanoparticles results in enhanced contrast for PAT. The ability of the new PAT method to utilize strongly absorbing chromophores for signal generation allowed us to convert a highly fluorescent dye into an exceptionally high PA contrast material. Spectroscopic characterization of the developed NIR dye-loaded perfluorocarbon-based nanoparticles for combined fluorescence and PA imaging revealed distinct dye-dependent photophysical behavior. We demonstrate that the enhanced contrast allows detection of regional lymph nodes of rats in vivo with time-domain optical and photoacoustic imaging methods. The results further show that the use of fluorescence lifetime (FLT) imaging, which is less dependent on fluorescence intensity, provides a strategic approach to bridge the disparate contrast reporting mechanisms of fluorescence and PA imaging methods. PMID:21171567

Optimisation of fluorescence guidance during robot-assisted laparoscopic sentinel node biopsy for prostate cancer.

PubMed

KleinJan, Gijs H; van den Berg, Nynke S; Brouwer, Oscar R; de Jong, Jeroen; Acar, Cenk; Wit, Esther M; Vegt, Erik; van der Noort, Vincent; Valdés Olmos, Renato A; van Leeuwen, Fijs W B; van der Poel, Henk G

2014-12-01

The hybrid tracer was introduced to complement intraoperative radiotracing towards the sentinel nodes (SNs) with fluorescence guidance. Improve in vivo fluorescence-based SN identification for prostate cancer by optimising hybrid tracer preparation, injection technique, and fluorescence imaging hardware. Forty patients with a Briganti nomogram-based risk >10% of lymph node (LN) metastases were included. After intraprostatic tracer injection, SN mapping was performed (lymphoscintigraphy and single-photon emission computed tomography with computed tomography (SPECT-CT)). In groups 1 and 2, SNs were pursued intraoperatively using a laparoscopic gamma probe followed by fluorescence imaging (FI). In group 3, SNs were initially located via FI. Compared with group 1, in groups 2 and 3, a new tracer formulation was introduced that had a reduced total injected volume (2.0 ml vs. 3.2 ml) but increased particle concentration. For groups 1 and 2, the Tricam SLII with D-Light C laparoscopic FI (LFI) system was used. In group 3, the LFI system was upgraded to an Image 1 HUB HD with D-Light P system. Hybrid tracer-based SN biopsy, extended pelvic lymph node dissection, and robot-assisted radical prostatectomy. Number and location of the preoperatively identified SNs, in vivo fluorescence-based SN identification rate, tumour status of SNs and LNs, postoperative complications, and biochemical recurrence (BCR). Mean fluorescence-based SN identification improved from 63.7% (group 1) to 85.2% and 93.5% for groups 2 and 3, respectively (p=0.012). No differences in postoperative complications were found. BCR occurred in three pN0 patients. Stepwise optimisation of the hybrid tracer formulation and the LFI system led to a significant improvement in fluorescence-assisted SN identification. Preoperative SPECT-CT remained essential for guiding intraoperative SN localisation. Intraoperative fluorescence-based SN visualisation can be improved by enhancing the hybrid tracer formulation and laparoscopic fluorescence imaging system. Copyright © 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.

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

PubMed Central

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

1989-01-01

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

A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices

PubMed Central

Abdelfattah, Ahmed S.; Farhi, Samouil L.; Zhao, Yongxin; Brinks, Daan; Zou, Peng; Ruangkittisakul, Araya; Platisa, Jelena; Pieribone, Vincent A.; Ballanyi, Klaus; Cohen, Adam E.

2016-01-01

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation. SIGNIFICANCE STATEMENT Fluorescent-protein-based voltage indicators enable imaging of the electrical activity of many genetically targeted neurons with high spatial and temporal resolution. Here, we describe the engineering of a bright red fluorescent protein-based voltage indicator designated as FlicR1 (fluorescent indicator for voltage imaging red). FlicR1 has sufficient speed and sensitivity to report single action potentials and voltage fluctuations at frequencies up to 100 Hz in single-trial recordings with wide-field microscopy. Because it is excitable with yellow light, FlicR1 can be used in conjunction with blue-light-activated optogenetic actuators. However, spatially distinct patterns of optogenetic activation and voltage imaging are required to avoid fluorescence artifacts due to photoactivation of the FlicR1 chromophore. PMID:26911693

Ultra-sensitive fluorescent imaging-biosensing using biological photonic crystals

NASA Astrophysics Data System (ADS)

Squire, Kenny; Kong, Xianming; Wu, Bo; Rorrer, Gregory; Wang, Alan X.

2018-02-01

Optical biosensing is a growing area of research known for its low limits of detection. Among optical sensing techniques, fluorescence detection is among the most established and prevalent. Fluorescence imaging is an optical biosensing modality that exploits the sensitivity of fluorescence in an easy-to-use process. Fluorescence imaging allows a user to place a sample on a sensor and use an imager, such as a camera, to collect the results. The image can then be processed to determine the presence of the analyte. Fluorescence imaging is appealing because it can be performed with as little as a light source, a camera and a data processor thus being ideal for nontrained personnel without any expensive equipment. Fluorescence imaging sensors generally employ an immunoassay procedure to selectively trap analytes such as antigens or antibodies. When the analyte is present, the sensor fluoresces thus transducing the chemical reaction into an optical signal capable of imaging. Enhancement of this fluorescence leads to an enhancement in the detection capabilities of the sensor. Diatoms are unicellular algae with a biosilica shell called a frustule. The frustule is porous with periodic nanopores making them biological photonic crystals. Additionally, the porous nature of the frustule allows for large surface area capable of multiple analyte binding sites. In this paper, we fabricate a diatom based ultra-sensitive fluorescence imaging biosensor capable of detecting the antibody mouse immunoglobulin down to a concentration of 1 nM. The measured signal has an enhancement of 6× when compared to sensors fabricated without diatoms.

Near-infrared fluorescence goggle system with complementary metal–oxide–semiconductor imaging sensor and see-through display

PubMed Central

Liu, Yang; Njuguna, Raphael; Matthews, Thomas; Akers, Walter J.; Sudlow, Gail P.; Mondal, Suman; Tang, Rui

2013-01-01

Abstract. We have developed a near-infrared (NIR) fluorescence goggle system based on the complementary metal–oxide–semiconductor active pixel sensor imaging and see-through display technologies. The fluorescence goggle system is a compact wearable intraoperative fluorescence imaging and display system that can guide surgery in real time. The goggle is capable of detecting fluorescence of indocyanine green solution in the picomolar range. Aided by NIR quantum dots, we successfully used the fluorescence goggle to guide sentinel lymph node mapping in a rat model. We further demonstrated the feasibility of using the fluorescence goggle in guiding surgical resection of breast cancer metastases in the liver in conjunction with NIR fluorescent probes. These results illustrate the diverse potential use of the goggle system in surgical procedures. PMID:23728180

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.

Applications of two-photon fluorescence microscopy in deep-tissue imaging

NASA Astrophysics Data System (ADS)

Dong, Chen-Yuan; Yu, Betty; Hsu, Lily L.; Kaplan, Peter D.; Blankschstein, D.; Langer, Robert; So, Peter T. C.

2000-07-01

Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

Near-infrared imaging of face transplants: are both pedicles necessary?

PubMed

Nguyen, John T; Ashitate, Yoshitomo; Venugopal, Vivek; Neacsu, Florin; Kettenring, Frank; Frangioni, John V; Gioux, Sylvain; Lee, Bernard T

2013-09-01

Facial transplantation is a complex procedure that corrects severe facial defects due to traumas, burns, and congenital disorders. Although face transplantation has been successfully performed clinically, potential risks include tissue ischemia and necrosis. The vascular supply is typically based on the bilateral neck vessels. As it remains unclear whether perfusion can be based off a single pedicle, this study was designed to assess perfusion patterns of facial transplant allografts using near-infrared (NIR) fluorescence imaging. Upper facial composite tissue allotransplants were created using both carotid artery and external jugular vein pedicles in Yorkshire pigs. A flap validation model was created in n = 2 pigs and a clamp occlusion model was performed in n = 3 pigs. In the clamp occlusion models, sequential clamping of the vessels was performed to assess perfusion. Animals were injected with indocyanine green and imaged with NIR fluorescence. Quantitative metrics were assessed based on fluorescence intensity. With NIR imaging, arterial perforators emitted fluorescence indicating perfusion along the surface of the skin. Isolated clamping of one vascular pedicle showed successful perfusion across the midline based on NIR fluorescence imaging. This perfusion extended into the facial allograft within 60 s and perfused the entire contralateral side within 5 min. Determination of vascular perfusion is important in microsurgical constructs as complications can lead to flap loss. It is still unclear if facial transplants require both pedicles. This initial pilot study using intraoperative NIR fluorescence imaging suggests that facial flap models can be adequately perfused from a single pedicle. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Multispectral Fluorescence Imaging During Robot-assisted Laparoscopic Sentinel Node Biopsy: A First Step Towards a Fluorescence-based Anatomic Roadmap.

PubMed

van den Berg, Nynke S; Buckle, Tessa; KleinJan, Gijs H; van der Poel, Henk G; van Leeuwen, Fijs W B

2017-07-01

During (robot-assisted) sentinel node (SN) biopsy procedures, intraoperative fluorescence imaging can be used to enhance radioguided SN excision. For this combined pre- and intraoperative SN identification was realized using the hybrid SN tracer, indocyanine green- 99m Tc-nanocolloid. Combining this dedicated SN tracer with a lymphangiographic tracer such as fluorescein may further enhance the accuracy of SN biopsy. Clinical evaluation of a multispectral fluorescence guided surgery approach using the dedicated SN tracer ICG- 99m Tc-nanocolloid, the lymphangiographic tracer fluorescein, and a commercially available fluorescence laparoscope. Pilot study in ten patients with prostate cancer. Following ICG- 99m Tc-nanocolloid administration and preoperative lymphoscintigraphy and single-photon emission computed tomograpy imaging, the number and location of SNs were determined. Fluorescein was injected intraprostatically immediately after the patient was anesthetized. A multispectral fluorescence laparoscope was used intraoperatively to identify both fluorescent signatures. Multispectral fluorescence imaging during robot-assisted radical prostatectomy with extended pelvic lymph node dissection and SN biopsy. (1) Number and location of preoperatively identified SNs. (2) Number and location of SNs intraoperatively identified via ICG- 99m Tc-nanocolloid imaging. (3) Rate of intraoperative lymphatic duct identification via fluorescein imaging. (4) Tumor status of excised (sentinel) lymph node(s). (5) Postoperative complications and follow-up. Near-infrared fluorescence imaging of ICG- 99m Tc-nanocolloid visualized 85.3% of the SNs. In 8/10 patients, fluorescein imaging allowed bright and accurate identification of lymphatic ducts, although higher background staining and tracer washout were observed. The main limitation is the small patient population. Our findings indicate that a lymphangiographic tracer can provide additional information during SN biopsy based on ICG- 99m Tc-nanocolloid. The study suggests that multispectral fluorescence image-guided surgery is clinically feasible. We evaluated the concept of surgical fluorescence guidance using differently colored dyes that visualize complementary features. In the future this concept may provide better guidance towards diseased tissue while sparing healthy tissue, and could thus improve functional and oncologic outcomes. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Compressive hyperspectral time-resolved wide-field fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Pian, Qi; Yao, Ruoyang; Sinsuebphon, Nattawut; Intes, Xavier

2017-07-01

Spectrally resolved fluorescence lifetime imaging and spatial multiplexing have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (∼40 ps temporal resolution) using fewer than N2 optical measurements. We demonstrate the potential of this new imaging platform by quantitatively imaging near-infrared (NIR) Förster resonance energy transfer (FRET) both in vitro and in vivo. The technique is well suited for quantitative hyperspectral lifetime imaging with a high sensitivity and paves the way for many important biomedical applications.

Spatially selective photonic crystal enhanced fluorescence and application to background reduction for biomolecule detection assays

PubMed Central

Chaudhery, Vikram; Huang, Cheng-Sheng; Pokhriyal, Anusha; Polans, James; Cunningham, Brian T.

2011-01-01

By combining photonic crystal label-free biosensor imaging with photonic crystal enhanced fluorescence, it is possible to selectively enhance the fluorescence emission from regions of the PC surface based upon the density of immobilized capture molecules. A label-free image of the capture molecules enables determination of optimal coupling conditions of the laser used for fluorescence imaging of the photonic crystal surface on a pixel-by-pixel basis, allowing maximization of fluorescence enhancement factor from regions incorporating a biomolecule capture spot and minimization of background autofluorescence from areas between capture spots. This capability significantly improves the contrast of enhanced fluorescent images, and when applied to an antibody protein microarray, provides a substantial advantage over conventional fluorescence microscopy. Using the new approach, we demonstrate detection limits as low as 0.97 pg/ml for a representative protein biomarker in buffer. PMID:22109210

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

Spatially selective photonic crystal enhanced fluorescence and application to background reduction for biomolecule detection assays.

PubMed

Chaudhery, Vikram; Huang, Cheng-Sheng; Pokhriyal, Anusha; Polans, James; Cunningham, Brian T

2011-11-07

By combining photonic crystal label-free biosensor imaging with photonic crystal enhanced fluorescence, it is possible to selectively enhance the fluorescence emission from regions of the PC surface based upon the density of immobilized capture molecules. A label-free image of the capture molecules enables determination of optimal coupling conditions of the laser used for fluorescence imaging of the photonic crystal surface on a pixel-by-pixel basis, allowing maximization of fluorescence enhancement factor from regions incorporating a biomolecule capture spot and minimization of background autofluorescence from areas between capture spots. This capability significantly improves the contrast of enhanced fluorescent images, and when applied to an antibody protein microarray, provides a substantial advantage over conventional fluorescence microscopy. Using the new approach, we demonstrate detection limits as low as 0.97 pg/ml for a representative protein biomarker in buffer.

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

PubMed

Jiang, Shihong; Walker, John

2010-01-20

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

Chip-scale fluorescence microscope based on a silo-filter complementary metal-oxide semiconductor image sensor.

PubMed

Ah Lee, Seung; Ou, Xiaoze; Lee, J Eugene; Yang, Changhuei

2013-06-01

We demonstrate a silo-filter (SF) complementary metal-oxide semiconductor (CMOS) image sensor for a chip-scale fluorescence microscope. The extruded pixel design with metal walls between neighboring pixels guides fluorescence emission through the thick absorptive filter to the photodiode of a pixel. Our prototype device achieves 13 μm resolution over a wide field of view (4.8 mm × 4.4 mm). We demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

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.

High-contrast fluorescence imaging based on the polarization dependence of the fluorescence enhancement using an optical interference mirror slide.

PubMed

Yasuda, Mitsuru; Akimoto, Takuo

2015-01-01

High-contrast fluorescence imaging using an optical interference mirror (OIM) slide that enhances the fluorescence from a fluorophore located on top of the OIM surface is reported. To enhance the fluorescence and reduce the background light of the OIM, transverse-electric-polarized excitation light was used as incident light, and the transverse-magnetic-polarized fluorescence signal was detected. As a result, an approximate 100-fold improvement in the signal-to-noise ratio was achieved through a 13-fold enhancement of the fluorescence signal and an 8-fold reduction of the background light.

Electron beam dispersion measurements in nitrogen using two-dimensional imaging of N2(+) fluorescence

NASA Technical Reports Server (NTRS)

Clapp, L. H.; Twiss, R. G.; Cattolica, R. J.

1991-01-01

Experimental results are presented related to the radial spread of fluorescence excited by 10 and 20 KeV electron beams passing through nonflowing rarefied nitrogen at 293 K. An imaging technique for obtaining species distributions from measured beam-excited fluorescence is described, based on a signal inversion scheme mathematically equivalent to the inversion of the Abel integral equation. From fluorescence image data, measurements of beam radius, integrated signal intensity, and spatially resolved distributions of N2(+) first-negative-band fluorescence-emitting species have been made. Data are compared with earlier measurements and with an heuristic beam spread model.

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

Cutaneous porphyrins exhibit anti-stokes fluorescence that is detectable in sebum (Conference Presentation)

NASA Astrophysics Data System (ADS)

Tian, Giselle; Zeng, Haishan; Zhao, Jianhua; Wu, Zhenguo; Al Jasser, Mohammed; Lui, Harvey; Mclean, David I.

2016-02-01

Porphyrins produced by Propionibacterium acnes represent the principal fluorophore associated with acne, and appear as orange-red luminescence under the Wood's lamp. Assessment of acne based on Wood's lamp (UV) or visible light illumination is limited by photon penetration depth and has limited sensitivity for earlier stage lesions. Inducing fluorescence with near infrared (NIR) excitation may provide an alternative way to assess porphyrin-related skin disorders. We discovered that under 785 nm CW laser excitation PpIX powder exhibits fluorescence emission in the shorter wavelength range of 600-715 nm with an intensity that is linearly dependent on the excitation power. We attribute this shorter wavelength emission to anti-Stokes fluorescence. Similar anti-Stokes fluorescence was also detected focally in all skin-derived samples containing porphyrins. Regular (Stokes) fluorescence was present under UV and visible light excitation on ex vivo nasal skin and sebum from uninflamed acne, but not on nose surface smears or sebum from inflamed acne. Co-registered CW laser-excited anti-Stokes fluorescence and fs laser-excited multi-photon fluorescence images of PpIX powder showed similar features. In the skin samples because of the anti-Stokes effect, the NIR-induced fluorescence was presumably specific for porphyrins since there appeared to be no anti-Stokes emission signals from other typical skin fluorophores such as lipids, keratins and collagen. Anti-Stokes fluorescence under NIR CW excitation is more sensitive and specific for porphyrin detection than UV- or visible light-excited regular fluorescence and fs laser-excited multi-photon fluorescence. This approach also has higher image contrast compared to NIR fs laser-based multi-photon fluorescence imaging. The anti-Stokes fluorescence of porphyrins within sebum could potentially be applied to detecting and targeting acne lesions for treatment via fluorescence image guidance.

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells

NASA Astrophysics Data System (ADS)

Li, Ke; Zhang, Zhi-Ping; Luo, Ming; Yu, Xiang; Han, Yu; Wei, Hong-Ping; Cui, Zong-Qiang; Zhang, Xian-En

2011-12-01

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging αvβ3 integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging αvβ3 integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging. Electronic supplementary information (ESI) available. See DOI: 10.1039/c1nr11132a

CMOS image sensor-based implantable glucose sensor using glucose-responsive fluorescent hydrogel.

PubMed

Tokuda, Takashi; Takahashi, Masayuki; Uejima, Kazuhiro; Masuda, Keita; Kawamura, Toshikazu; Ohta, Yasumi; Motoyama, Mayumi; Noda, Toshihiko; Sasagawa, Kiyotaka; Okitsu, Teru; Takeuchi, Shoji; Ohta, Jun

2014-11-01

A CMOS image sensor-based implantable glucose sensor based on an optical-sensing scheme is proposed and experimentally verified. A glucose-responsive fluorescent hydrogel is used as the mediator in the measurement scheme. The wired implantable glucose sensor was realized by integrating a CMOS image sensor, hydrogel, UV light emitting diodes, and an optical filter on a flexible polyimide substrate. Feasibility of the glucose sensor was verified by both in vitro and in vivo experiments.

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

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.

Fluorescence-based enhanced reality (FLER) for real-time estimation of bowel perfusion in minimally invasive surgery

NASA Astrophysics Data System (ADS)

Diana, Michele

2016-03-01

Pre-anastomotic bowel perfusion is a key factor for a successful healing process. Clinical judgment has limited accuracy to evaluate intestinal microperfusion. Fluorescence videography is a promising tool for image-guided intraoperative assessment of the bowel perfusion at the future anastomotic site in the setting of minimally invasive procedures. The standard configuration for fluorescence videography includes a Near-Infrared endoscope able to detect the signal emitted by a fluorescent dye, more frequently Indocyanine Green (ICG), which is administered by intravenous injection. Fluorescence intensity is proportional to the amount of fluorescent dye diffusing in the tissue and consequently is a surrogate marker of tissue perfusion. However, fluorescence intensity alone remains a subjective approach and an integrated computer-based analysis of the over-time evolution of the fluorescence signal is required to obtain quantitative data. We have developed a solution integrating computer-based analysis for intra-operative evaluation of the optimal resection site, based on the bowel perfusion as determined by the dynamic fluorescence intensity. The software can generate a "virtual perfusion cartography", based on the "fluorescence time-to-peak". The virtual perfusion cartography can be overlapped onto real-time laparoscopic images to obtain the Enhanced Reality effect. We have defined this approach FLuorescence-based Enhanced Reality (FLER). This manuscript describes the stepwise development of the FLER concept.

Development of a QDots 800 based fluorescent solid phantom for validation of NIRF imaging platforms

NASA Astrophysics Data System (ADS)

Zhu, Banghe; Sevick-Muraca, Eva M.

2013-02-01

Over the past decade, we developed near-infrared fluorescence (NIRF) devices for non-invasive lymphatic imaging using microdosages of ICG in humans and for detection of lymph node metastasis in animal models mimicking metastatic human prostate cancer. To validate imaging, a NIST traceable phantom is needed so that developed "first-inhumans" drugs may be used with different luorescent imaging platforms. In this work, we developed a QDots 800 based fluorescent solid phantom for installation and operational qualification of clinical and preclinical, NIRF imaging devices. Due to its optical clearance, polyurethane was chosen as the base material. Titanium dioxide was used as the scattering agent because of its miscibility in polyurethane. QDots 800 was chosen owing to its stability and NIR emission spectra. A first phantom was constructed for evaluation of the noise floor arising from excitation light leakage, a phenomenon that can be minimized during engineering and design of fluorescent imaging systems. A second set of phantoms were constructed to enable quantification of device sensitivity associated with our preclinical and clinical devices. The phantoms have been successfully applied for installation and operational qualification of our preclinical and clinical devices. Assessment of excitation light leakage provides a figure of merit for "noise floor" and imaging sensitivity can be used to benchmark devices for specific imaging agents.

Synthesis and optical properties of water-soluble biperylene-based dendrimers.

PubMed

Shao, Pin; Jia, Ningyang; Zhang, Shaojuan; Bai, Mingfeng

2014-05-30

We report the synthesis and photophysical properties of three biperylene-based dendrimers, which show red fluorescence in water. A fluorescence microscopy study demonstrated uptake of biperylene-based dendrimers in living cells. Our results indicate that these biperylene-based dendrimers are promising candidates in fluorescence imaging applications with the potential as therapeutic carriers.

Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

PubMed

Agrawalla, Bikram Keshari; Chandran, Yogeswari; Phue, Wut-Hmone; Lee, Sung-Chan; Jeong, Yun-Mi; Wan, Si Yan Diana; Kang, Nam-Young; Chang, Young-Tae

2015-04-29

Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

Multifunctional fluorescent and magnetic nanoparticles for biomedical applications

NASA Astrophysics Data System (ADS)

Selvan, Subramanian T.

2012-03-01

Hybrid multifunctional nanoparticles (NPs) are emerging as useful probes for magnetic based targeting, delivery, cell separation, magnetic resonance imaging (MRI), and fluorescence-based bio-labeling applications. Assessing from the literature, the development of multifunctional NPs for multimodality imaging is still in its infancy state. This report focuses on our recent work on quantum dots (QDs), magnetic NPs (MNPs) and bi-functional NPs (composed of either QDs or rare-earth NPs, and magnetic NPs - iron oxide or gadolinium oxide) for multimodality imaging based biomedical applications. The combination of MRI and fluorescence would ally each other in improving the sensitivity and resolution, resulting in improved and early diagnosis of the disease. The challenges in this area are discussed.

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 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.

A Dual Modality System for Simultaneous Fluorescence and Positron Emission Tomography Imaging of Small Animals

NASA Astrophysics Data System (ADS)

Liu, Shuangquan; Zhang, Bin; Wang, Xin; Li, Lin; Chen, Yan; Liu, Xin; Liu, Fei; Shan, Baoci; Bai, Jing

2011-02-01

A dual-modality imaging system for simultaneous fluorescence molecular tomography (FMT) and positron emission tomography (PET) of small animals has been developed. The system consists of a noncontact 360°-projection FMT module and a flat panel detector pair based PET module, which are mounted orthogonally for the sake of eliminating cross interference. The FMT images and PET data are simultaneously acquired by employing dynamic sampling mode. Phantom experiments, in which the localization and range of radioactive and fluorescence probes are exactly indicated, have been carried out to verify the feasibility of the system. An experimental tumor-bearing mouse is also scanned using the dual-modality simultaneous imaging system, the preliminary fluorescence tomographic images and PET images demonstrate the in vivo performance of the presented dual-modality system.

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.

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow.

PubMed

Wang, Sheng; Ding, Miao; Xue, Boxin; Hou, Yingping; Sun, Yujie

2018-05-18

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

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

Molecular engineering of two-photon fluorescent probes for bioimaging applications

NASA Astrophysics Data System (ADS)

Liu, Hong-Wen; Liu, Yongchao; Wang, Peng; Zhang, Xiao-Bing

2017-03-01

During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

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.

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.

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.

Enhancing analysis of cells and proteins by fluorescence imaging on silk-based biomaterials: modulating the autofluorescence of silk.

PubMed

Neo, Puay Yong; Tan, Daryl Jian-An; Shi, Pujiang; Toh, Siew Lok; Goh, James Cho-Hong

2015-02-01

Silk is a versatile and established biomaterial for various tissue engineering purposes. However, it also exhibits strong autofluorescence signals-thereby hindering fluorescence imaging analysis of cells and proteins on silk-derived biomaterials. Sudan Black B (SB) is a lysochrome dye commonly used to stain lipids in histology. It has also been reported to be able to quench autofluorescence of tissues in histology and has been tested on artificial biomedical polymers in recent years. It was hypothesized that SB would exert similar quenching effects on silk, modulating the autofluorescence signals, and thereby enabling improved imaging analysis of cells and molecules of interests. The quenching effect of SB on the intrinsic fluorescence properties of silk and on commercial fluorescent dyes were first investigated in this study. SB was then incorporated into typical fluorescence-based staining protocols to study its effectiveness in improving fluorescence-based imaging of the cells and proteins residing with the silk-based biomaterials. Silk processed into various forms of biomaterials (e.g., films, sponges, fibers, and electrospun mats) was seeded with cells and cultured in vitro. At sacrificial time points, specimens were harvested, fixed, and prepared for fluorescence staining. SB, available commercially as a powder, was dissolved in 70% ethanol (0.3% [w/v]) to form staining solutions. SB treatment was introduced at the last step of typical immunofluorescence staining protocols for 15-120 min. For actin staining protocols by phalloidin toxin, SB staining solutions were added before and after permeabilization with Triton-X for 15-30 min. Results showed that ideal SB treatment duration is about 15 min. Apart from being able to suppress the autofluorescence of silk, this treatment duration was also not too long to adversely affect the fluorescent labeling probes used. The relative improvement brought about by SB treatment was most evident in the blue and green emission wavelengths compared with the red emission wavelength. This study has showed that the use of SB is a cost and time effective approach to enhance fluorescence-based imaging analyses of cell-seeded silk biomaterials, which otherwise would have been hindered by the unmodulated autofluorescence signals.

Intracellular distribution and stability of a luminescent rhenium(i) tricarbonyl tetrazolato complex using epifluorescence microscopy in conjunction with X-ray fluorescence imaging.

PubMed

Wedding, J L; Harris, H H; Bader, C A; Plush, S E; Mak, R; Massi, M; Brooks, D A; Lai, B; Vogt, S; Werrett, M V; Simpson, P V; Skelton, B W; Stagni, S

2017-04-19

Optical epifluorescence microscopy was used in conjunction with X-ray fluorescence imaging to monitor the stability and intracellular distribution of the luminescent rhenium(i) complex fac-[Re(CO) 3 (phen)L], where phen = 1,10-phenathroline and L = 5-(4-iodophenyl)tetrazolato, in 22Rv1 cells. The rhenium complex showed no signs of ancillary ligand dissociation, a conclusion based on data obtained via X-ray fluorescence imaging aligning iodine and rhenium distributions. A diffuse reticular localisation was detected for the complex in the nuclear/perinuclear region of cells, by either optical or X-ray fluorescence imaging techniques. X-ray fluorescence also showed that the rhenium complex disrupted the homeostasis of some biologically relevant elements, such as chlorine, potassium and zinc.

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.

Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast.

PubMed

Kao, Ya-Ting; Zhu, Xinxin; Xu, Fang; Min, Wei

2012-08-01

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.

Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast

PubMed Central

Kao, Ya-Ting; Zhu, Xinxin; Xu, Fang; Min, Wei

2012-01-01

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes. PMID:22876358

CMOS image sensor-based implantable glucose sensor using glucose-responsive fluorescent hydrogel

PubMed Central

Tokuda, Takashi; Takahashi, Masayuki; Uejima, Kazuhiro; Masuda, Keita; Kawamura, Toshikazu; Ohta, Yasumi; Motoyama, Mayumi; Noda, Toshihiko; Sasagawa, Kiyotaka; Okitsu, Teru; Takeuchi, Shoji; Ohta, Jun

2014-01-01

A CMOS image sensor-based implantable glucose sensor based on an optical-sensing scheme is proposed and experimentally verified. A glucose-responsive fluorescent hydrogel is used as the mediator in the measurement scheme. The wired implantable glucose sensor was realized by integrating a CMOS image sensor, hydrogel, UV light emitting diodes, and an optical filter on a flexible polyimide substrate. Feasibility of the glucose sensor was verified by both in vitro and in vivo experiments. PMID:25426316

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.

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

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.

Mueller matrix signature in advanced fluorescence microscopy imaging

NASA Astrophysics Data System (ADS)

Mazumder, Nirmal; Qiu, Jianjun; Kao, Fu-Jen; Diaspro, Alberto

2017-02-01

We have demonstrated the measurement and characterization of the polarization properties of a fluorescence signal using four-channel photon counting based Stokes-Mueller polarization microscopy. Thus, Lu-Chipman decomposition was applied to extract the critical polarization properties such as depolarization, linear retardance and the optical rotation of collagen type I fiber. We observed the spatial distribution of anisotropic and helical molecules of collagen from the reconstructed 2D Mueller images based on the fluorescence signal in a pixel-by-pixel manner.

Organic Dots Based on AIEgens for Two-Photon Fluorescence Bioimaging.

PubMed

Lou, Xiaoding; Zhao, Zujin; Tang, Ben Zhong

2016-12-01

Two-photon fluorescence imaging technique is a powerful bioanalytical approach in terms of high photostability, low photodamage, high spatiotemporal resolution. Recently, fluorescent organic dots comprised of organic emissive cores and a polymeric matrix are emerging as promising contrast reagents for two-photon fluorescence imaging, owing to their numerous merits of high and tunable fluorescence, good biocompatibility, strong photobleaching resistance, and multiple surface functionality. The emissive core is crucial for organic dots to get high brightness but many conventional chromophores often encounter a severe problem of fluorescence quenching when they form aggregates. To solve this problem, fluorogens featuring aggregation-induced emission (AIE) can fluoresce strongly in aggregates, and thus become ideal candidates for fluorescent organic dots. In addition, two-photon absorption property of the dots can be readily improved by just increase loading contents of AIE fluorogen (AIEgen). Hence, organic dots based on AIEgens have exhibited excellent performances in two-photon fluorescence in vitro cellular imaging, and in vivo vascular architecture visualization of mouse skin, muscle, brain and skull bone. In view of the rapid advances in this important research field, here, we highlight representative fluorescent organic dots with an emissive core of AIEgen aggregate, and discuss their great potential in bioimaging applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Deep-red to near-infrared fluorescent dyes: Synthesis, photophysical properties, and application in cell imaging

NASA Astrophysics Data System (ADS)

Li, Qi; Liu, Weimin; Wu, Jiasheng; Zhou, Bingjiang; Niu, Guangle; Zhang, Hongyan; Ge, Jiechao; Wang, Pengfei

2016-07-01

More and more attention has been paid to the design of new fluorescent imaging agents with good photostability and water solubility, especially those with emissions in the deep-red and near-infrared regions. In this work, we designed and synthesized four novel fluorescent dyes with deep-red or NIR fluorescence by hybridizing coumarin and pyronin moieties based on our previous work. Introduction of carboxylic acid in the dyes not only imparted the dyes with water solubility but also provided a versatile sensing platform for designing the fluorescent probes and sensors of biomolecules. The photophysical properties of these new dyes were investigated through absorption and fluorescence spectroscopy. Cell imaging experiments showed that esterification products could selectively stain lysosomes with good photostability, thereby indicating that they could be useful in the development of fluorescent probes for bioimaging.

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

NASA Astrophysics Data System (ADS)

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-03-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

Red Fluorescent Carbon Nanoparticle-Based Cell Imaging Probe.

PubMed

Ali, Haydar; Bhunia, Susanta Kumar; Dalal, Chumki; Jana, Nikhil R

2016-04-13

Fluorescent carbon nanoparticle-based probes with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. However, synthesis of red fluorescent carbon nanoparticles and their transformation into functional nanoparticles are very challenging. Here we report red fluorescent carbon nanoparticle-based nanobioconjugates of <25 nm hydrodynamic size and their application as fluorescent cell labels. Hydrophobic carbon nanoparticles are synthesized via high temperature colloid-chemical approach and transformed into water-soluble functional nanoparticles via coating with amphiphilic polymer followed by covalent linking with desired biomolecules. Following this approach, carbon nanoparticles are functionalized with polyethylene glycol, primary amine, glucose, arginine, histidine, biotin and folic acid. These functional nanoparticles can be excited with blue/green light (i.e., 400-550 nm) to capture their emission spanning from 550 to 750 nm. Arginine and folic acid functionalized nanoparticles have been demonstrated as fluorescent cell labels where blue and green excitation has been used for imaging of labeled cells. The presented method can be extended for the development of carbon nanoparticle-based other bioimaging probes.

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

PubMed Central

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-01-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy. PMID:28300146

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.

A highly selective fluorescent probe based on coumarin for the imaging of N2H4 in living cells

NASA Astrophysics Data System (ADS)

Chen, Song; Hou, Peng; Wang, Jing; Liu, Lei; Zhang, Qi

2017-02-01

A turn-on fluorescence probe for highly sensitive and selective detection of N2H4 was developed based on hydrazine-triggered a substitution- cyclization-elimination cascade. Upon the treatment with N2H4, probe 1, 4-methyl-coumarin-7-yl bromobutanoate, displayed a remarkable fluorescence enhancement (25-fold) with a maximum at 450 nm. This probe can quantitatively detect N2H4 with a extremely low detection limit as 7 × 10- 8 M. Moreover, cell imaging experiments have indicated that probe 1 has potential ability to detect and image N2H4 in biological systems.

A near-infrared fluorescence-based surgical navigation system imaging software for sentinel lymph node detection

NASA Astrophysics Data System (ADS)

Ye, Jinzuo; Chi, Chongwei; Zhang, Shuang; Ma, Xibo; Tian, Jie

2014-02-01

Sentinel lymph node (SLN) in vivo detection is vital in breast cancer surgery. A new near-infrared fluorescence-based surgical navigation system (SNS) imaging software, which has been developed by our research group, is presented for SLN detection surgery in this paper. The software is based on the fluorescence-based surgical navigation hardware system (SNHS) which has been developed in our lab, and is designed specifically for intraoperative imaging and postoperative data analysis. The surgical navigation imaging software consists of the following software modules, which mainly include the control module, the image grabbing module, the real-time display module, the data saving module and the image processing module. And some algorithms have been designed to achieve the performance of the software, for example, the image registration algorithm based on correlation matching. Some of the key features of the software include: setting the control parameters of the SNS; acquiring, display and storing the intraoperative imaging data in real-time automatically; analysis and processing of the saved image data. The developed software has been used to successfully detect the SLNs in 21 cases of breast cancer patients. In the near future, we plan to improve the software performance and it will be extensively used for clinical purpose.

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

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.

Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging.

PubMed

Nakamura, Akinobu; Takigawa, Kazumasa; Kurishita, Yasutaka; Kuwata, Keiko; Ishida, Manabu; Shimoda, Yasushi; Hamachi, Itaru; Tsukiji, Shinya

2014-06-11

We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.

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.

Image navigation as a means to expand the boundaries of fluorescence-guided surgery

NASA Astrophysics Data System (ADS)

Brouwer, Oscar R.; Buckle, Tessa; Bunschoten, Anton; Kuil, Joeri; Vahrmeijer, Alexander L.; Wendler, Thomas; Valdés-Olmos, Renato A.; van der Poel, Henk G.; van Leeuwen, Fijs W. B.

2012-05-01

Hybrid tracers that are both radioactive and fluorescent help extend the use of fluorescence-guided surgery to deeper structures. Such hybrid tracers facilitate preoperative surgical planning using (3D) scintigraphic images and enable synchronous intraoperative radio- and fluorescence guidance. Nevertheless, we previously found that improved orientation during laparoscopic surgery remains desirable. Here we illustrate how intraoperative navigation based on optical tracking of a fluorescence endoscope may help further improve the accuracy of hybrid surgical guidance. After feeding SPECT/CT images with an optical fiducial as a reference target to the navigation system, optical tracking could be used to position the tip of the fluorescence endoscope relative to the preoperative 3D imaging data. This hybrid navigation approach allowed us to accurately identify marker seeds in a phantom setup. The multispectral nature of the fluorescence endoscope enabled stepwise visualization of the two clinically approved fluorescent dyes, fluorescein and indocyanine green. In addition, the approach was used to navigate toward the prostate in a patient undergoing robot-assisted prostatectomy. Navigation of the tracked fluorescence endoscope toward the target identified on SPECT/CT resulted in real-time gradual visualization of the fluorescent signal in the prostate, thus providing an intraoperative confirmation of the navigation accuracy.

Multimodal Spectral Imaging of Cells Using a Transmission Diffraction Grating on a Light Microscope

PubMed Central

Isailovic, Dragan; Xu, Yang; Copus, Tyler; Saraswat, Suraj; Nauli, Surya M.

2011-01-01

A multimodal methodology for spectral imaging of cells is presented. The spectral imaging setup uses a transmission diffraction grating on a light microscope to concurrently record spectral images of cells and cellular organelles by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Initially, the setup was applied for fluorescence spectral imaging of yeast and mammalian cells labeled with multiple fluorophores. Fluorescence signals originating from fluorescently labeled biomolecules in cells were collected through triple or single filter cubes, separated by the grating, and imaged using a charge-coupled device (CCD) camera. Cellular components such as nuclei, cytoskeleton, and mitochondria were spatially separated by the fluorescence spectra of the fluorophores present in them, providing detailed multi-colored spectral images of cells. Additionally, the grating-based spectral microscope enabled measurement of scattering and absorption spectra of unlabeled cells and stained tissue sections using darkfield and brightfield or DIC spectral microscopy, respectively. The presented spectral imaging methodology provides a readily affordable approach for multimodal spectral characterization of biological cells and other specimens. PMID:21639978

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

NASA Astrophysics Data System (ADS)

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

2011-07-01

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

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

Flexible imaging payload for real-time fluorescent biological imaging in parabolic, suborbital and space analog environments

NASA Astrophysics Data System (ADS)

Bamsey, Matthew T.; Paul, Anna-Lisa; Graham, Thomas; Ferl, Robert J.

2014-10-01

Fluorescent imaging offers the ability to monitor biological functions, in this case biological responses to space-related environments. For plants, fluorescent imaging can include general health indicators such as chlorophyll fluorescence as well as specific metabolic indicators such as engineered fluorescent reporters. This paper describes the Flex Imager a fluorescent imaging payload designed for Middeck Locker deployment and now tested on multiple flight and flight-related platforms. The Flex Imager and associated payload elements have been developed with a focus on 'flexibility' allowing for multiple imaging modalities and change-out of individual imaging or control components in the field. The imaging platform is contained within the standard Middeck Locker spaceflight form factor, with components affixed to a baseplate that permits easy rearrangement and fine adjustment of components. The Flex Imager utilizes standard software packages to simplify operation, operator training, and evaluation by flight provider flight test engineers, or by researchers processing the raw data. Images are obtained using a commercial cooled CCD image sensor, with light-emitting diodes for excitation and a suite of filters that allow biological samples to be imaged over wavelength bands of interest. Although baselined for the monitoring of green fluorescent protein and chlorophyll fluorescence from Arabidopsis samples, the Flex Imager payload permits imaging of any biological sample contained within a standard 10 cm by 10 cm square Petri plate. A sample holder was developed to secure sample plates under different flight profiles while permitting sample change-out should crewed operations be possible. In addition to crew-directed imaging, autonomous or telemetric operation of the payload is also a viable operational mode. An infrared camera has also been integrated into the Flex Imager payload to allow concurrent fluorescent and thermal imaging of samples. The Flex Imager has been utilized to assess, in real-time, the response of plants to novel environments including various spaceflight analogs, including several parabolic flight environments as well as hypobaric plant growth chambers. Basic performance results obtained under these operational environments, as well as laboratory-based tests are described. The Flex Imager has also been designed to be compatible with emerging suborbital platforms.

Light-driven transformable optical agent with adaptive functions for boosting cancer surgery outcomes.

PubMed

Qi, Ji; Chen, Chao; Zhang, Xiaoyan; Hu, Xianglong; Ji, Shenglu; Kwok, Ryan T K; Lam, Jacky W Y; Ding, Dan; Tang, Ben Zhong

2018-05-10

Fluorescence and photoacoustic imaging have different advantages in cancer diagnosis; however, combining effects in one agent normally requires a trade-off as the mechanisms interfere. Here, based on rational molecular design, we introduce a smart organic nanoparticle whose absorbed excitation energy can be photo-switched to the pathway of thermal deactivation for photoacoustic imaging, or to allow opposed routes for fluorescence imaging and photodynamic therapy. The molecule is made of a dithienylethene (DTE) core with two surrounding 2-(1-(4-(1,2,2-triphenylvinyl)phenyl)ethylidene)malononitrile (TPECM) units (DTE-TPECM). The photosensitive molecule changes from a ring-closed, for photoacoustic imaging, to a ring-opened state for fluorescence and photodynamic effects upon an external light trigger. The nanoparticles' photoacoustic and fluorescence imaging properties demonstrate the advantage of the switch. The use of the nanoparticles improves the outcomes of in vivo cancer surgery using preoperative photoacoustic imaging and intraoperative fluorescent visualization/photodynamic therapy of residual tumours to ensure total tumour removal.

Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

NASA Astrophysics Data System (ADS)

Taik Lim, Yong; Cho, Mi Young; Noh, Young-Woock; Chung, Jin Woong; Chung, Bong Hyun

2009-11-01

This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

Toxicological evaluation of Cd-based fluorescent nanoprobes by means of in vivo studies

NASA Astrophysics Data System (ADS)

Farias, Patricia M. A.; Ma-Hock, Lan; Landsiedel, Robert; van Ravenzwaay, Bennard

2018-02-01

Cadmium still represents a stigma for many research- and/or industrial applications. Some deleterious effects are attributed to Cadmium. In the present work, highly fluorescent Cadmium sulfide quantum dots are investigated by e.g. physical-chemical characterization. Most important however is their application as fluorescent probes for bio-imaging in living cells and tissues. This work presents their toxicological evaluation by means of in vivo studies. Bio-imaging experiments are performed without any pre-treatment. The toxicological studies performed, strongly indicate that the use of Cadmium based nanoparticles as fluorescent probes may be nonhazardous and not induce side effects for cells/tissues.

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.

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.

Time-Domain Fluorescence Lifetime Imaging Techniques Suitable for Solid-State Imaging Sensor Arrays

PubMed Central

Li, David Day-Uei; Ameer-Beg, Simon; Arlt, Jochen; Tyndall, David; Walker, Richard; Matthews, Daniel R.; Visitkul, Viput; Richardson, Justin; Henderson, Robert K.

2012-01-01

We have successfully demonstrated video-rate CMOS single-photon avalanche diode (SPAD)-based cameras for fluorescence lifetime imaging microscopy (FLIM) by applying innovative FLIM algorithms. We also review and compare several time-domain techniques and solid-state FLIM systems, and adapt the proposed algorithms for massive CMOS SPAD-based arrays and hardware implementations. The theoretical error equations are derived and their performances are demonstrated on the data obtained from 0.13 μm CMOS SPAD arrays and the multiple-decay data obtained from scanning PMT systems. In vivo two photon fluorescence lifetime imaging data of FITC-albumin labeled vasculature of a P22 rat carcinosarcoma (BD9 rat window chamber) are used to test how different algorithms perform on bi-decay data. The proposed techniques are capable of producing lifetime images with enough contrast. PMID:22778606

A multi-analytical investigation of semi-conductor pigments with time-resolved spectroscopy and imaging

NASA Astrophysics Data System (ADS)

Nevin, A.; Cesaratto, A.; D'Andrea, C.; Valentini, Gianluca; Comelli, D.

2013-05-01

We present the non-invasive study of historical and modern Zn- and Cd-based pigments with time-resolved fluorescence spectroscopy, fluorescence multispectral imaging and fluorescence lifetime imaging (FLIM). Zinc oxide and Zinc sulphide are semiconductors which have been used as white pigments in paintings, and the luminescence of these pigments from trapped states is strongly dependent on the presence of impurities and crystal defects. Cadmium sulphoselenide pigments vary in hue from yellow to deep red based on their composition, and are another class of semiconductor pigments which emit both in the visible and the near infrared. The Fluorescence lifetime of historical and modern pigments has been measured using both an Optical Multichannel Analyser (OMA) coupled with a Nd:YAG nslaser, and a streak camera coupled with a ps-laser for spectrally-resolved fluorescence lifetime measurements. For Znbased pigments we have also employed Fluorescence Lifetime Imaging (FLIM) for the measurement of luminescence. A case study of FLIM applied to the analysis of the painting by Vincent Van Gogh on paper - "Les Bretonnes et le pardon de Pont-Aven" (1888) is presented. Through the integration of complementary, portable and non-invasive spectroscopic techniques, new insights into the optical properties of Zn- and Cd-based pigments have been gained which will inform future analysis of late 19th] and early 20th C. paintings.

Ratiometric, visual, dual-signal fluorescent sensing and imaging of pH/copper ions in real samples based on carbon dots-fluorescein isothiocyanate composites.

PubMed

Zhu, Xinxin; Jin, Hui; Gao, Cuili; Gui, Rijun; Wang, Zonghua

2017-01-01

In this article, a facile aqueous synthesis of carbon dots (CDs) was developed by using natural kelp as a new carbon source. Through hydrothermal carbonization of kelp juice, fluorescent CDs were prepared and the CDs' surface was modified with polyethylenimine (PEI). The PEI-modified CDs were conjugated with fluorescein isothiocyanate (FITC) to fabricate CDs-FITC composites. To exploit broad applications, the CDs-FITC composites were developed as fluorescent sensing or imaging platforms of pH and Cu 2+ . Analytical performances of the composites-based fluorescence (FL) sensors were evaluated, including visual FL imaging of pH in glass bottle, ratiometric FL sensing of pH in yogurt samples, visual FL latent fingerprint and leaf imaging detection of [Cu 2+ ], dual-signal FL sensing of [Cu 2+ ] in yogurt and human serum samples. Experimental results from ratiometric, visual, dual-signal FL sensing and imaging applications confirmed the high feasibility, accuracy, stabilization and simplicity of CDs-FITC composites-based FL sensors for the detection of pH and Cu 2+ ions in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Study on excitation and fluorescence spectrums of Japanese citruses to construct machine vision systems for acquiring fluorescent images

NASA Astrophysics Data System (ADS)

Momin, Md. Abdul; Kondo, Naoshi; Kuramoto, Makoto; Ogawa, Yuichi; Shigi, Tomoo

2011-06-01

Research was conducted to acquire knowledge of the ultraviolet and visible spectrums from 300 -800 nm of some common varieties of Japanese citrus, to investigate the best wave-lengths for fluorescence excitation and the resulting fluorescence wave-lengths and to provide a scientific background for the best quality fluorescent imaging technique for detecting surface defects of citrus. A Hitachi U-4000 PC-based microprocessor controlled spectrophotometer was used to measure the absorption spectrum and a Hitachi F-4500 spectrophotometer was used for the fluorescence and excitation spectrums. We analyzed the spectrums and the selected varieties of citrus were categorized into four groups of known fluorescence level, namely strong, medium, weak and no fluorescence.The level of fluorescence of each variety was also examined by using machine vision system. We found that around 340-380 nm LEDs or UV lamps are appropriate as lighting devices for acquiring the best quality fluorescent image of the citrus varieties to examine their fluorescence intensity. Therefore an image acquisition device was constructed with three different lighting panels with UV LED at peak 365 nm, Blacklight blue lamps (BLB) peak at 350 nm and UV-B lamps at peak 306 nm. The results from fluorescent images also revealed that the findings of the measured spectrums worked properly and can be used for practical applications such as for detecting rotten, injured or damaged parts of a wide variety of citrus.

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

A Single-Photon Avalanche Diode Array for Fluorescence Lifetime Imaging Microscopy.

PubMed

Schwartz, David Eric; Charbon, Edoardo; Shepard, Kenneth L

2008-11-21

We describe the design, characterization, and demonstration of a fully integrated single-photon avalanche diode (SPAD) imager for use in time-resolved fluorescence imaging. The imager consists of a 64-by-64 array of active SPAD pixels and an on-chip time-to-digital converter (TDC) based on a delay-locked loop (DLL) and calibrated interpolators. The imager can perform both standard time-correlated single-photon counting (TCSPC) and an alternative gated-window detection useful for avoiding pulse pile-up when measuring bright signal levels. To illustrate the use of the imager, we present measurements of the decay lifetimes of fluorescent dyes of several types with a timing resolution of 350 ps.

A Single-Photon Avalanche Diode Array for Fluorescence Lifetime Imaging Microscopy

PubMed Central

Schwartz, David Eric; Charbon, Edoardo; Shepard, Kenneth L.

2013-01-01

We describe the design, characterization, and demonstration of a fully integrated single-photon avalanche diode (SPAD) imager for use in time-resolved fluorescence imaging. The imager consists of a 64-by-64 array of active SPAD pixels and an on-chip time-to-digital converter (TDC) based on a delay-locked loop (DLL) and calibrated interpolators. The imager can perform both standard time-correlated single-photon counting (TCSPC) and an alternative gated-window detection useful for avoiding pulse pile-up when measuring bright signal levels. To illustrate the use of the imager, we present measurements of the decay lifetimes of fluorescent dyes of several types with a timing resolution of 350 ps. PMID:23976789

A coumarin based Schiff base probe for selective fluorescence detection of Al3 + and its application in live cell imaging

NASA Astrophysics Data System (ADS)

Sen, Bhaskar; Sheet, Sanjoy Kumar; Thounaojam, Romita; Jamatia, Ramen; Pal, Amarta Kumar; Aguan, Kripamoy; Khatua, Snehadrinarayan

2017-02-01

A new coumarin based Schiff base compound, CSB-1 has been synthesized to detect metal ion based on the chelation enhanced fluorescence (CHEF). The cation binding properties of CSB-1 was thoroughly examined in UV-vis and fluorescence spectroscopy. In fluorescence spectroscopy the compound showed high selectivity toward Al3 + ion and the Al3 + can be quantified in mixed aqueous buffer solution (MeOH: 0.01 M HEPES Buffer; 9:1; v/v) at pH 7.4 as well as in BSA media. The fluorescence intensity of CSB-1 was enhanced by 24 fold after addition of only five equivalents of Al3 +. The fluorescence titration of CSB-1 with Al3 + in mixed aqueous buffer afforded a binding constant, Ka = (1.06 ± 0.2) × 104 M- 1. The colour change from light yellow to colourless and the appearance of blue fluorescence, which can be observed by the naked eye, provides a real-time method for Al3 + sensing. Further the live cell imaging study indicated that the detection of intracellular Al3 + ions are also readily possible in living cell.

Field portable mobile phone based fluorescence microscopy for detection of Giardia lamblia cysts in water samples

NASA Astrophysics Data System (ADS)

Ceylan Koydemir, Hatice; Gorocs, Zoltan; McLeod, Euan; Tseng, Derek; Ozcan, Aydogan

2015-03-01

Giardia lamblia is a waterborne parasite that causes an intestinal infection, known as giardiasis, and it is found not only in countries with inadequate sanitation and unsafe water but also streams and lakes of developed countries. Simple, sensitive, and rapid detection of this pathogen is important for monitoring of drinking water. Here we present a cost-effective and field portable mobile-phone based fluorescence microscopy platform designed for automated detection of Giardia lamblia cysts in large volume water samples (i.e., 10 ml) to be used in low-resource field settings. This fluorescence microscope is integrated with a disposable water-sampling cassette, which is based on a flow-through porous polycarbonate membrane and provides a wide surface area for fluorescence imaging and enumeration of the captured Giardia cysts on the membrane. Water sample of interest, containing fluorescently labeled Giardia cysts, is introduced into the absorbent pads that are in contact with the membrane in the cassette by capillary action, which eliminates the need for electrically driven flow for sample processing. Our fluorescence microscope weighs ~170 grams in total and has all the components of a regular microscope, capable of detecting individual fluorescently labeled cysts under light-emitting-diode (LED) based excitation. Including all the sample preparation, labeling and imaging steps, the entire measurement takes less than one hour for a sample volume of 10 ml. This mobile phone based compact and cost-effective fluorescent imaging platform together with its machine learning based cyst counting interface is easy to use and can even work in resource limited and field settings for spatio-temporal monitoring of water quality.

Colorful protein-based fluorescent probes for collagen imaging.

PubMed

Aper, Stijn J A; van Spreeuwel, Ariane C C; van Turnhout, Mark C; van der Linden, Ardjan J; Pieters, Pascal A; van der Zon, Nick L L; de la Rambelje, Sander L; Bouten, Carlijn V C; Merkx, Maarten

2014-01-01

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

Nanoscale Ultrasound-Switchable FRET-Based Liposomes for Near-Infrared Fluorescence Imaging in Optically Turbid Media.

PubMed

Zhang, Qimei; Morgan, Stephen P; Mather, Melissa L

2017-09-01

A new approach for fluorescence imaging in optically turbid media centered on the use of nanoscale ultrasound-switchable FRET-based liposome contrast agents is reported. Liposomes containing lipophilic carbocyanine dyes as FRET pairs with emission wavelengths located in the near-infrared window are prepared. The efficacy of FRET and self-quenching for liposomes with a range of fluorophore concentrations is first calculated from measurement of the liposome emission spectra. Exposure of the liposomes to ultrasound results in changes in the detected fluorescent signal, the nature of which depends on the fluorophores used, detection wavelength, and the fluorophore concentration. Line scanning of a tube containing the contrast agents with 1 mm inner diameter buried at a depth of 1 cm in a heavily scattering tissue phantom demonstrates an improvement in image spatial resolution by a factor of 6.3 as compared with images obtained in the absence of ultrasound. Improvements are also seen in image contrast with the highest obtained being 9% for a liposome system containing FRET pairs. Overall the results obtained provide evidence of the potential the nanoscale ultrasound-switchable FRET-based liposomes studied here have for in vivo fluorescence imaging. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photon-Counting H33D Detector for Biological Fluorescence Imaging

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Millaud, J.E.; Weiss, S.

2010-01-01

We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25 mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single-molecule imaging and high-throughput study of biomolecular interactions. PMID:20151021

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.

Use of acousto-optic tunable filter in fluorescence imaging endoscopy

NASA Astrophysics Data System (ADS)

Bouhifd, Mounir; Whelan, Maurice; Aprahamian, Marc

2003-10-01

A prototype instrument for fluorescence-based medical diagnostics in vivo is described. The system consists of a rigid endoscope comprising a UV laser-source for fluorescence excitation and a white light source for direct imaging. An acousto-optic tuneable filter (AOTF) is employed as a full-field tuneable bandpass filter. This allows fast continuous or random-access tuning with high filtering efficiency. A study of the diagnostic potential of fluorescence imaging for pancreatitis was conducted on a rat model. In particular, the aim was to detect autofluorescence of endogenous protoporphyrin IX (PpIX) that has been shown to accumulate in early-stage diseased tissue undergoing an inflammatory response.

Optical/MRI Multimodality Molecular Imaging

NASA Astrophysics Data System (ADS)

Ma, Lixin; Smith, Charles; Yu, Ping

2007-03-01

Multimodality molecular imaging that combines anatomical and functional information has shown promise in development of tumor-targeted pharmaceuticals for cancer detection or therapy. We present a new multimodality imaging technique that combines fluorescence molecular tomography (FMT) and magnetic resonance imaging (MRI) for in vivo molecular imaging of preclinical tumor models. Unlike other optical/MRI systems, the new molecular imaging system uses parallel phase acquisition based on heterodyne principle. The system has a higher accuracy of phase measurements, reduced noise bandwidth, and an efficient modulation of the fluorescence diffuse density waves. Fluorescent Bombesin probes were developed for targeting breast cancer cells and prostate cancer cells. Tissue phantom and small animal experiments were performed for calibration of the imaging system and validation of the targeting probes.

Quantitative comparison between full-spectrum and filter-based imaging in hyperspectral fluorescence microscopy

PubMed Central

GAO, L.; HAGEN, N.; TKACZYK, T.S.

2012-01-01

Summary We implement a filterless illumination scheme on a hyperspectral fluorescence microscope to achieve full-range spectral imaging. The microscope employs polarisation filtering, spatial filtering and spectral unmixing filtering to replace the role of traditional filters. Quantitative comparisons between full-spectrum and filter-based microscopy are provided in the context of signal dynamic range and accuracy of measured fluorophores’ emission spectra. To show potential applications, a five-colour cell immunofluorescence imaging experiment is theoretically simulated. Simulation results indicate that the use of proposed full-spectrum imaging technique may result in three times improvement in signal dynamic range compared to that can be achieved in the filter-based imaging. PMID:22356127

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.

Fluorescent rhenium-naphthalimide conjugates as cellular imaging agents.

PubMed

Langdon-Jones, Emily E; Symonds, Nadine O; Yates, Sara E; Hayes, Anthony J; Lloyd, David; Williams, Rebecca; Coles, Simon J; Horton, Peter N; Pope, Simon J A

2014-04-07

A range of biologically compatible, fluorescent rhenium-naphthalimide conjugates, based upon the rhenium fac-tricarbonyl core, has been synthesized. The fluorescent ligands are based upon a N-functionalized, 4-amino-derived 1,8-naphthalimide core and incorporate a dipicolyl amine binding unit to chelate Re(I); the structural variations accord to the nature of the alkylated imide with ethyl ester glycine (L(1)), 3-propanol (L(2)), diethylene glycol (L(3)), and benzyl alcohol (L(4)) variants. The species are fluorescent in the visible region between 505 and 537 nm through a naphthalimide-localized intramolecular charge transfer, with corresponding fluorescent lifetimes of up to 9.8 ns. The ligands and complexes were investigated for their potential as imaging agents for human osteoarthritic cells and protistan fish parasite Spironucleus vortens using confocal fluorescence microscopy. The results show that the specific nature of the naphthalimide structure serves to control the uptake and intracellular localization of these imaging agents. Significant differences were noted between the free ligands and complexes, with the Re(I) complex of L(2) showing hydrogenosomal localization in S. vortens.

Holographic techniques for cellular fluorescence microscopy

NASA Astrophysics Data System (ADS)

Kim, Myung K.

2017-04-01

We have constructed a prototype instrument for holographic fluorescence microscopy (HFM) based on self-interference incoherent digital holography (SIDH) and demonstrate novel imaging capabilities such as differential 3D fluorescence microscopy and optical sectioning by compressive sensing.

Synthesis and evaluation of new NIR-fluorescent probes for cathepsin B: ICT versus FRET as a turn-ON mode-of-action.

PubMed

Kisin-Finfer, Einat; Ferber, Shiran; Blau, Rachel; Satchi-Fainaro, Ronit; Shabat, Doron

2014-06-01

Recent years have seen tremendous progress in the design and study of molecular imaging geared towards biological and biomedical applications. The expression or activity of specific enzymes including proteases can be monitored by cutting edge molecular imaging techniques. Cathepsin B plays key roles in tumor progression via controlled degradation of extracellular matrix. Consequently, this protease has been attracting significant attention in cancer research, and many imaging probes targeting its activity have been developed. Here, we describe the design, synthesis and evaluation of two novel near infrared (NIR) fluorescent probes for detection of cathepsin B activity with different turn-ON mechanisms. One probe is based on an ICT activation mechanism of a donor-two-acceptor π-electron dye system, while the other is based on the FRET mechanism obtained by a fluorescent dye and a quencher. The two probes exhibit significant fluorescent turn-ON response upon cleavage by cathepsin B. The NIR fluorescence of the ICT probe in its OFF state was significantly lower than that of the FRET-based probe. This effect results in a higher signal-to-noise ratio and consequently increased sensitivity and better image contrast. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

PubMed Central

Zhang, Ming; Chakraborty, Subhasish K.; Sampath, Padma; Rojas, Juan J.; Hou, Weizhou; Saurabh, Saumya; Thorne, Steve H.; Bruchez, Marcel P.; Waggoner, Alan S.

2015-01-01

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule–based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter–tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene. PMID:26348895

Nano-Gap Embedded Plasmonic Gratings for Surface Plasmon Enhanced Fluorescence

NASA Astrophysics Data System (ADS)

Bhatnagar, Kunal; Bok, Sangho; Korampally, Venumadhav; Gangopadhyay, Shubhra

2012-02-01

Plasmonic nanostructures have been extensively used in the past few decades for applications in sub-wavelength optics, data storage, optoelectronic circuits, microscopy and bio-photonics. The enhanced electromagnetic field produced at the metal/dielectric interface by the excitation of surface plasmons via incident radiation can be used for signal enhancement in fluorescence and surface enhanced Raman scattering studies. Novel plasmonic structures on the sub wavelength scale have been shown to provide very efficient and extreme light concentration at the nano-scale. The enhanced electric field produced within a few hundred nanometers of these structures can be used to excite fluorophores in the surrounding environment. Fluorescence based bio-detection and bio-imaging are two of the most important tools in the life sciences. Improving the qualities and capabilities of fluorescence based detectors and imaging equipment has been a big challenge to the industry manufacturers. We report the novel fabrication of nano-gap embedded periodic grating substrates on the nanoscale using micro-contact printing and polymethylsilsesquioxane (PMSSQ) polymer. Fluorescence enhancement of up to 118 times was observed with these silver nanostructures in conjugation with Rhodamine-590 fluorescent dye. These substrates are ideal candidates for low-level fluorescence detection and single molecule imaging.

Wide-field spectrally resolved quantitative fluorescence imaging system: toward neurosurgical guidance in glioma resection

NASA Astrophysics Data System (ADS)

Xie, Yijing; Thom, Maria; Ebner, Michael; Wykes, Victoria; Desjardins, Adrien; Miserocchi, Anna; Ourselin, Sebastien; McEvoy, Andrew W.; Vercauteren, Tom

2017-11-01

In high-grade glioma surgery, tumor resection is often guided by intraoperative fluorescence imaging. 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) provides fluorescent contrast between normal brain tissue and glioma tissue, thus achieving improved tumor delineation and prolonged patient survival compared with conventional white-light-guided resection. However, commercially available fluorescence imaging systems rely solely on visual assessment of fluorescence patterns by the surgeon, which makes the resection more subjective than necessary. We developed a wide-field spectrally resolved fluorescence imaging system utilizing a Generation II scientific CMOS camera and an improved computational model for the precise reconstruction of the PpIX concentration map. In our model, the tissue's optical properties and illumination geometry, which distort the fluorescent emission spectra, are considered. We demonstrate that the CMOS-based system can detect low PpIX concentration at short camera exposure times, while providing high-pixel resolution wide-field images. We show that total variation regularization improves the contrast-to-noise ratio of the reconstructed quantitative concentration map by approximately twofold. Quantitative comparison between the estimated PpIX concentration and tumor histopathology was also investigated to further evaluate the system.

Fluorescence spectroscopy using indocyanine green for lymph node mapping

NASA Astrophysics Data System (ADS)

Haj-Hosseini, Neda; Behm, Pascal; Shabo, Ivan; Wârdell, Karin

2014-02-01

The principles of cancer treatment has for years been radical resection of the primary tumor. In the oncologic surgeries where the affected cancer site is close to the lymphatic system, it is as important to detect the draining lymph nodes for metastasis (lymph node mapping). As a replacement for conventional radioactive labeling, indocyanine green (ICG) has shown successful results in lymph node mapping; however, most of the ICG fluorescence detection techniques developed are based on camera imaging. In this work, fluorescence spectroscopy using a fiber-optical probe was evaluated on a tissue-like ICG phantom with ICG concentrations of 6-64 μM and on breast tissue from five patients. Fiber-optical based spectroscopy was able to detect ICG fluorescence at low intensities; therefore, it is expected to increase the detection threshold of the conventional imaging systems when used intraoperatively. The probe allows spectral characterization of the fluorescence and navigation in the tissue as opposed to camera imaging which is limited to the view on the surface of the tissue.

Differences in the intensity of light-induced fluorescence emitted by resin composites.

PubMed

Kim, Bo-Ra; Kang, Si-Mook; Kim, Gyung-Min; Kim, Baek-Il

2016-03-01

The aims of this study were to compare the intensities of fluorescence emitted by different resin composites as detected using quantitative light-induced fluorescence (QLF) technology, and to compare the fluorescence intensity contrast with the color contrast between a restored composite and the adjacent region of the tooth. Six brands of light-cured resin composites (shade A2) were investigated. The composites were used to prepare composite discs, and fill holes that had been prepared in extracted human teeth. White-light and fluorescence images of all specimens were obtained using a fluorescence camera based on QLF technology (QLF-D) and converted into 8-bit grayscale images. The fluorescence intensity of the discs as well as the fluorescence intensity contrast and the color contrast between the composite restoration and adjacent tooth region were calculated as grayscale levels. The grayscale levels for the composite discs differed significantly with the brand (p<0.001): DenFil (10.84±0.35, mean±SD), Filtek Z350 (58.28±1.37), Premisa (156.94±1.58), Grandio (177.20±0.81), Charisma (207.05±0.77), and Gradia direct posterior (211.52±1.66). The difference in grayscale levels between a resin restoration and the adjacent tooth was significantly greater in fluorescence images for each brand than in white-light images, except for the Filtek Z350 (p<0.05). However, the Filtek Z350 restoration was distinguishable from the adjacent tooth in a fluorescence image. The intensities of fluorescence detected from the resin composites varied. The differences between the composite and adjacent tooth were greater for the fluorescence intensity contrast than for the colors observed in the white-light images. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells.

PubMed

Li, Ke; Zhang, Zhi-Ping; Luo, Ming; Yu, Xiang; Han, Yu; Wei, Hong-Ping; Cui, Zong-Qiang; Zhang, Xian-En

2012-01-07

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging α(v)β(3) integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.

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.

A Quninolylthiazole Derivatives as an ICT-Based Fluorescent Probe of Hg(II) and its Application in Ratiometric Imaging in Live HeLa Cells.

PubMed

Bai, Jian-Ying; Xie, Yu-Zhong; Wang, Chang-Jiang; Fang, Shu-Qing; Cao, Lin-Nan; Wang, Ling-Li; Jin, Jing-Yi

2018-05-28

As a structural analogue of pyridylthiazole, 2-(2-benzothiazoyl)-phenylethynylquinoline (QBT) was designed as a fluorescent probe for Hg(II) based on an intramolecular charge transfer (ICT) mechanism. The compound was synthesized in three steps starting from 6-bromo-2-methylquinoline, with moderate yield. Corresponding studies on the optical properties of QBT indicate that changes in the fluorescence ratio of QBT in response to Hg(II) could be quantified based on dual-emission changes. More specifically, the emission spectrum of QBT before and after interactions with Hg(II) exhibited a remarkable red shift of about 120 nm, which is rarely reported in ICT-based fluorescent sensors. Finally, QBT was applied in the two-channel imaging of Hg(II) in live HeLa cells.

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.

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.

M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors.

PubMed

Yi, Hyunjung; Ghosh, Debadyuti; Ham, Moon-Ho; Qi, Jifa; Barone, Paul W; Strano, Michael S; Belcher, Angela M

2012-03-14

Second near-infrared (NIR) window light (950-1400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to 4-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control nontargeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas. © 2012 American Chemical Society

M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors

PubMed Central

HAM, MOON-HO; QI, JIFA; BARONE, PAUL W.; STRANO, MICHAEL S.; BELCHER, ANGELA M.

2014-01-01

Second near-infrared (NIR) window light (950-1,400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to four-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control non-targeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas. PMID:22268625

Nanogap embedded silver gratings for surface plasmon enhanced fluorescence

NASA Astrophysics Data System (ADS)

Bhatnagar, Kunal

Plasmonic nanostructures have been extensively used in the past few decades for applications in sub-wavelength optics, data storage, optoelectronic circuits, microscopy and bio-photonics. The enhanced electromagnetic field produced at the metal and dielectric interface by the excitation of surface plasmons via incident radiation can be used for signal enhancement in fluorescence and surface enhanced Raman scattering studies. Novel plasmonic structures have shown to provide very efficient and extreme light concentration at the nano-scale in recent years. The enhanced electric field produced within a few hundred nanometers of these surfaces can be used to excite fluorophores in the surrounding environment. Fluorescence based bio-detection and bio-imaging are two of the most important tools in the life sciences and improving the qualities and capabilities of fluorescence based detectors and imaging equipment remains a big challenge for industry manufacturers. We report a novel fabrication technique for producing nano-gap embedded periodic grating substrates on the nanoscale using a store bought HD-DVD and conventional soft lithography procedures. Polymethylsilsesquioxane (PMSSQ) polymer is used as the ink for the micro-contact printing process with PDMS stamps obtained from the inexpensive HD-DVDs as master molds. Fluorescence enhancement factors of up to 118 times were observed with these silver nanostructures in conjugation with Rhodamine-590 fluorescent dye. These substrates are ideal candidates for a robust and inexpensive optical system with applications such as low-level fluorescence based analyte detection, single molecule imaging, and surface enhanced Raman studies. Preliminary results in single molecule experiments have also been obtained by imaging individual 3 nm and 20 nm dye-doped nanoparticles attached to the silver plasmonic gratings using epi-fluorescence microscopy.

Optofluidic Fluorescent Imaging Cytometry on a Cell Phone

PubMed Central

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F.; Yaglidere, Oguzhan; Ozcan, Aydogan

2012-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings. PMID:21774454

Optofluidic fluorescent imaging cytometry on a cell phone.

PubMed

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F; Yaglidere, Oguzhan; Ozcan, Aydogan

2011-09-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings.

Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.

PubMed Central

Marriott, G.; Heidecker, M.; Diamandis, E. P.; Yan-Marriott, Y.

1994-01-01

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found. Through the coupling of SBMC to streptavidin,a plethora of biotin-based tracer molecules are available for immunocytochemical studies. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:7811952

Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability.

PubMed

Hirabayashi, Kazuhisa; Hanaoka, Kenjiro; Egawa, Takahiro; Kobayashi, Chiaki; Takahashi, Shodai; Komatsu, Toru; Ueno, Tasuku; Terai, Takuya; Ikegaya, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-10-01

Fluorescence imaging of calcium ions (Ca(2+)) has become an essential technique for investigation of signaling pathways involving Ca(2+) as a second messenger. But, Ca(2+) signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca(2+) and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca(2+) that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca(2+) concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca(2+) red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Parallel detection experiment of fluorescence confocal microscopy using DMD.

PubMed

Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

2016-05-01

Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

Selective imaging of cancer cells with a pH-activatable lysosome-targeting fluorescent probe.

PubMed

Shi, Rongguang; Huang, Lu; Duan, Xiaoxue; Sun, Guohao; Yin, Gui; Wang, Ruiyong; Zhu, Jun-Jie

2017-10-02

Fluorescence imaging with tumor-specific fluorescent probe has emerged as a tool to aid surgeons in the identification and removal of tumor tissue. We report here a new lysosome-targeting fluorescent probe (NBOH) with BODIPY fluorephore to distinguish tumor tissue out of normal tissue based on different pH environment. The probe exhibited remarkable pH-dependent fluorescence behavior in a wide pH range from 3.0 to 11.0, especially a sensitive pH-dependent fluorescence change at pH range between 3.5 and 5.5, corresponding well to the acidic microenvironment of tumor cells, in aqueous solution. The response time of NBOH was extremely short and the photostability was proved to be good. Toxicity test and fluorescence cell imaging together with a sub-cellular localization study were carried out revealing its low biotoxicity and good cell membrane permeability. And NBOH was successfully applied to the imaging of tumor tissue in tumor-bearing mice suggesting potential application to surgery as a tumor-specific probe. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

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.

Identifying Novel Regulators of Vacuolar Trafficking by Combining Fluorescence Imaging-Based Forward Genetic Screening and In Vitro Pollen Germination.

PubMed

Feng, Qiang-Nan; Zhang, Yan

2017-01-01

Subcellular targeting of vacuolar proteins depends on cellular machinery regulating vesicular trafficking. Plant-specific vacuolar trafficking routes have been reported. However, regulators mediating these processes are obscure. By combining a fluorescence imaging-based forward genetic approach and in vitro pollen germination system, we show an efficient protocol of identifying regulators of plant-specific vacuolar trafficking routes.

Wireless fluorescence capsule for endoscopy using single photon-based detection

NASA Astrophysics Data System (ADS)

Al-Rawhani, Mohammed A.; Beeley, James; Cumming, David R. S.

2015-12-01

Fluorescence Imaging (FI) is a powerful technique in biological science and clinical medicine. Current FI devices that are used either for in-vivo or in-vitro studies are expensive, bulky and consume substantial power, confining the technique to laboratories and hospital examination rooms. Here we present a miniaturised wireless fluorescence endoscope capsule with low power consumption that will pave the way for future FI systems and applications. With enhanced sensitivity compared to existing technology we have demonstrated that the capsule can be successfully used to image tissue autofluorescence and targeted fluorescence via fluorophore labelling of tissues. The capsule incorporates a state-of-the-art complementary metal oxide semiconductor single photon avalanche detector imaging array, miniaturised optical isolation, wireless technology and low power design. When in use the capsule consumes only 30.9 mW, and deploys very low-level 468 nm illumination. The device has the potential to replace highly power-hungry intrusive optical fibre based endoscopes and to extend the range of clinical examination below the duodenum. To demonstrate the performance of our capsule, we imaged fluorescence phantoms incorporating principal tissue fluorophores (flavins) and absorbers (haemoglobin). We also demonstrated the utility of marker identification by imaging a 20 μM fluorescein isothiocyanate (FITC) labelling solution on mammalian tissue.

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.

Low-cost fluorescence microscopy for point-of-care cell imaging

NASA Astrophysics Data System (ADS)

Lochhead, Michael J.; Ives, Jeff; Givens, Monique; Delaney, Marie; Moll, Kevin; Myatt, Christopher J.

2010-02-01

Fluorescence microscopy has long been a standard tool in laboratory medicine. Implementation of fluorescence microscopy for near-patient diagnostics, however, has been limited due to cost and complexity associated with traditional fluorescence microscopy techniques. There is a particular need for robust, low-cost imaging in high disease burden areas in the developing world, where access to central laboratory facilities and trained staff is limited. Here we describe a point-of-care assay that combines a disposable plastic cartridge with an extremely low cost fluorescence imaging instrument. Based on a novel, multi-mode planar waveguide configuration, the system capitalizes on advances in volume-manufactured consumer electronic components to deliver an imaging system with minimal moving parts and low power requirements. A two-color cell imager is presented, with magnification optimized for enumeration of immunostained human T cells. To demonstrate the system, peripheral blood mononuclear cells were stained with fluorescently labeled anti-human-CD4 and anti-human-CD3 antibodies. Registered images were used to generate fractional CD4+ and CD3+ staining and enumeration results that show excellent correlation with flow cytometry. The cell imager is under development as a very low cost CD4+ T cell counter for HIV disease management in limited resource settings.

Wide-field fluorescent microscopy on a cell-phone.

PubMed

Zhu, Hongying; Yaglidere, Oguzhan; Su, Ting-Wei; Tseng, Derek; Ozcan, Aydogan

2011-01-01

We demonstrate wide-field fluorescent imaging on a cell-phone, using compact and cost-effective optical components that are mechanically attached to the existing camera unit of the cell-phone. Battery powered light-emitting diodes (LEDs) are used to side-pump the sample of interest using butt-coupling. The pump light is guided within the sample cuvette to excite the specimen uniformly. The fluorescent emission from the sample is then imaged with an additional lens that is put in front of the existing lens of the cell-phone camera. Because the excitation occurs through guided waves that propagate perpendicular to the detection path, an inexpensive plastic color filter is sufficient to create the dark-field background needed for fluorescent imaging. The imaging performance of this light-weight platform (~28 grams) is characterized with red and green fluorescent microbeads, achieving an imaging field-of-view of ~81 mm(2) and a spatial resolution of ~10 μm, which is enhanced through digital processing of the captured cell-phone images using compressive sampling based sparse signal recovery. We demonstrate the performance of this cell-phone fluorescent microscope by imaging labeled white-blood cells separated from whole blood samples as well as water-borne pathogenic protozoan parasites such as Giardia Lamblia cysts.

Noninvasive Optical Tracking of Red Fluorescent Protein-Expressing Cancer Cells in a Model of Metastatic Breast Cancer 1*

PubMed Central

Winnard, Paul T; Kluth, Jessica B; Raman, Venu

2006-01-01

Abstract We have evaluated the use of the Xenogen IVIS 200 imaging system for real-time fluorescence protein-based optical imaging of metastatic progression in live animals. We found that green fluorescent protein-expressing cells (100 x 106) were not detectable in a mouse cadaver phantom experiment. However, a 10-fold lower number of tdTomato-expressing cells were easily detected. Mammary fat pad xenografts of stable MDA-MB-231-tdTomato cells were generated for the imaging of metastatic progression. At 2 weeks postinjection, barely palpable tumor burdens were easily detected at the sites of injection. At 8 weeks, a small contralateral mammary fat pad metastasis was imaged and, by 13 weeks, metastases to lymph nodes were detectable. Metastases with nodular composition were detectable within the rib cage region at 15 weeks. 3-D image reconstructions indicated that the detection of fluorescence extended to approximately 1 cm below the surface. A combination of intense tdTomato fluorescence, imaging at ≥ 620 nm (where autofluorescence is minimized), and the sensitivity of the Xenogen imager made this possible. This study demonstrates the utility of the noninvasive optical tracking of cancer cells during metastatic progression with endogenously expressed fluorescence protein reporters using detection wavelengths of ≥ 620 nm. PMID:17032496

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

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.

Estimating background-subtracted fluorescence transients in calcium imaging experiments: a quantitative approach.

PubMed

Joucla, Sébastien; Franconville, Romain; Pippow, Andreas; Kloppenburg, Peter; Pouzat, Christophe

2013-08-01

Calcium imaging has become a routine technique in neuroscience for subcellular to network level investigations. The fast progresses in the development of new indicators and imaging techniques call for dedicated reliable analysis methods. In particular, efficient and quantitative background fluorescence subtraction routines would be beneficial to most of the calcium imaging research field. A background-subtracted fluorescence transients estimation method that does not require any independent background measurement is therefore developed. This method is based on a fluorescence model fitted to single-trial data using a classical nonlinear regression approach. The model includes an appropriate probabilistic description of the acquisition system's noise leading to accurate confidence intervals on all quantities of interest (background fluorescence, normalized background-subtracted fluorescence time course) when background fluorescence is homogeneous. An automatic procedure detecting background inhomogeneities inside the region of interest is also developed and is shown to be efficient on simulated data. The implementation and performances of the proposed method on experimental recordings from the mouse hypothalamus are presented in details. This method, which applies to both single-cell and bulk-stained tissues recordings, should help improving the statistical comparison of fluorescence calcium signals between experiments and studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

Visualization of Porphyrin-Based Photosensitizer Distribution from Fluorescence Images In Vivo Using an Optimized RGB Camera

NASA Astrophysics Data System (ADS)

Liu, L.; Huang, Zh.; Qiu, Zh.; Li, B.

2018-01-01

A handheld RGB camera was developed to monitor the in vivo distribution of porphyrin-based photosensitizer (PS) hematoporphyrin monomethyl ether (HMME) in blood vessels during photodynamic therapy (PDT). The focal length, f-number, International Standardization Organization (ISO) sensitivity, and shutter speed of the camera were optimized for the solution sample with various HMME concentrations. After the parameter optimization, it was found that the red intensity value of the fluorescence image was linearly related to the fluorescence intensity under investigated conditions. The RGB camera was then used to monitor the in vivo distribution of HMME in blood vessels in a skin-fold window chamber model. The red intensity value of the recorded RGB fluorescence image was found to be linearly correlated to HMME concentrations in the range 0-24 μM. Significant differences in the red to green intensity ratios were observed between the blood vessels and the surrounding tissue.

Fluorescent magnetic hybrid nanoprobe for multimodal bioimaging

PubMed Central

Bright, Vanessa

2011-01-01

A fluorescent magnetic hybrid imaging nanoprobe (HINP) was fabricated by conjugation of superparamagnetic Fe3O4 nanoparticles and visible light-emitting (~600 nm) fluorescent CdTe/CdS quantum dots (QDs). The assembly strategy used the covalent linking of the oxidized dextran shell of magnetic particles to the glutathione ligands of QDs. Synthesized HINP formed stable water-soluble colloidal dispersions. The structure and properties of the particles were characterized by transmission electron and atomic force microscopy, energy dispersive X-ray analysis and inductively coupled plasma optical emission spectroscopy, dynamic light scattering analysis, optical absorption and photoluminescence spectroscopy, and fluorescent imaging. The luminescence imaging region of the nanoprobe was extended to the near-infrared (NIR) (~800 nm) by conjugation of superparamagnetic nanoparticles with synthesized CdHgTe/CdS QDs. Cadmium, mercury based QDs in HINP can be easily replaced by novel water soluble glutathione stabilized AgInS2/ZnS QDs to present a new class of cadmium-free multimodal imaging agents. Observed NIR photoluminescence of fluorescent magnetic nanocomposites supports their use for bioimaging. The developed HINP provides dual-imaging channels for simultaneous optical and magnetic resonance imaging. PMID:21597146

Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging.

PubMed

Zhao, Qiaole; Schelen, Ben; Schouten, Raymond; van den Oever, Rein; Leenen, René; van Kuijk, Harry; Peters, Inge; Polderdijk, Frank; Bosiers, Jan; Raspe, Marcel; Jalink, Kees; Geert Sander de Jong, Jan; van Geest, Bert; Stoop, Karel; Young, Ian Ted

2012-12-01

We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

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.

A Neuron-Based Screening Platform for Optimizing Genetically-Encoded Calcium Indicators

PubMed Central

Schreiter, Eric R.; Hasseman, Jeremy P.; Tsegaye, Getahun; Fosque, Benjamin F.; Behnam, Reza; Shields, Brenda C.; Ramirez, Melissa; Kimmel, Bruce E.; Kerr, Rex A.; Jayaraman, Vivek; Looger, Loren L.; Svoboda, Karel; Kim, Douglas S.

2013-01-01

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude. PMID:24155972

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.

Synthesis of fluorescent carbon dots by a microwave heating process: structural characterization and cell imaging applications

NASA Astrophysics Data System (ADS)

Stefanakis, Dimitrios; Philippidis, Aggelos; Sygellou, Labrini; Filippidis, George; Ghanotakis, Demetrios; Anglos, Demetrios

2014-10-01

Two types of highly fluorescent carbon dots (C-dots) were prepared by a single-step procedure based on microwave heating citric acid and 6-aminocaproic acid or citric acid and urea in an aqueous solution. The small size of the isolated carbon dots along with their strong absorption in the UV and their excitation wavelength-dependent fluorescence render them ideal nanomaterials for biomedical applications (imaging and sensing). The structure and properties of the two types of C-dot materials were studied using a series of spectroscopic techniques. The ability of the C-dots to be internalized by HeLa cells was demonstrated via 3-photon fluorescence microscopy imaging.

Whole-body multicolor spectrally resolved fluorescence imaging for development of target-specific optical contrast agents using genetically engineered probes

NASA Astrophysics Data System (ADS)

Kobayashi, Hisataka; Hama, Yukihiro; Koyama, Yoshinori; Barrett, Tristan; Urano, Yasuteru; Choyke, Peter L.

2007-02-01

Target-specific contrast agents are being developed for the molecular imaging of cancer. Optically detectable target-specific agents are promising for clinical applications because of their high sensitivity and specificity. Pre clinical testing is needed, however, to validate the actual sensitivity and specificity of these agents in animal models, and involves both conventional histology and immunohistochemistry, which requires large numbers of animals and samples with costly handling. However, a superior validation tool takes advantage of genetic engineering technology whereby cell lines are transfected with genes that induce the target cell to produce fluorescent proteins with characteristic emission spectra thus, identifying them as cancer cells. Multicolor fluorescence imaging of these genetically engineered probes can provide rapid validation of newly developed exogenous probes that fluoresce at different wavelengths. For example, the plasmid containing the gene encoding red fluorescent protein (RFP) was transfected into cell lines previously developed to either express or not-express specific cell surface receptors. Various antibody-based or receptor ligand-based optical contrast agents with either green or near infrared fluorophores were developed to concurrently target and validate cancer cells and their positive and negative controls, such as β-D-galactose receptor, HER1 and HER2 in a single animal/organ. Spectrally resolved fluorescence multicolor imaging was used to detect separate fluorescent emission spectra from the exogenous agents and RFP. Therefore, using this in vivo imaging technique, we were able to demonstrate the sensitivity and specificity of the target-specific optical contrast agents, thus reducing the number of animals needed to conduct these experiments.

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.

Fluorescent water-Soluble Probes Based on Ammonium Cation Peg Substituted Perylenepisimides: Synthesis, Photophysical Properties, and Live Cell Images

NASA Astrophysics Data System (ADS)

Yang, Wei; Cai, Jiaxuan; Zhang, Shuchen; Yi, Xuegang; Gao, Baoxiang

2018-01-01

To synthesize perylenbisimides (PBI) fluorescent probes that will improve the water-soluble ability and the cytocompatibility, the synthesis and properties of fluorescent water-soluble probes based on dendritic ammonium cation polyethylene glycol (PEG) substituted perylenebisimides(GPDIs) are presented. As we expected, with increased ammonium cation PEG, the aggregation of the PBI in an aqueous solution is completely suppressed by the hydrophilic ammonium cation PEG groups. And the fluorescence quantum yield increases from 25% for GPDI-1 to 62% for GPDI-2. When incubated with Hela cells for 48 h, the viabilities are 71% (for GPDI-1) and 76% (for GPDI-2). Live cell imaging shows that these probes are efficiently internalized by HeLa cells. The study of the photophysical properties indicated increasing the ammonium cation PEG generation can increase the fluorescence quantum yield. Live cell imaging shows that with the ammonium cation PEG chains of perylenebisimides has high biocompatibility. The exceptionally low cytotoxicity is ascribed to the ammonium cation PEG chains, which protect the dyes from nonspecifically interacting with the extracellular proteins. Live cell imaging shows that ammonium cations PEG chains can promote the internalization of these probes.

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

PubMed

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

2015-03-01

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

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells.

PubMed

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip.

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells

PubMed Central

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W.; Gautier, Virginie W.

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip. PMID:26485569

Detection of fecal contamination on beef meat surfaces using handheld fluorescence imaging device (HFID)

USDA-ARS?s Scientific Manuscript database

Current meat inspection in slaughter plants, for food safety and quality attributes including potential fecal contamination, is conducted through by visual examination human inspectors. A handheld fluorescence-based imaging device (HFID) was developed to be an assistive tool for human inspectors by ...

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...

Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

PubMed Central

Hsieh, Ya-Ju; Ke, Chien-Chih; Yeh, Skye Hsin-Hsien; Lin, Chien-Feng; Chen, Fu-Du; Lin, Kang-Ping; Chen, Ran-Chou; Liu, Ren-Shyan

2014-01-01

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications. PMID:24809057

A Simple BODIPY-Based Viscosity Probe for Imaging of Cellular Viscosity in Live Cells

PubMed Central

Su, Dongdong; Teoh, Chai Lean; Gao, Nengyue; Xu, Qing-Hua; Chang, Young-Tae

2016-01-01

Intracellular viscosity is a fundamental physical parameter that indicates the functioning of cells. In this work, we developed a simple boron-dipyrromethene (BODIPY)-based probe, BTV, for cellular mitochondria viscosity imaging by coupling a simple BODIPY rotor with a mitochondria-targeting unit. The BTV exhibited a significant fluorescence intensity enhancement of more than 100-fold as the solvent viscosity increased. Also, the probe showed a direct linear relationship between the fluorescence lifetime and the media viscosity, which makes it possible to trace the change of the medium viscosity. Furthermore, it was demonstrated that BTV could achieve practical applicability in the monitoring of mitochondrial viscosity changes in live cells through fluorescence lifetime imaging microscopy (FLIM). PMID:27589762

A Simple BODIPY-Based Viscosity Probe for Imaging of Cellular Viscosity in Live Cells.

PubMed

Su, Dongdong; Teoh, Chai Lean; Gao, Nengyue; Xu, Qing-Hua; Chang, Young-Tae

2016-08-31

Intracellular viscosity is a fundamental physical parameter that indicates the functioning of cells. In this work, we developed a simple boron-dipyrromethene (BODIPY)-based probe, BTV, for cellular mitochondria viscosity imaging by coupling a simple BODIPY rotor with a mitochondria-targeting unit. The BTV exhibited a significant fluorescence intensity enhancement of more than 100-fold as the solvent viscosity increased. Also, the probe showed a direct linear relationship between the fluorescence lifetime and the media viscosity, which makes it possible to trace the change of the medium viscosity. Furthermore, it was demonstrated that BTV could achieve practical applicability in the monitoring of mitochondrial viscosity changes in live cells through fluorescence lifetime imaging microscopy (FLIM).

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).

Upconverting rare-earth nanoparticles with a paramagnetic lanthanide complex shell for upconversion fluorescent and magnetic resonance dual-modality imaging

NASA Astrophysics Data System (ADS)

Wang, Yan; Ji, Lei; Zhang, Bingbo; Yin, Peihao; Qiu, Yanyan; Song, Daqian; Zhou, Juying; Li, Qi

2013-05-01

Multi-modal imaging based on multifunctional nanoparticles is a promising alternative approach to improve the sensitivity of early cancer diagnosis. In this study, highly upconverting fluorescence and strong relaxivity rare-earth nanoparticles coated with paramagnetic lanthanide complex shells and polyethylene glycol (PEGylated UCNPs@DTPA-Gd3+) are synthesized as dual-modality imaging contrast agents (CAs) for upconverting fluorescent and magnetic resonance dual-modality imaging. PEGylated UCNPs@DTPA-Gd3+ with sizes in the range of 32-86 nm are colloidally stable. They exhibit higher longitudinal relaxivity and transverse relaxivity in water (r1 and r2 values are 7.4 and 27.8 s-1 per mM Gd3+, respectively) than does commercial Gd-DTPA (r1 and r2 values of 3.7 and 4.6 s-1 per mM Gd3+, respectively). They are found to be biocompatible. In vitro cancer cell imaging shows good imaging contrast of PEGylated UCNPs@DTPA-Gd3+. In vivo upconversion fluorescent imaging and T1-weighted MRI show excellent enhancement of both fluorescent and MR signals in the livers of mice administered PEGylated UCNPs@DTPA-Gd3+. All the experimental results indicate that the synthesized PEGylated UCNPs@DTPA-Gd3+ present great potential for biomedical upconversion of fluorescent and magnetic resonance dual-modality imaging applications.

A benzothiazole-based fluorescent probe for hypochlorous acid detection and imaging in living cells

NASA Astrophysics Data System (ADS)

Nguyen, Khac Hong; Hao, Yuanqiang; Zeng, Ke; Fan, Shengnan; Li, Fen; Yuan, Suke; Ding, Xuejing; Xu, Maotian; Liu, You-Nian

2018-06-01

A benzothiazole-based turn-on fluorescent probe with a large Stokes shift (190 nm) has been developed for hypochlorous acid detection. The probe displays prompt fluorescence response for HClO with excellent selectivity over other reactive oxygen species as well as a low detection limit of 0.08 μM. The sensing mechanism involves the HClO-induced specific oxidation of oxime moiety of the probe to nitrile oxide, which was confirmed by HPLC-MS technique. Furthermore, imaging studies demonstrated that the probe is cell permeable and can be applied to detect HClO in living cells.

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

Improving confocal microscopy with solid-state semiconductor excitation sources

NASA Astrophysics Data System (ADS)

Sivers, Nelson L.

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

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

NASA Astrophysics Data System (ADS)

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

2006-03-01

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

Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.

PubMed

Heppert, Jennifer K; Dickinson, Daniel J; Pani, Ariel M; Higgins, Christopher D; Steward, Annette; Ahringer, Julie; Kuhn, Jeffrey R; Goldstein, Bob

2016-11-07

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments. © 2016 Heppert et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

GCaMP expression in retinal ganglion cells characterized using a low-cost fundus imaging system

NASA Astrophysics Data System (ADS)

Chang, Yao-Chuan; Walston, Steven T.; Chow, Robert H.; Weiland, James D.

2017-10-01

Objective. Virus-transduced, intracellular-calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. Due to the existence of an optimal time window for the expression of calcium indicators, a suitable tool for tracking GECI expression in vivo following transduction is highly desirable. Approach. We developed a noninvasive imaging approach based on a custom-modified, low-cost fundus viewing system that allowed us to monitor and characterize in vivo bright-field and fluorescence images of the mouse retina. AAV2-CAG-GCaMP6f was injected into a mouse eye. The fundus imaging system was used to measure fluorescence at several time points post injection. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array and used calcium imaging to evaluate the responsiveness of retinal ganglion cells (RGCs) to external electrical stimulation. Main results. The noninvasive fundus imaging system clearly resolves individual (RGCs and axons. RGC fluorescence intensity and the number of observable fluorescent cells show a similar rising trend from week 1 to week 3 after viral injection, indicating a consistent increase of GCaMP6f expression. Analysis of the in vivo fluorescence intensity trend and in vitro neurophysiological responsiveness shows that the slope of intensity versus days post injection can be used to estimate the optimal time for calcium imaging of RGCs in response to external electrical stimulation. Significance. The proposed fundus imaging system enables high-resolution digital fundus imaging in the mouse eye, based on off-the-shelf components. The long-term tracking experiment with in vitro calcium imaging validation demonstrates the system can serve as a powerful tool monitoring the level of genetically-encoded calcium indicator expression, further determining the optimal time window for following experiment.

Vital-dye-enhanced multimodal imaging of neoplastic progression in a mouse model of oral carcinogenesis

NASA Astrophysics Data System (ADS)

Hellebust, Anne; Rosbach, Kelsey; Wu, Jessica Keren; Nguyen, Jennifer; Gillenwater, Ann; Vigneswaran, Nadarajah; Richards-Kortum, Rebecca

2013-12-01

In this longitudinal study, a mouse model of 4-nitroquinoline 1-oxide chemically induced tongue carcinogenesis was used to assess the ability of optical imaging with exogenous and endogenous contrast to detect neoplastic lesions in a heterogeneous mucosal surface. Widefield autofluorescence and fluorescence images of intact 2-NBDG-stained and proflavine-stained tissues were acquired at multiple time points in the carcinogenesis process. Confocal fluorescence images of transverse fresh tissue slices from the same specimens were acquired to investigate how changes in tissue microarchitecture affect widefield fluorescence images of intact tissue. Widefield images were analyzed to develop and evaluate an algorithm to delineate areas of dysplasia and cancer. A classification algorithm for the presence of neoplasia based on the mean fluorescence intensity of 2-NBDG staining and the standard deviation of the fluorescence intensity of proflavine staining was found to separate moderate dysplasia, severe dysplasia, and cancer from non-neoplastic regions of interest with 91% sensitivity and specificity. Results suggest this combination of noninvasive optical imaging modalities can be used in vivo to discriminate non-neoplastic from neoplastic tissue in this model with the potential to translate this technology to the clinic.

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.

Efficacy of NGR peptide-modified PEGylated quantum dots for crossing the blood-brain barrier and targeted fluorescence imaging of glioma and tumor vasculature.

PubMed

Huang, Ning; Cheng, Si; Zhang, Xiang; Tian, Qi; Pi, Jiangli; Tang, Jun; Huang, Qing; Wang, Feng; Chen, Jin; Xie, Zongyi; Xu, Zhongye; Chen, Weifu; Zheng, Huzhi; Cheng, Yuan

2017-01-01

Delivery of imaging agents to brain glioma is challenging because the blood-brain barrier (BBB) functions as a physiological checkpoint guarding the central nervous system from circulating large molecules. Moreover, the ability of existing probes to target glioma has been insufficient and needs to be improved. In present study, PEG-based long circulation, CdSe/ZnS quantum dots (QDs)-based nanoscale and fluorescence, asparagines-glycine-arginine peptides (NGR)-based specific CD13 recognition were integrated to design and synthesize a novel nanoprobe by conjugating biotinylated NGR peptides to avidin-PEG-coated QDs. Our data showed that the NGR-PEG-QDs were nanoscale with less than 100 nm and were stable in various pH (4.0~8.0). These nanomaterials with non-toxic concentrations could cross the BBB and target CD13-overexpressing glioma and tumor vasculature in vitro and in vivo, contributing to fluorescence imaging of this brain malignancy. These achievements allowed groundbreaking technological advances in targeted fluorescence imaging for the diagnosis and surgical removal of glioma, facilitating potential transformation toward clinical nanomedicine. Copyright © 2016 Elsevier Inc. All rights reserved.

Multistage Spatial Property Based Segmentation for Quantification of Fluorescence Distribution in Cells

NASA Astrophysics Data System (ADS)

Zhang, Guangyun; Jia, Xiuping; Pham, Tuan D.; Crane, Denis I.

2010-01-01

The interpretation of the distribution of fluorescence in cells is often by simple visualization of microscope-derived images for qualitative studies. In other cases, however, it is desirable to be able to quantify the distribution of fluorescence using digital image processing techniques. In this paper, the challenges of fluorescence segmentation due to the noise present in the data are addressed. We report that intensity measurements alone do not allow separation of overlapping data between target and background. Consequently, spatial properties derived from neighborhood profile were included. Mathematical Morphological operations were implemented for cell boundary extraction and a window based contrast measure was developed for fluorescence puncta identification. All of these operations were applied in the proposed multistage processing scheme. The testing results show that the spatial measures effectively enhance the target separability.

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

Facile synthesis of a two-photon fluorescent probe based on pyrimidine 2-isothiocyanate and its application in bioimaging.

PubMed

Yang, Jie; Hu, Wei; Li, Huirong; Hou, Hanna; Tu, Yi; Liu, Bo

2018-04-18

Two-photon microscopy imaging has been widely applied in biological imaging, but the development of two-photon absorption probes is obviously lagging behind in the development of imaging technology. In this paper, a two-photon fluorescent probe (1) based on pyrimidine 2-isothiocyanate has been designed and synthesized through a simple method for two-photon biological imaging. Probe 1 was able to couple effectively with the amino groups on biomolecules. To verify the reactivity of the isothiocyanate group on probe 1 and the amine groups on the biomolecules, d-glucosamine was chosen as a model biomolecule to conjugate with probe 1. The result showed that probe 1 could effectively conjugate with d-glucosamine to synthesize probe 2, and the yield of probe 2 was 83%. After conjugating with d-glucosamine, linear absorption spectra, single-photon fluorescence spectra, and two-photon fluorescence spectra of probes 1 and 2 did not present significant changes. Probes 1 and 2 exhibited high fluorescence quantum yields (0.71-0.79) in toluene and chloroform. They also exhibited different photo-physical properties in solvents with different polarities. The two-photon absorption cross-section of probe 1 was 953 GM in toluene. In addition, probe 1 could be effectively conjugated with transferrin, and the conjugated probe (Tf-1) could be transported into Hep G2 cells through a receptor-mediated process for biological imaging. These results demonstrate that such probes are expected to have great potential applications in two-photon fluorescence bioimaging.

Preparation of Microkernel-Based Mesoporous (SiO2-CdTe-SiO2)@SiO2 Fluorescent Nanoparticles for Imaging Screening and Enrichment of Heat Shock Protein 90 Inhibitors from Tripterygium Wilfordii.

PubMed

Hu, Yue; Miao, Zhao-Yi; Zhang, Xiao-Jing; Yang, Xiao-Tong; Tang, Ying-Ying; Yu, Sheng; Shan, Chen-Xiao; Wen, Hong-Mei; Zhu, Dong

2018-05-01

The currently utilized ligand fishing for bioactive molecular screening from complex matrixes cannot perform imaging screening. Here, we developed a new solid-phase ligand fishing coupled with an in situ imaging protocol for the specific enrichment and identification of heat shock protein 90 (Hsp 90) inhibitors from Tripterygium wilfordii, utilizing a multiple-layer and microkernel-based mesoporous nanostructure composed of a protective silica coating CdTe quantum dot (QD) core and a mesoporous silica shell, i.e., microkernel-based mesoporous (SiO 2 -CdTe-SiO 2 )@SiO 2 fluorescent nanoparticles (MMFNPs) as extracting carries and fluorescent probes. The prepared MMFNPs showed a highly uniform spherical morphology, retention of fluorescence emission, and great chemical stability. The fished ligands by Hsp 90α-MMFNPs were evaluated via the preliminary bioactivity based on real-time cellular morphology imaging by confocal laser scanning microscopy (CLSM) and then identified by mass spectrometry (MS). Celastrol was successfully isolated as an Hsp 90 inhibitor, and two other specific components screened by Hsp 90α-MMFNPs, i.e., demecolcine and wilforine, were preliminarily identified as potential Hsp 90 inhibitors through the verification of strong affinity to Hsp 90 and antitumor bioactivity. The approach based on the MMFNPs provides a strong platform for imaging screening and discovery of plant-derived biologically active molecules with high efficiency and selectivity.

Characterizing the Utility and Limitations of Repurposing an Open-Field Optical Imaging Device for Fluorescence-Guided Surgery in Head and Neck Cancer Patients.

PubMed

Moore, Lindsay S; Rosenthal, Eben L; Chung, Thomas K; de Boer, Esther; Patel, Neel; Prince, Andrew C; Korb, Melissa L; Walsh, Erika M; Young, E Scott; Stevens, Todd M; Withrow, Kirk P; Morlandt, Anthony B; Richman, Joshua S; Carroll, William R; Zinn, Kurt R; Warram, Jason M

2017-02-01

The purpose of this study was to assess the potential of U.S. Food and Drug Administration-cleared devices designed for indocyanine green-based perfusion imaging to identify cancer-specific bioconjugates with overlapping excitation and emission wavelengths. Recent clinical trials have demonstrated potential for fluorescence-guided surgery, but the time and cost of the approval process may impede clinical translation. To expedite this translation, we explored the feasibility of repurposing existing optical imaging devices for fluorescence-guided surgery. Consenting patients (n = 15) scheduled for curative resection were enrolled in a clinical trial evaluating the safety and specificity of cetuximab-IRDye800 (NCT01987375). Open-field fluorescence imaging was performed preoperatively and during the surgical resection. Fluorescence intensity was quantified using integrated instrument software, and the tumor-to-background ratio characterized fluorescence contrast. In the preoperative clinic, the open-field device demonstrated potential to guide preoperative mapping of tumor borders, optimize the day of surgery, and identify occult lesions. Intraoperatively, the device demonstrated robust potential to guide surgical resections, as all peak tumor-to-background ratios were greater than 2 (range, 2.2-14.1). Postresection wound bed fluorescence was significantly less than preresection tumor fluorescence (P < 0.001). The repurposed device also successfully identified positive margins. The open-field imaging device was successfully repurposed to distinguish cancer from normal tissue in the preoperative clinic and throughout surgical resection. This study illuminated the potential for existing open-field optical imaging devices with overlapping excitation and emission spectra to be used for fluorescence-guided surgery. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Non-invasive imaging of prostate cancer progression in nude mice using iRFP gene reporter

NASA Astrophysics Data System (ADS)

Zhu, Banghe; Wu, Grace; Robinson, Holly; Wilganowski, Nathaniel; Sevick-Muraca, Eva M.

2013-03-01

Prostate cancer (PCa) is the second most common cancer in US men. Metastasis is the final step of tumor progression and remains the primary cause of PCa death. Hence preclinical, orthotopic models of PCa metastasis are necessary to develop new therapeutics against metastatic disease. Yet unlike irrelevant subcutaneous tumor models, the deployment of orthotopic models of cancer metastasis in drug research and development is limited by the inability to longitudinally monitor cancer progression/regression in response to administration of experimental pharmaceuticals. Recently, a nearinfrared fluorescent protein (iRFP) was created for deeper imaging [1]. Imaging prostate tumor growth and lymph node metastasis in nude mice therefore becomes possible using this new fluorescent gene reporter. In this study, we first developed an intensified CCD (ICCD)-based iRFP fluorescence imaging device. Then human PCa PC3 cell lines expressing iRFP gene reporter were orthotopically implanted in male Nu/Nu mice at 8-10 weeks old. After 6-10 weeks, in vivo, in situ and ex vivo fluorescence imaging was performed. In vivo iRFP fluorescence imaging showed that the detected fluorescence concentrated at the prostate and became stronger over time, indicating the growth of implanted PCa. Fluorescence was non-invasively detected at locations of prostate-draining lymph nodes as early as 5 weeks post implantation, indicating the metastasis to lymph nodes. In situ and ex vivo fluorescence imaging demonstrated that the detected signals from PCa and lymph nodes were correlated with cancer positive status of tissues as assessed through standard pathology.

Intraoperative Near-infrared Imaging for Parathyroid Gland Identification by Auto-fluorescence: A Feasibility Study.

PubMed

De Leeuw, Frederic; Breuskin, Ingrid; Abbaci, Muriel; Casiraghi, Odile; Mirghani, Haïtham; Ben Lakhdar, Aïcha; Laplace-Builhé, Corinne; Hartl, Dana

2016-09-01

Parathyroid glands (PGs) can be particularly hard to distinguish from surrounding tissue and thus can be damaged or removed during thyroidectomy. Postoperative hypoparathyroidism is the most common complication after thyroidectomy. Very recently, it has been found that the parathyroid tissue shows near-infrared (NIR) auto-fluorescence which could be used for intraoperative detection, without any use of contrast agents. The work described here presents a histological validation ex vivo of the NIR imaging procedure and evaluates intraoperative PG detection by NIR auto-fluorescence using for the first time to our knowledge a commercially available clinical NIR imaging device. Ex vivo study on resected operative specimens combined with a prospective in vivo study of consecutive patients who underwent total or partial thyroid, or parathyroid surgery at a comprehensive cancer center. During surgery, any tissue suspected to be a potential PG by the surgeon was imaged with the Fluobeam 800 (®) system. NIR imaging was compared to conventional histology (ex vivo) and/or visual identification by the surgeon (in vivo). We have validated NIR auto-fluorescence with an ex vivo study including 28 specimens. Sensitivity and specificity were 94.1 and 80 %, respectively. Intraoperative NIR imaging was performed in 35 patients and 81 parathyroids were identified. In 80/81 cases, the fluorescence signal was subjectively obvious on real-time visualization. We determined that PG fluorescence is 2.93 ± 1.59 times greater than thyroid fluorescence in vivo. Real-time NIR imaging based on parathyroid auto-fluorescence is fast, safe, and non-invasive and shows very encouraging results, for intraoperative parathyroid identification.

Synthesis and characterization of citrate-based fluorescent small molecules and biodegradable polymers.

PubMed

Xie, Zhiwei; Kim, Jimin P; Cai, Qing; Zhang, Yi; Guo, Jinshan; Dhami, Ranjodh S; Li, Li; Kong, Bin; Su, Yixue; Schug, Kevin A; Yang, Jian

2017-03-01

Novel citric acid based photoluminescent dyes and biodegradable polymers are synthesized via a facile "one-pot" reaction. A comprehensive understanding of the fluorescence mechanisms of the resulting citric acid-based fluorophores is reported. Two distinct types of fluorophores are identified: a thiozolopyridine family with high quantum yield, long lifetime, and exceptional photostability, and a dioxopyridine family with relatively lower quantum yield, multiple lifetimes, and solvent-dependent band shifting behavior. Applications in molecular labeling and cell imaging were demonstrated. The above discoveries contribute to the field of fluorescence chemistry and have laid a solid foundation for further development of new fluorophores and materials that show promise in a diversity of fluorescence-based applications. Photoluminescent materials are pivotal for fluorescence based imaging, labeling and sensing applications. Understanding their fluorescence mechanism is challenging and imperative. We develop a new class of citric acid-derived fluorescent materials in forms of polymers and small molecular dyes by a one-step solvent free reaction. We discovered two different classes of citric acid-derived fluorophores. A two-ring thiozolopyridine structure demonstrates strong fluorescence and exceptional resistance to photo-bleaching. A one-ring dioxopyridine exhibits relative weak fluorescence but with intriguing excitation and solvent-dependent emission wavelength shifting. Our methodology of synthesizing citric acid-derived fluorophores and the understanding on their luminescence are instrumental to the design and production of a large number of new photoluminescent materials for biological and biomedical applications. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Understanding the Biological Basis of Autofluorescence Imaging for Oral Cancer Detection: High-Resolution Fluorescence Microscopy in Viable Tissue

PubMed Central

Pavlova, Ina; Williams, Michelle; El-Naggar, Adel; Richards-Kortum, Rebecca; Gillenwater, Ann

2009-01-01

Purpose Autofluorescence imaging is increasingly used to noninvasively identify neoplastic oral cavity lesions. Improving the diagnostic accuracy of these techniques requires a better understanding of the biological basis for optical changes associated with neoplastic transformation in oral tissue. Experimental Design A total of 49 oral biopsies were considered in this study. The autofluorescence patterns of viable normal, benign, and neoplastic oral tissue were imaged using high-resolution confocal fluorescence microscopy. Results The autofluorescence properties of oral tissue vary significantly based on anatomic site and pathologic diagnosis. In normal oral tissue, most of the epithelial autofluorescence originates from the cytoplasm of cells in the basal and intermediate regions, whereas structural fibers are responsible for most of the stromal fluorescence. A strongly fluorescent superficial layer was observed in tissues from the palate and the gingiva, which contrasts with the weakly fluorescent superficial layer found in other oral sites. Upon UV excitation, benign inflammation shows decreased epithelial fluorescence, whereas dysplasia displays increased epithelial fluorescence compared with normal oral tissue. Stromal fluorescence in both benign inflammation and dysplasia drops significantly at UV and 488 nm excitation. Conclusion Imaging oral lesions with optical devices/probes that sample mostly stromal fluorescence may result in a similar loss of fluorescence intensity and may fail to distinguish benign from precancerous lesions. Improved diagnostic accuracy may be achieved by designing optical probes/devices that distinguish epithelial fluorescence from stromal fluorescence and by using excitation wavelengths in the UV range. PMID:18413830

Pyridine Based Fluorescence Probe: Simultaneous Detection and Removal of Arsenate from Real Samples with Living Cell Imaging Properties.

PubMed

Nandi, Sandip; Sahana, Animesh; Sarkar, Bidisha; Mukhopadhyay, Subhra Kanti; Das, Debasis

2015-09-01

Pyridine based fluorescence probe, DFPPIC and its functionalized Merrifield polymer has been synthesized, characterized and used as an arsenate selective fluorescence sensor. Arsenate induced fluorescence enhancement is attributed to inter-molecular H-bonding assisted CHEF process. The detection limit for arsenate is 0.001 μM, much below the WHO recommended tolerance level in drinking water. DFPPIC can detect intracellular arsenate in drinking water of Purbasthali, West Bengal, India efficiently. Graphical Abstract DFPPIC and its Merrifield conjugate polymer are used for selective determination and removal of arsenate from real drinking water samples of Purbasthali, a highly arsenic contaminated region of West Bengal, India. DFPPIC is very promising to imaging arsenate in living cells.

Fluorescent nanoparticles based on AIE fluorogens for bioimaging.

PubMed

Yan, Lulin; Zhang, Yan; Xu, Bin; Tian, Wenjing

2016-02-07

Fluorescent nanoparticles (FNPs) have recently attracted increasing attention in the biomedical field because of their unique optical properties, easy fabrication and outstanding performance in imaging. Compared with conventional molecular probes including small organic dyes and fluorescent proteins, FNPs based on aggregation-induced emission (AIE) fluorogens have shown significant advantages in tunable emission and brightness, good biocompatibility, superb photo- and physical stability, potential biodegradability and facile surface functionalization. In this review, we summarize the latest advances in the development of fluorescent nanoparticles based on AIE fluorogens including polymer nanoparticles and silica nanoparticles over the past few years, and the various biomedical applications based on these fluorescent nanoparticles are also elaborated.

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

PubMed Central

Fruhwirth, Gilbert O.; Ameer-Beg, Simon; Cook, Richard; Watson, Timothy; Ng, Tony; Festy, Frederic

2010-01-01

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications. PMID:20588974

Fluorescent Imaging of Single Nanoparticles and Viruses on a Smart Phone

PubMed Central

Wei, Qingshan; Qi, Hangfei; Luo, Wei; Tseng, Derek; Ki, So Jung; Wan, Zhe; Göröcs, Zoltán; Bentolila, Laurent A.; Wu, Ting-Ting; Sun, Ren; Ozcan, Aydogan

2014-01-01

Optical imaging of nanoscale objects, whether it is based on scattering or fluorescence, is a challenging task due to reduced detection signal-to-noise ratio and contrast at subwavelength dimensions. Here, we report a field-portable fluorescence microscopy platform installed on a smart phone for imaging of individual nanoparticles as well as viruses using a lightweight and compact opto-mechanical attachment to the existing camera module of the cell phone. This hand-held fluorescent imaging device utilizes (i) a compact 450 nm laser diode that creates oblique excitation on the sample plane with an incidence angle of ~75°, (ii) a long-pass thin-film interference filter to reject the scattered excitation light, (iii) an external lens creating 2× optical magnification, and (iv) a translation stage for focus adjustment. We tested the imaging performance of this smart-phone-enabled microscopy platform by detecting isolated 100 nm fluorescent particles as well as individual human cytomegaloviruses that are fluorescently labeled. The size of each detected nano-object on the cell phone platform was validated using scanning electron microscopy images of the same samples. This field-portable fluorescence microscopy attachment to the cell phone, weighing only ~186 g, could be used for specific and sensitive imaging of subwavelength objects including various bacteria and viruses and, therefore, could provide a valuable platform for the practice of nanotechnology in field settings and for conducting viral load measurements and other biomedical tests even in remote and resource-limited environments. PMID:24016065

Fluorenyl benzothiadiazole and benzoselenadiazole near-IR fluorescent probes for two-photon fluorescence imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Belfield, Kevin D.; Yao, Sheng; Kim, Bosung; Yue, Xiling

2016-03-01

Imaging biological samples with two-photon fluorescence (2PF) microscopy has the unique advantage of resulting high contrast 3D resolution subcellular image that can reach up to several millimeters depth. 2PF probes that absorb and emit at near IR region need to be developed. Two-photon excitation (2PE) wavelengths are less concerned as 2PE uses wavelengths doubles the absorption wavelength of the probe, which means 2PE wavelengths for probes even with absorption at visible wavelength will fall into NIR region. Therefore, probes that fluoresce at near IR region with high quantum yields are needed. A series of dyes based on 5-thienyl-2, 1, 3-benzothiadiazole and 5-thienyl-2, 1, 3-benzoselenadiazole core were synthesized as near infrared two-photon fluorophores. Fluorescence maxima wavelengths as long as 714 nm and fluorescence quantum yields as high as 0.67 were achieved. The fluorescence quantum yields of the dyes were nearly constant, regardless of solvents polarity. These diazoles exhibited large Stokes shift (<114nm), high two-photon absorption cross sections (up to 2,800 GM), and high two-photon fluorescence figure of merit (FM , 1.04×10-2 GM). Cells incubated on a 3D scaffold with one of the new probes (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.

Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection

PubMed Central

Bates, Mark; Dempsey, Graham T; Chen, Kok Hao; Zhuang, Xiaowei

2012-01-01

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution. PMID:22213647

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

PubMed

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

2015-12-01

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

Inverse transport problems in quantitative PAT for molecular imaging

NASA Astrophysics Data System (ADS)

Ren, Kui; Zhang, Rongting; Zhong, Yimin

2015-12-01

Fluorescence photoacoustic tomography (fPAT) is a molecular imaging modality that combines photoacoustic tomography with fluorescence imaging to obtain high-resolution imaging of fluorescence distributions inside heterogeneous media. The objective of this work is to study inverse problems in the quantitative step of fPAT where we intend to reconstruct physical coefficients in a coupled system of radiative transport equations using internal data recovered from ultrasound measurements. We derive uniqueness and stability results on the inverse problems and develop some efficient algorithms for image reconstructions. Numerical simulations based on synthetic data are presented to validate the theoretical analysis. The results we present here complement these in Ren K and Zhao H (2013 SIAM J. Imaging Sci. 6 2024-49) on the same problem but in the diffusive regime.

Open-air multispectral fluorescence-guided surgery platform for intraoperative detection of malignant tissue under ambient lighting conditions

NASA Astrophysics Data System (ADS)

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

2017-02-01

Intraoperative resection of tumors currently relies upon the surgeon's ability to visually locate and palpate tumor nodules. Undetected residual malignant tissue often results in the need for additional treatment or surgical intervention. The Solaris platform is a multispectral open-air fluorescence imaging system designed for translational fluorescence-guided surgery. Solaris supports video-rate imaging in four fixed fluorescence channels ranging from visible to near infrared, and a multispectral channel equipped with a liquid crystal tunable filter (LCTF) for multispectral image acquisition (520-620 nm). Identification of tumor margins using reagents emitting in the visible spectrum (400-650 nm), such as fluorescein isothiocyanate (FITC), present challenges considering the presence of auto-fluorescence from tissue and food in the gastrointestinal (GI) tract. To overcome this, Solaris acquires LCTF-based multispectral images, and by applying an automated spectral unmixing algorithm to the data, separates reagent fluorescence from tissue and food auto-fluorescence. The unmixing algorithm uses vertex component analysis to automatically extract the primary pure spectra, and resolves the reagent fluorescent signal using non-negative least squares. For validation, intraoperative in vivo studies were carried out in tumor-bearing rodents injected with FITC-dextran reagent that is primarily residing in malignant tissue 24 hours post injection. In the absence of unmixing, fluorescence from tumors is not distinguishable from that of surrounding tissue. Upon spectral unmixing, the FITC-labeled malignant regions become well defined and detectable. The results of these studies substantiate the multispectral power of Solaris in resolving FITC-based agent signal in deep tumor masses, under ambient and surgical light, and enhancing the ability to surgically resect them.

PET and NIR Optical Imaging Using Self-Illuminating 64Cu-Doped Chelator-Free Gold Nanoclusters

PubMed Central

Hu, Hao; Huang, Peng; Weiss, Orit Jacobson; Yan, Xuefeng; Yue, Xuyi; Zhang, Molly Gu; Tang, Yuxia; Nie, Liming; Ma, Ying; Niu, Gang; Wu, Kaichun; Chen, Xiaoyuan

2014-01-01

Self-illuminating fluorescence imaging without autofluorescence background interference has recently aroused more research interests in molecular imaging. Currently, only a few self-illuminating probes were developed, based mainly on toxic quantum dots such as CdSe, CdTe. Herein, we report a novel design of nontoxic self-illuminating gold nanocluster (64Cu-doped AuNCs) for dual-modality positron emission tomography (PET) and near-infrared (NIR) fluorescence imaging based on Cerenkov resonance energy transfer (CRET). PET radionuclide 64Cu was introduced by a chelator-free doping method, which played dual roles as the energy donor and the PET imaging source. Meanwhile, AuNCs acted as the energy acceptor for NIR fluorescence imaging. 64Cu-doped AuNCs exhibited efficient CRET-NIR and PET imaging both in vitro and in vivo. In a U87MG glioblastoma xenograft model, 64Cu-doped AuNCs showed high tumor uptake (14.9%ID/g at 18 h) and produced satisfactory tumor self-illuminating NIR images in the absence of external excitation. This self-illuminating nanocluster with non-toxicity and good biocompatibility can be employed as a novel imaging contrast agent for biomedical applications, especially for molecular imaging. PMID:25224367

PET and NIR optical imaging using self-illuminating (64)Cu-doped chelator-free gold nanoclusters.

PubMed

Hu, Hao; Huang, Peng; Weiss, Orit Jacobson; Yan, Xuefeng; Yue, Xuyi; Zhang, Molly Gu; Tang, Yuxia; Nie, Liming; Ma, Ying; Niu, Gang; Wu, Kaichun; Chen, Xiaoyuan

2014-12-01

Self-illuminating fluorescence imaging without autofluorescence background interference has recently aroused more research interests in molecular imaging. Currently, only a few self-illuminating probes were developed, based mainly on toxic quantum dots such as CdSe, CdTe. Herein, we report a novel design of nontoxic self-illuminating gold nanocluster ((64)Cu-doped AuNCs) for dual-modality positron emission tomography (PET) and near-infrared (NIR) fluorescence imaging based on Cerenkov resonance energy transfer (CRET). PET radionuclide (64)Cu was introduced by a chelator-free doping method, which played dual roles as the energy donor and the PET imaging source. Meanwhile, AuNCs acted as the energy acceptor for NIR fluorescence imaging. (64)Cu-doped AuNCs exhibited efficient CRET-NIR and PET imaging both in vitro and in vivo. In a U87MG glioblastoma xenograft model, (64)Cu-doped AuNCs showed high tumor uptake (14.9 %ID/g at 18 h) and produced satisfactory tumor self-illuminating NIR images in the absence of external excitation. This self-illuminating nanocluster with non-toxicity and good biocompatibility can be employed as a novel imaging contrast agent for biomedical applications, especially for molecular imaging. Published by Elsevier Ltd.

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.

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.

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.

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.

One-step fabrication of PEGylated fluorescent nanodiamonds through the thiol-ene click reaction and their potential for biological imaging

NASA Astrophysics Data System (ADS)

Huang, Hongye; Liu, Meiying; Tuo, Xun; Chen, Junyu; Mao, Liucheng; Wen, Yuanqing; Tian, Jianwen; Zhou, Naigen; Zhang, Xiaoyong; Wei, Yen

2018-05-01

Over the past years, fluorescent carbon nanoparticles have got growing interest for biological imaging. Fluorescent nanodiamonds (FNDs) are novel fluorescent carbon nanoparticles with multitudinous useful properties, including remarkable fluorescence properties, extremely low toxicity and high refractive index. However, facile preparation of FNDs with designable properties and functions from non-fluorescent detonation nanodiamonds (DNDs) has demonstrated to be challengeable. In this work, we reported for the first time that preparation of Polyethylene glycol (PEG) functionalized FNDs through a one-step thiol-ene click reaction using thiol containing PEG (PEG-SH) as the coating agent. Based on the characterization results, we demonstrated that PEG-SH could be efficiently introduced on DNDs to obtain FNDs through the thiol-ene click chemistry. The resultant FND-PEG composites showed high water dispersibility, strong fluorescence and low cytotoxicity. Moreover, FND-PEG composites could be internalized by cells and displayed good cell dyeing performance. All of these features implied that FND-PEG composites are of great potential for biological imaging. Taken together, a facile one-step strategy based on the one-step thiol-ene click reaction has been developed for efficient preparation of FND-PEG composites from non-fluorescent DNDs. The strategy should be also useful for fabrication of many other functional FNDs via using different thiol containing compounds for the universality of thiol-ene click reaction.

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.

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.

Visualizing tributyltin (TBT) in bacterial aggregates by specific rhodamine-based fluorescent probes.

PubMed

Jin, Xilang; Hao, Likai; She, Mengyao; Obst, Martin; Kappler, Andreas; Yin, Bing; Liu, Ping; Li, Jianli; Wang, Lanying; Shi, Zhen

2015-01-01

Here we present the first examples of fluorescent and colorimetric probes for microscopic TBT imaging. The fluorescent probes are highly selective and sensitive to TBT and have successfully been applied for imaging of TBT in bacterial Rhodobacter ferrooxidans sp. strain SW2 cell-EPS-mineral aggregates and in cell suspensions of the marine cyanobacterium Synechococcus PCC 7002 by using confocal laser scanning microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

Chip-based wide field-of-view nanoscopy

NASA Astrophysics Data System (ADS)

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

2017-04-01

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

Study of Fluorescent Imaging of Se (IV) in Living Cells Using a Turn-on Fluorescent Probe Based on a Rhodamine Spirolactame Derivative.

PubMed

Guan, Mingming; Mi, Hongyu; Xu, Hui; Fei, Qiang; Shan, Hongyan; Huan, Yanfu; Lv, Shaowu; Feng, Guodong

2017-03-01

A highly selective fluorescent probe 2-(2-(2-aminoethylamino)ethyl)-3',6'-bis(ethylamino)-2',7'-dimethylspiro[isoindoline-1,9'-xanthen]-3-one (ABDO) for Se (IV) had been synthesized in our earlier report. In this study, this fluorescent sensor is applied on analysis fluorescent imaging of Se (IV) in Hela cells. The experiment conditions, such as the MTT assay, different concentration of saline, incubated time of Hela cells with ABDO and Se (IV), and intracellular action position of Se (IV), are investigated. Through a series of experiments, the fluorescent image of Se (IV) in Hela cells can be observed when the cells cultured by 2 μM ABDO and 2 μM Se (IV) for 210 min. And the intracellular action position of Se (IV) is verified after the co-localization experiments are done. It is mitochondria. These experimental results show that ABDO will be an eagerly anticipated sensor for fluorescent imaging analysis of selenium ion in living cells. Besides, we also can use the complexes of ABDO-Se to observe morphology and distribution of mitochondria in cells like JG-B.

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.

Manganese-containing Prussian blue nanoparticles for imaging of pediatric brain tumors

PubMed Central

Dumont, Matthieu F; Yadavilli, Sridevi; Sze, Raymond W; Nazarian, Javad; Fernandes, Rohan

2014-01-01

Pediatric brain tumors (PBTs) are a leading cause of death in children. For an improved prognosis in patients with PBTs, there is a critical need to develop molecularly-specific imaging agents to monitor disease progression and response to treatment. In this paper, we describe manganese-containing Prussian blue nanoparticles as agents for molecular magnetic resonance imaging (MRI) and fluorescence-based imaging of PBTs. Our core-shell nanoparticles consist of a core lattice structure that incorporates and retains paramagnetic Mn2+ ions, and generates MRI contrast (both negative and positive). The biofunctionalized shell is comprised of fluorescent avidin, which serves the dual purpose of enabling fluorescence imaging and functioning as a platform for the attachment of biotinylated ligands that target PBTs. The surfaces of our nanoparticles are modified with biotinylated antibodies targeting neuron-glial antigen 2 or biotinylated transferrin. Both neuron-glial antigen 2 and the transferrin receptor are protein markers overexpressed in PBTs. We describe the synthesis, biofunctionalization, and characterization of these multimodal nanoparticles. Further, we demonstrate the MRI and fluorescence imaging capabilities of manganese-containing Prussian blue nanoparticles in vitro. Finally, we demonstrate the potential of these nanoparticles as PBT imaging agents by measuring their organ and brain biodistribution in an orthotopic mouse model of PBTs using ex vivo fluorescence imaging. PMID:24920896

A projective surgical navigation system for cancer resection

NASA Astrophysics Data System (ADS)

Gan, Qi; Shao, Pengfei; Wang, Dong; Ye, Jian; Zhang, Zeshu; Wang, Xinrui; Xu, Ronald

2016-03-01

Near infrared (NIR) fluorescence imaging technique can provide precise and real-time information about tumor location during a cancer resection surgery. However, many intraoperative fluorescence imaging systems are based on wearable devices or stand-alone displays, leading to distraction of the surgeons and suboptimal outcome. To overcome these limitations, we design a projective fluorescence imaging system for surgical navigation. The system consists of a LED excitation light source, a monochromatic CCD camera, a host computer, a mini projector and a CMOS camera. A software program is written by C++ to call OpenCV functions for calibrating and correcting fluorescence images captured by the CCD camera upon excitation illumination of the LED source. The images are projected back to the surgical field by the mini projector. Imaging performance of this projective navigation system is characterized in a tumor simulating phantom. Image-guided surgical resection is demonstrated in an ex-vivo chicken tissue model. In all the experiments, the projected images by the projector match well with the locations of fluorescence emission. Our experimental results indicate that the proposed projective navigation system can be a powerful tool for pre-operative surgical planning, intraoperative surgical guidance, and postoperative assessment of surgical outcome. We have integrated the optoelectronic elements into a compact and miniaturized system in preparation for further clinical validation.

Fluorescence Imaging Assisted Photodynamic Therapy Using Photosensitizer-Linked Gold Quantum Clusters.

PubMed

Nair, Lakshmi V; Nazeer, Shaiju S; Jayasree, Ramapurath S; Ajayaghosh, Ayyappanpillai

2015-06-23

Fluorescence imaging assisted photodynamic therapy (PDT) is a viable two-in-one clinical tool for cancer treatment and follow-up. While the surface plasmon effect of gold nanorods and nanoparticles has been effective for cancer therapy, their emission properties when compared to gold nanoclusters are weak for fluorescence imaging guided PDT. In order to address the above issues, we have synthesized a near-infrared-emitting gold quantum cluster capped with lipoic acid (L-AuC with (Au)18(L)14) based nanoplatform with excellent tumor reduction property by incorporating a tumor-targeting agent (folic acid) and a photosensitizer (protoporphyrin IX), for selective PDT. The synthesized quantum cluster based photosensitizer PFL-AuC showed 80% triplet quantum yield when compared to that of the photosensitizer alone (63%). PFL-AuC having 60 μg (0.136 mM) of protoporphyrin IX was sufficient to kill 50% of the tumor cell population. Effective destruction of tumor cells was evident from the histopathology and fluorescence imaging, which confirm the in vivo PDT efficacy of PFL-AuC.

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

A fluorescence-based imaging approach to pharmacokinetic analysis of intracochlear drug delivery.

PubMed

Ayoob, Andrew M; Peppi, Marcello; Tandon, Vishal; Langer, Robert; Borenstein, Jeffrey T

2018-04-05

Advances in microelectromechanical systems (MEMS) technologies are enhancing the development of intracochlear delivery devices for the treatment of hearing loss with emerging pharmacological therapies. Direct intracochlear delivery addresses the limitations of systemic and intratympanic delivery. However, optimization of delivery parameters for these devices requires pharmacokinetic assessment of the spatiotemporal drug distribution inside the cochlea. Robust methods of measuring drug concentration in the perilymph have been developed, but lack spatial resolution along the tonotopic axis or require complex physiological measurements. Here we describe an approach for quantifying distribution of fluorescent drug-surrogate probe along the cochlea's sensory epithelium with high spatial resolution enabled by confocal fluorescence imaging. Fluorescence from FM 1-43 FX, a fixable endocytosis marker, was quantified using confocal fluorescence imaging of whole mount sections of the organ of Corti from cochleae resected and fixed at several time points after intracochlear delivery. Intracochlear delivery of FM 1-43 FX near the base of the cochlea produces a base-apex gradient of fluorescence in the row of inner hair cells after 1 h post-delivery that is consistent with diffusion-limited transport along the scala tympani. By 3 h post-delivery there is approximately an order of magnitude decrease in peak average fluorescence intensity, suggesting FM 1-43 FX clearance from both the perilymph and inner hair cells. The increase in fluorescence intensity at 72 h post-delivery compared to 3 h post-delivery may implicate a potential radial transport pathway into the scala media. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Imaging workflow and calibration for CT-guided time-domain fluorescence tomography

PubMed Central

Tichauer, Kenneth M.; Holt, Robert W.; El-Ghussein, Fadi; Zhu, Qun; Dehghani, Hamid; Leblond, Frederic; Pogue, Brian W.

2011-01-01

In this study, several key optimization steps are outlined for a non-contact, time-correlated single photon counting small animal optical tomography system, using simultaneous collection of both fluorescence and transmittance data. The system is presented for time-domain image reconstruction in vivo, illustrating the sensitivity from single photon counting and the calibration steps needed to accurately process the data. In particular, laser time- and amplitude-referencing, detector and filter calibrations, and collection of a suitable instrument response function are all presented in the context of time-domain fluorescence tomography and a fully automated workflow is described. Preliminary phantom time-domain reconstructed images demonstrate the fidelity of the workflow for fluorescence tomography based on signal from multiple time gates. PMID:22076264

Tracking multiple particles in fluorescence time-lapse microscopy images via probabilistic data association.

PubMed

Godinez, William J; Rohr, Karl

2015-02-01

Tracking subcellular structures as well as viral structures displayed as 'particles' in fluorescence microscopy images yields quantitative information on the underlying dynamical processes. We have developed an approach for tracking multiple fluorescent particles based on probabilistic data association. The approach combines a localization scheme that uses a bottom-up strategy based on the spot-enhancing filter as well as a top-down strategy based on an ellipsoidal sampling scheme that uses the Gaussian probability distributions computed by a Kalman filter. The localization scheme yields multiple measurements that are incorporated into the Kalman filter via a combined innovation, where the association probabilities are interpreted as weights calculated using an image likelihood. To track objects in close proximity, we compute the support of each image position relative to the neighboring objects of a tracked object and use this support to recalculate the weights. To cope with multiple motion models, we integrated the interacting multiple model algorithm. The approach has been successfully applied to synthetic 2-D and 3-D images as well as to real 2-D and 3-D microscopy images, and the performance has been quantified. In addition, the approach was successfully applied to the 2-D and 3-D image data of the recent Particle Tracking Challenge at the IEEE International Symposium on Biomedical Imaging (ISBI) 2012.

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

PubMed

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

2016-04-01

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

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.

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

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.

Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle.

PubMed

Santos, Silvia; Chu, Kengyeh K; Lim, Daryl; Bozinovic, Nenad; Ford, Tim N; Hourtoule, Claire; Bartoo, Aaron C; Singh, Satish K; Mertz, Jerome

2009-01-01

We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.

Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle

NASA Astrophysics Data System (ADS)

Santos, Silvia; Chu, Kengyeh K.; Lim, Daryl; Bozinovic, Nenad; Ford, Tim N.; Hourtoule, Claire; Bartoo, Aaron C.; Singh, Satish K.; Mertz, Jerome

2009-05-01

We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.

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.

Thermally activated delayed fluorescence of fluorescein derivative for time-resolved and confocal fluorescence imaging.

PubMed

Xiong, Xiaoqing; Song, Fengling; Wang, Jingyun; Zhang, Yukang; Xue, Yingying; Sun, Liangliang; Jiang, Na; Gao, Pan; Tian, Lu; Peng, Xiaojun

2014-07-09

Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔE(ST) was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Depletion-based techniques for super-resolution imaging of NV-diamond

NASA Astrophysics Data System (ADS)

Jaskula, Jean-Christophe; Trifonov, Alexei; Glenn, David; Walsworth, Ronald

2012-06-01

We discuss the development and application of depletion-based techniques for super-resolution imaging of NV centers in diamond: stimulated emission depletion (STED), metastable ground state depletion (GSD), and dark state depletion (DSD). NV centers in diamond do not bleach under optical excitation, are not biotoxic, and have long-lived electronic spin coherence and spin-state-dependent fluorescence. Thus NV-diamond has great potential as a fluorescent biomarker and as a magnetic biosensor.

A novel peptide-based fluorescence chemosensor for selective imaging of hydrogen sulfide both in living cells and zebrafish.

PubMed

Wang, Peng; Wu, Jiang; Di, Cuixia; Zhou, Rong; Zhang, Hong; Su, Pingru; Xu, Cong; Zhou, Panpan; Ge, Yushu; Liu, Dan; Liu, Weisheng; Tang, Yu

2017-06-15

Hydrogen sulfide (H 2 S) plays an important role as a signaling compound (gasotransmitter) in living systems. However, the development of an efficient imaging chemosensor of H 2 S in live animals is a challenging field for chemists. Herein, a novel peptide-based fluorescence chemosensor L-Cu was designed and synthesized on the basis of the copper chelating with the peptide ligand (FITC-Ahx-Ser-Pro-Gly-His-NH 2 , L), and its H 2 S sensing ability has been evaluated both in living cells and zebrafish. The peptide backbone and Cu 2+ -removal sensing mechanism are used to deliver rapid response time, high sensitivity, and good biocompatibility. After a fast fluorescence quench by Cu 2+ coordinated with L, the fluorescence of L is recovered by adding S 2- to form insoluble copper sulfide in aqueous solution with a detection limit for hydrogen sulfide measured to be 31nM. Furthermore, the fluorescence chemosensor L-Cu showed excellent cell permeation and low biotoxicity to realize the intracellular biosensing, L-Cu has also been applied to image hydrogen sulfide in live zebrafish larvae. We expect that this peptide-based fluorescence chemosensor L-Cu can be used to study H 2 S-related chemical biology in physiological and pathological events. Copyright © 2016 Elsevier B.V. All rights reserved.

The Quality of In Vivo Upconversion Fluorescence Signals Inside Different Anatomic Structures.

PubMed

Wang, Lijiang; Draz, Mohamed Shehata; Wang, Wei; Liao, Guodong; Xu, Yuhong

2015-02-01

Fluorescence imaging is a broadly interesting and rapidly growing strategy for non-invasive clinical applications. However, because of interference from light scattering, absorbance, and tissue autofluorescence, the images can exhibit low sensitivity and poor quality. Upconversion fluorescence imaging, which is based on the use of near-infrared (NIR) light for excitation, has recently been introduced as an improved approach to minimize the effects of light scattering and tissue autofluorescence. This strategy is promising for ultrasensitive and deep tissue imaging applications. However, the emitted upconversion fluorescence signals are primarily in the visible range and are likely to be absorbed and scattered by tissues. Therefore, different anatomic structures could impose various effects on the quality of the images. In this study, we used upconversion-core/silica-shell nanoprobes to evaluate the quality of upconversion fluorescence at different anatomic locations in athymic nude mice. The nanoprobe contained an upconversion core, which was green (β-NaYF4:Yb3+/Ho3+) or red (β-NaYF4:Yb3+/Er3+), and a nonporous silica shell to allow for multicolor imaging. High-quality upconversion fluorescence signals were detected with signal-to-noise ratios of up to 170 at tissue depths of up to - 1.0 cm when a 980 nm laser excitation source and a bandpass emission filter were used. The presence of dense tissue structures along the imaging path reduced the signal intensity and imaging quality, and nanoprobes with longer-wavelength emission spectra were therefore preferable. This study offers a detailed analysis of the quality of upconversion signals in vivo inside different anatomic structures. Such information could be essential for the analysis of upconversion fluorescence images in any in vivo biodiagnostic and microbial tracking applications.

Experimental assessment and analysis of super-resolution in fluorescence microscopy based on multiple-point spread function fitting of spectrally demultiplexed images

NASA Astrophysics Data System (ADS)

Nishimura, Takahiro; Kimura, Hitoshi; Ogura, Yusuke; Tanida, Jun

2018-06-01

This paper presents an experimental assessment and analysis of super-resolution microscopy based on multiple-point spread function fitting of spectrally demultiplexed images using a designed DNA structure as a test target. For the purpose, a DNA structure was designed to have binding sites at a certain interval that is smaller than the diffraction limit. The structure was labeled with several types of quantum dots (QDs) to acquire their spatial information as spectrally encoded images. The obtained images are analyzed with a point spread function multifitting algorithm to determine the QD locations that indicate the binding site positions. The experimental results show that the labeled locations can be observed beyond the diffraction-limited resolution using three-colored fluorescence images that were obtained with a confocal fluorescence microscope. Numerical simulations show that labeling with eight types of QDs enables the positions aligned at 27.2-nm pitches on the DNA structure to be resolved with high accuracy.

Tracers for fluorescence-guided surgery: how elongation of the polymethine chain in cyanine dyes alters the pharmacokinetics of a (bimodal) c[RGDyK] tracer.

PubMed

Buckle, Tessa; van Willigen, Danny M; Spa, Silvia J; Hensbergen, Albertus W; van der Wal, Steffen; de Korne, Clarize M; Welling, Mick M; van der Poel, Henk G; Hardwick, James C H; van Leeuwen, Fijs W B

2018-02-15

Objectives: The potential of (receptor-mediated) fluorescence-based image-guided surgery tracers is generally linked to the near-infrared emission profile and good manufacturing production (GMP) availability of fluorescent dyes. Surprisingly, little is known about the critical interaction between the structural composition of the dye and the pharmacokinetics of the tracers. In this study, a bimodal/hybrid tracer design was used to systematically and quantitatively evaluate the influence of elongation of the polymethine chain in a fluorescent cyanine (Cy) dye on the imaging potential of a targeted (RGD-based) tracer. Methods: As model system, the integrin marker ανβ3 was targeted using c[RGDyK] vectors functionalized with a ( 111 In-)DTPA chelate and a fluorescent dye (Cy3-(SO3)Methyl-COOH (λem 580nm), Cy5-(SO3)Methyl-COOH (λem 680nm), or Cy7-(SO3)Methyl-COOH (λem 780nm)). Tracers were analyzed for differences in (photo-) physical properties, serum protein binding, chemical/optical stability and signal penetration through tissue. Receptor affinities (KD) were evaluated using saturation and competition experiments. In vivo biodistribution (SPECT imaging and percentage injected dose per gram of tissue (%ID/g)) was assessed in tumor-bearing mice and complimented with in- and ex vivo fluorescence images obtained using a clinical grade multispectral fluorescence laparoscope. Results: Two carbon-atom-step variations in the polymethine chain of the fluorescent Cy-dyes were shown to significantly influence the chemical and photophysical characteristics e.g. stability, brightness and tissue penetration of the hybrid RGD-tracers. Herein DTPA-Cy5-(SO3)Methyl-COOH-c[RGDyK] systematically outperformed its Cy3- and Cy7- derivatives. Radioactivity-based evaluation of in vivo tracer pharmacokinetics yielded the lowest non-specific uptake and highest tumor-to-background ratio (T/B) for DTPA-Cy5-(SO3)Methyl-COOH-c[RGDyK] (13.2 ± 1.7), with the Cy3- and Cy7- analogs trailing at a respective T/B of 5.7 ± 0.7 and 4.7 ± 0.7. Fluorescence-based assessment of the tumor visibility revealed a similar trend. Conclusion: These findings underline that variations in the polymethine chain lengths of Cy dyes have a profound influence on the photophysical properties, stability and in vivo targeting capabilities of fluorescent imaging tracers. In a direct comparison the intermediate length dye (Cy5) yielded a superior c[RGDyK] -tracer compared to the shorter (Cy3-) and longer (Cy7-) analogs. Copyright © 2018 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

PubMed Central

Zhu, Hongying; Ozcan, Aydogan

2013-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 μm over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water. PMID:23603893

Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2013-04-11

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

PubMed Central

Warren, Sean C.; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M. W.

2015-01-01

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. PMID:26133241

Reaction-based small-molecule fluorescent probes for dynamic detection of ROS and transient redox changes in living cells and small animals.

PubMed

Lü, Rui

2017-09-01

Dynamic detection of transient redox changes in living cells and animals has broad implications for human health and disease diagnosis, because intracellular redox homeostasis regulated by reactive oxygen species (ROS) plays important role in cell functions, normal physiological functions and some serious human diseases (e.g., cancer, Alzheimer's disease, diabetes, etc.) usually have close relationship with the intracellular redox status. Small-molecule ROS-responsive fluorescent probes can act as powerful tools for dynamic detection of ROS and redox changes in living cells and animals through fluorescence imaging techniques; and great advances have been achieved recently in the design and synthesis of small-molecule ROS-responsive fluorescent probes. This article highlights up-to-date achievements in designing and using the reaction-based small-molecule fluorescent probes (with high sensitivity and selectivity to ROS and redox cycles) in the dynamic detection of ROS and transient redox changes in living cells and animals through fluorescence imaging. Copyright © 2017. Published by Elsevier Ltd.

Plant Cell Imaging Based on Nanodiamonds with Excitation-Dependent Fluorescence

NASA Astrophysics Data System (ADS)

Su, Li-Xia; Lou, Qing; Jiao, Zhen; Shan, Chong-Xin

2016-09-01

Despite extensive work on fluorescence behavior stemming from color centers of diamond, reports on the excitation-dependent fluorescence of nanodiamonds (NDs) with a large-scale redshift from 400 to 620 nm under different excitation wavelengths are so far much fewer, especially in biological applications. The fluorescence can be attributed to the combined effects of the fraction of sp2-hybridized carbon atoms among the surface of the fine diamond nanoparticles and the defect energy trapping states on the surface of the diamond. The excitation-dependent fluorescent NDs have been applied in plant cell imaging for the first time. The results reported in this paper may provide a promising route to multiple-color bioimaging using NDs.

Plant Cell Imaging Based on Nanodiamonds with Excitation-Dependent Fluorescence.

PubMed

Su, Li-Xia; Lou, Qing; Jiao, Zhen; Shan, Chong-Xin

2016-12-01

Despite extensive work on fluorescence behavior stemming from color centers of diamond, reports on the excitation-dependent fluorescence of nanodiamonds (NDs) with a large-scale redshift from 400 to 620 nm under different excitation wavelengths are so far much fewer, especially in biological applications. The fluorescence can be attributed to the combined effects of the fraction of sp(2)-hybridized carbon atoms among the surface of the fine diamond nanoparticles and the defect energy trapping states on the surface of the diamond. The excitation-dependent fluorescent NDs have been applied in plant cell imaging for the first time. The results reported in this paper may provide a promising route to multiple-color bioimaging using NDs.

Advanced fluorescence imaging endoscopy using an acousto-optic tuneable filter

NASA Astrophysics Data System (ADS)

Whelan, Maurice P.; Bouhifd, Mounir; Aprahamian, Marc

2004-07-01

Two novel prototype instruments for in vivo fluorescence-based medical diagnostics are described. The devices are based on an acousto-optic tuneable filter (AOTF) and can be easily attached to the eyepiece of most commercially available endoscopes. The instruments developed offer significant advantages over typical fixed-filter or filter-wheel fluorescence imaging systems in terms of flexibility, performance and diagnostic potential. Any filtering center-wavelength in the range from 450 to 700 nm can be rapidly selected either by random access or sequential tuning using simple commands delivered over a PC serial interface. In addition, both filtered and unfiltered light can be imaged to facilitate the direct association of fluorescence signals with specific anatomical sites. To demonstrate the system in vivo, a study of the diagnostic potential of fluorescence imaging for pancreatitis was conducted on rats. The aim was to detect extremely low-levels of endogenous protoporphyrin IX (PpIX) that has been shown to accumulate in early-stage diseased tissue undergoing an inflammatory response. Results show clearly that the device is effective in diagnosing mild pancreatitis in rats without the necessity of administering PpIX promoting agents such as ALA. Planning of human clinical trials is currently underway to demonstrate its potential as a tool for non-invasive early diagnosis of gastroenterological diseases.

Simple and robust image-based autofocusing for digital microscopy.

PubMed

Yazdanfar, Siavash; Kenny, Kevin B; Tasimi, Krenar; Corwin, Alex D; Dixon, Elizabeth L; Filkins, Robert J

2008-06-09

A simple image-based autofocusing scheme for digital microscopy is demonstrated that uses as few as two intermediate images to bring the sample into focus. The algorithm is adapted to a commercial inverted microscope and used to automate brightfield and fluorescence imaging of histopathology tissue sections.

A study of MRI-guided diffuse fluorescence molecular tomography for monitoring PDT effects in pancreas cancer

NASA Astrophysics Data System (ADS)

Samkoe, Kimberley S.; Davis, Scott C.; Srinivasan, Subhadra; O'Hara, Julia A.; Hasan, Tayyaba; Pogue, Brian W.

2009-06-01

Over the last several decades little progress has been made in the therapy and treatment monitoring of pancreas adenocarcinoma, a devastating and aggressive form of cancer that has a 5-year patient survival rate of 3%. Currently, investigations for the use of interstitial Verteporfin photodynamic therapy (PDT) are being undertaken in both orthotopic xenograft mouse models and in human clinical trials. In the mouse models, magnetic resonance (MR) imaging has been used as a measure of surrogate response to Verteporfin PDT; however, MR imaging alone lacks the molecular information required to assess the metabolic function and growth rates of the tumor immediately after treatment. We propose the implementation of MR-guided fluorescence tomography in conjunction with a fluorescently labeled (IR-Dye 800 CW, LI-COR) epidermal growth factor (EGF) as a molecular measure of surrogate response. To demonstrate the effectiveness of MR-guided diffuse fluorescence tomography for molecular imaging, we have used the AsPC-1 (+EGFR) human pancreatic adenocarcinoma in an orthotopic mouse model. EGF IRDye 800CW was injected 48 hours prior to imaging. MR image sequences were collected simultaneously with the fluorescence data using a MR-coupled diffuse optical tomography system. Image reconstruction was performed multiple times with varying abdominal organ segmentation in order to obtain a optimal tomographic image. It is shown that diffuse fluorescence tomography of the orthotopic pancreas model is feasible, with consideration of confounding fluorescence signals from the multiple organs and tissues surrounding the pancreas. MR-guided diffuse fluorescence tomography will be used to monitor EGF response after photodynamic therapy. Additionally, it provide the opportunity to individualize subsequent therapies based on response to PDT as well as to evaluate the success of combination therapies, such as PDT with chemotherapy, antibody therapy or even radiation.

Innovative molecular-based fluorescent nanoparticles for multicolor single particle tracking in cells

NASA Astrophysics Data System (ADS)

Daniel, Jonathan; Godin, Antoine G.; Palayret, Matthieu; Lounis, Brahim; Cognet, Laurent; Blanchard-Desce, Mireille

2016-03-01

Based on an original molecular-based design, we present bright and photostable fluorescent organic nanoparticles (FONs) showing excellent colloidal stability in various aqueous environments. Complementary near-infrared emitting and green emitting FONs were prepared using a simple, fast and robust protocol. Both types of FONs could be simultaneously imaged at the single-particle level in solution as well as in biological environments using a monochromatic excitation and a dual-color fluorescence microscope. No evidence of acute cytotoxicity was found upon incubation of live cells with mixed solutions of FONs, and both types of nanoparticles were found internalized in the cells where their motion could be simultaneously tracked at video-rate up to minutes. These fluorescent organic nanoparticles open a novel non-toxic alternative to existing nanoparticles for imaging biological structures, compatible with live-cell experiments and specially fitted for multicolor single particle tracking.

Nitroolefin-based BODIPY as a novel water-soluble ratiometric fluorescent probe for detection of endogenous thiols

NASA Astrophysics Data System (ADS)

Kang, Jin; Huo, Fangjun; Chao, Jianbin; Yin, Caixia

2018-04-01

Small molecule biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play many crucial roles in physiological processes. In this work, we have prepared a nitroolefin-based BODIPY fluorescent probe with excellent water solubility for detection thiols, which displayed ratiometric fluorescent signal for thiols. Incorporation of a nitroolefin unit to the BODIPY dye would transform it into a strong Michael acceptor, which would be highly susceptible to sulfhydryl nucleophiles. This probe shows an obvious ratio change upon response with thiols, an increase of the emission at 517 nm along with a concomitant decrease of fluorescence peak at 573 nm. Moreover, these successes of intracellular imaging experiments in A549 cells indicated that this probe is suitable for imaging of ex-/endogenous thiols in living cells.

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

PubMed

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

2015-06-01

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

NeuronRead, an open source semi-automated tool for morphometric analysis of phase contrast and fluorescence neuronal images.

PubMed

Dias, Roberto A; Gonçalves, Bruno P; da Rocha, Joana F; da Cruz E Silva, Odete A B; da Silva, Augusto M F; Vieira, Sandra I

2017-12-01

Neurons are specialized cells of the Central Nervous System whose function is intricately related to the neuritic network they develop to transmit information. Morphological evaluation of this network and other neuronal structures is required to establish relationships between neuronal morphology and function, and may allow monitoring physiological and pathophysiologic alterations. Fluorescence-based microphotographs are the most widely used in cellular bioimaging, but phase contrast (PhC) microphotographs are easier to obtain, more affordable, and do not require invasive, complicated and disruptive techniques. Despite the various freeware tools available for fluorescence-based images analysis, few exist that can tackle the more elusive and harder-to-analyze PhC images. To surpass this, an interactive semi-automated image processing workflow was developed to easily extract relevant information (e.g. total neuritic length, average cell body area) from both PhC and fluorescence neuronal images. This workflow, named 'NeuronRead', was developed in the form of an ImageJ macro. Its robustness and adaptability were tested and validated on rat cortical primary neurons under control and differentiation inhibitory conditions. Validation included a comparison to manual determinations and to a golden standard freeware tool for fluorescence image analysis. NeuronRead was subsequently applied to PhC images of neurons at distinct differentiation days and exposed or not to DAPT, a pharmacological inhibitor of the γ-secretase enzyme, which cleaves the well-known Alzheimer's amyloid precursor protein (APP) and the Notch receptor. Data obtained confirms a neuritogenic regulatory role for γ-secretase products and validates NeuronRead as a time- and cost-effective useful monitoring tool. Copyright © 2017. Published by Elsevier Inc.

Effect of probe diffusion on the SOFI imaging accuracy.

PubMed

Vandenberg, Wim; Dedecker, Peter

2017-03-23

Live-cell super-resolution fluorescence imaging is becoming commonplace for exploring biological systems, though sample dynamics can affect the imaging quality. In this work we evaluate the effect of probe diffusion on super-resolution optical fluctuation imaging (SOFI), using a theoretical model and numerical simulations based on the imaging of live cells labelled with photochromic fluorescent proteins. We find that, over a range of physiological conditions, fluorophore diffusion results in a change in the amplitude of the SOFI signal. The magnitude of this change is approximately proportional to the on-time ratio of the fluorophores. However, for photochromic fluorescent proteins this effect is unlikely to present a significant distortion in practical experiments in biological systems. Due to this lack of distortions, probe diffusion strongly enhances the SOFI imaging by avoiding spatial undersampling caused by the limited labeling density.

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.

A CTRW-based model of time-resolved fluorescence lifetime imaging in a turbid medium

NASA Astrophysics Data System (ADS)

Chernomordik, Victor; Gandjbakhche, Amir H.; Hassan, Moinuddin; Pajevic, Sinisa; Weiss, George H.

2010-12-01

We develop an analytic model of time-resolved fluorescent imaging of photons migrating through a semi-infinite turbid medium bounded by an infinite plane in the presence of a single stationary point fluorophore embedded in the medium. In contrast to earlier models of fluorescent imaging in which photon motion is assumed to be some form of continuous diffusion process, the present analysis is based on a continuous-time random walk (CTRW) on a simple cubic lattice, the objective being to estimate the position and lifetime of the fluorophore. This can provide information related to local variations in pH and temperature with potential medical significance. Aspects of the theory were tested using time-resolved measurements of the fluorescence from small inclusions inside tissue-like phantoms. The experimental results were found to be in good agreement with theoretical predictions provided that the fluorophore was not located too close to the planar boundary, a common problem in many diffusive systems.

The Phasor Approach to Fluorescence Lifetime Imaging Analysis

PubMed Central

Digman, Michelle A.; Caiolfa, Valeria R.; Zamai, Moreno; Gratton, Enrico

2008-01-01

Changing the data representation from the classical time delay histogram to the phasor representation provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we can easily recognize the presence of different molecular species in a pixel or the occurrence of fluorescence resonance energy transfer. The analysis of the fluorescence lifetime imaging microscopy (FLIM) data in the phasor space is done observing clustering of pixels values in specific regions of the phasor plot rather than by fitting the fluorescence decay using exponentials. The analysis is instantaneous since is not based on calculations or nonlinear fitting. The phasor approach has the potential to simplify the way data are analyzed in FLIM, paving the way for the analysis of large data sets and, in general, making the FLIM technique accessible to the nonexpert in spectroscopy and data analysis. PMID:17981902

Fluorescence imaging of angiogenesis in green fluorescent protein-expressing tumors

NASA Astrophysics Data System (ADS)

Yang, Meng; Baranov, Eugene; Jiang, Ping; Li, Xiao-Ming; Wang, Jin W.; Li, Lingna; Yagi, Shigeo; Moossa, A. R.; Hoffman, Robert M.

2002-05-01

The development of therapeutics for the control of tumor angiogenesis requires a simple, reliable in vivo assay for tumor-induced vascularization. For this purpose, we have adapted the orthotopic implantation model of angiogenesis by using human and rodent tumors genetically tagged with Aequorea victoria green fluorescent protein (GFP) for grafting into nude mice. Genetically-fluorescent tumors can be readily imaged in vivo. The non-luminous induced capillaries are clearly visible against the bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. Fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. High-level GFP-expressing tumor cell lines made it possible to acquire the high-resolution real-time fluorescent optical images of angiogenesis in both primary tumors and their metastatic lesions in various human and rodent tumor models by means of a light-based imaging system. Intravital images of angiogenesis onset and development were acquired and quantified from a GFP- expressing orthotopically-growing human prostate tumor over a 19-day period. Whole-body optical imaging visualized vessel density increasing linearly over a 20-week period in orthotopically-growing, GFP-expressing human breast tumor MDA-MB-435. Vessels in an orthotopically-growing GFP- expressing Lewis lung carcinoma tumor were visualized through the chest wall via a reversible skin flap. These clinically-relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological micro- environments.

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.

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

The Value of 5-Aminolevulinic Acid in Low-grade Gliomas and High-grade Gliomas Lacking Glioblastoma Imaging Features: An Analysis Based on Fluorescence, Magnetic Resonance Imaging, 18F-Fluoroethyl Tyrosine Positron Emission Tomography, and Tumor Molecular Factors.

PubMed

Jaber, Mohammed; Wölfer, Johannes; Ewelt, Christian; Holling, Markus; Hasselblatt, Martin; Niederstadt, Thomas; Zoubi, Tarek; Weckesser, Matthias; Stummer, Walter

2016-03-01

Approximately 20% of grade II and most grade III gliomas fluoresce after 5-aminolevulinic acid (5-ALA) application. Conversely, approximately 30% of nonenhancing gliomas are actually high grade. The aim of this study was to identify preoperative factors (ie, age, enhancement, 18F-fluoroethyl tyrosine positron emission tomography [F-FET PET] uptake ratios) for predicting fluorescence in gliomas without typical glioblastomas imaging features and to determine whether fluorescence will allow prediction of tumor grade or molecular characteristics. Patients harboring gliomas without typical glioblastoma imaging features were given 5-ALA. Fluorescence was recorded intraoperatively, and biopsy specimens collected from fluorescing tissue. World Health Organization (WHO) grade, Ki-67/MIB-1 index, IDH1 (R132H) mutation status, O-methylguanine DNA methyltransferase (MGMT) promoter methylation status, and 1p/19q co-deletion status were assessed. Predictive factors for fluorescence were derived from preoperative magnetic resonance imaging and F-FET PET. Classification and regression tree analysis and receiver-operating-characteristic curves were generated for defining predictors. Of 166 tumors, 82 were diagnosed as WHO grade II, 76 as grade III, and 8 as glioblastomas grade IV. Contrast enhancement, tumor volume, and F-FET PET uptake ratio >1.85 predicted fluorescence. Fluorescence correlated with WHO grade (P < .001) and Ki-67/MIB-1 index (P < .001), but not with MGMT promoter methylation status, IDH1 mutation status, or 1p19q co-deletion status. The Ki-67/MIB-1 index in fluorescing grade III gliomas was higher than in nonfluorescing tumors, whereas in fluorescing and nonfluorescing grade II tumors, no differences were noted. Age, tumor volume, and F-FET PET uptake are factors predicting 5-ALA-induced fluorescence in gliomas without typical glioblastoma imaging features. Fluorescence was associated with an increased Ki-67/MIB-1 index and high-grade pathology. Whether fluorescence in grade II gliomas identifies a subtype with worse prognosis remains to be determined.

Highly Stable Near-Infrared Fluorescent Organic Nanoparticles with a Large Stokes Shift for Noninvasive Long-Term Cellular Imaging.

PubMed

Zhang, Jinfeng; Chen, Rui; Zhu, Zelin; Adachi, Chihaya; Zhang, Xiaohong; Lee, Chun-Sing

2015-12-02

Fluorescent organic nanoparticles based on small molecules have been regarded as promising candidates for bioimaging in recent years. In this study, we report a highly stable near-infrared (NIR) fluorescent organic nanoprobes based on nanoparticles of an anthraquinone derivate with strong aggregation-induced emission (AIE) characteristics and a large Stokes shift (>175 nm). These endow the nanoprobe with high fluorescent brightness and high signal-to-noise ratio. On the other hand, the nanoprobe also shows low cytotoxicity, good stability over a wide pH range, superior resistance against photodegradation and photobleaching comparing to typical commercial fluorescent organic dyes such as fluorescein sodium. Endowed with such merits in term of optical performance, biocompatibility, and stability, the nanoprobe is demonstrated to be an ideal fluorescent probe for noninvasive long-term cellular tracing and imaging applications. As an example, it is shown that strong red fluorescence from the nanoprobe can still be clearly observed in A549 human lung cancer cells after incubation for six generations over 15 days.

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

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

NASA Astrophysics Data System (ADS)

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

2007-02-01

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

Optical imaging probes in oncology

PubMed Central

Martelli, Cristina; Dico, Alessia Lo; Diceglie, Cecilia; Lucignani, Giovanni; Ottobrini, Luisa

2016-01-01

Cancer is a complex disease, characterized by alteration of different physiological molecular processes and cellular features. Keeping this in mind, the possibility of early identification and detection of specific tumor biomarkers by non-invasive approaches could improve early diagnosis and patient management. Different molecular imaging procedures provide powerful tools for detection and non-invasive characterization of oncological lesions. Clinical studies are mainly based on the use of computed tomography, nuclear-based imaging techniques and magnetic resonance imaging. Preclinical imaging in small animal models entails the use of dedicated instruments, and beyond the already cited imaging techniques, it includes also optical imaging studies. Optical imaging strategies are based on the use of luminescent or fluorescent reporter genes or injectable fluorescent or luminescent probes that provide the possibility to study tumor features even by means of fluorescence and luminescence imaging. Currently, most of these probes are used only in animal models, but the possibility of applying some of them also in the clinics is under evaluation. The importance of tumor imaging, the ease of use of optical imaging instruments, the commercial availability of a wide range of probes as well as the continuous description of newly developed probes, demonstrate the significance of these applications. The aim of this review is providing a complete description of the possible optical imaging procedures available for the non-invasive assessment of tumor features in oncological murine models. In particular, the characteristics of both commercially available and newly developed probes will be outlined and discussed. PMID:27145373

A multimodal imaging platform with integrated simultaneous photoacoustic microscopy, optical coherence tomography, optical Doppler tomography and fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dadkhah, Arash; Zhou, Jun; Yeasmin, Nusrat; Jiao, Shuliang

2018-02-01

Various optical imaging modalities with different optical contrast mechanisms have been developed over the past years. Although most of these imaging techniques are being used in many biomedical applications and researches, integration of these techniques will allow researchers to reach the full potential of these technologies. Nevertheless, combining different imaging techniques is always challenging due to the difference in optical and hardware requirements for different imaging systems. Here, we developed a multimodal optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissue in micrometer scale. This imaging system integrates photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and fluorescence microscopy in one platform. Optical-resolution PAM (OR-PAM) provides absorption-based imaging of biological tissues. Spectral domain OCT is able to provide structural information based on the scattering property of biological sample with no need for exogenous contrast agents. In addition, ODT is a functional extension of OCT with the capability of measurement and visualization of blood flow based on the Doppler effect. Fluorescence microscopy allows to reveal molecular information of biological tissue using autofluoresce or exogenous fluorophores. In-vivo as well as ex-vivo imaging studies demonstrated the capability of our multimodal imaging system to provide comprehensive microscopic information on biological tissues. Integrating all the aforementioned imaging modalities for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the near future.

Automated image-based phenotypic analysis in zebrafish embryos

PubMed Central

Vogt, Andreas; Cholewinski, Andrzej; Shen, Xiaoqiang; Nelson, Scott; Lazo, John S.; Tsang, Michael; Hukriede, Neil A.

2009-01-01

Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to utilizing the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)y1) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes. PMID:19235725

Aptamer-Based Dual-Functional Probe for Rapid and Specific Counting and Imaging of MCF-7 Cells.

PubMed

Yang, Bin; Chen, Beibei; He, Man; Yin, Xiao; Xu, Chi; Hu, Bin

2018-02-06

Development of multimodal detection technologies for accurate diagnosis of cancer at early stages is in great demand. In this work, we report a novel approach using an aptamer-based dual-functional probe for rapid, sensitive, and specific counting and visualization of MCF-7 cells by inductively coupled plasma-mass spectrometry (ICP-MS) and fluorescence imaging. The probe consists of a recognition unit of aptamer to catch cancer cells specifically, a fluorescent dye (FAM) moiety for fluorescence resonance energy transfer (FRET)-based "off-on" fluorescence imaging as well as gold nanoparticles (Au NPs) tag for both ICP-MS quantification and fluorescence quenching. Due to the signal amplification effect and low spectral interference of Au NPs in ICP-MS, an excellent linearity and sensitivity were achieved. Accordingly, a limit of detection of 81 MCF-7 cells and a relative standard deviation of 5.6% (800 cells, n = 7) were obtained. The dynamic linear range was 2 × 10 2 to 1.2 × 10 4 cells, and the recoveries in human whole blood were in the range of 98-110%. Overall, the established method provides quantitative and visualized information on MCF-7 cells with a simple and rapid process and paves the way for a promising strategy for biomedical research and clinical diagnostics.

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.

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.

An endoscopic fluorescence imaging system for simultaneous visual examination and photodetection of cancers

NASA Astrophysics Data System (ADS)

Wagnières, Georges A.; Studzinski, André P.; van den Bergh, Hubert E.

1997-01-01

We describe the design and performance tested during six years of clinical trials of a fluorescence endoscope for the detection and delineation of cancers in several hollow organs. The apparatus is based on the imaging of the laser-induced fluorescence that differs between a tumor and its surrounding normal tissue. The tests are carried out in the upper aerodigestive tract, the tracheobronchial tree, the esophagus, and the colon. In the three former cases an exogenous dye is used (Photofrin II), whereas in the latter case fluorescein molecules conjugated with monoclonal antibodies directed against carcinoembryonic antigen are injected. The decrease of native tissue autofluorescence observed in early cancers is also used for detecting lesions in the tracheobronchial tree. The fluorescence contrast between the tumor and surrounding normal tissue is enhanced by real time image processing. This is done by simultaneously recording the fluorescence image in two spectral domains, after which these two images are digitized and manipulated with a mathematical operator (look-up table) at video frequency. Moreover, the device that is described below allows for an immediate observation of the endoscopic area under white light illumination during fluorescence detection in order to localize the origin of the "positive" fluorescence signals. Typical results obtained in the tracheobronchial tree and in the colon are presented and the sources of false positives and false negatives are evaluated in terms of the fluorescent dye, tissue optical properties, and illumination optics.

Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

NASA Astrophysics Data System (ADS)

Nienhaus, Karin; Nienhaus, G. Ulrich

2016-11-01

Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.

A chromenoquinoline-based fluorescent off-on thiol probe for bioimaging.

PubMed

Kand, Dnyaneshwar; Kalle, Arunasree Marasanapalli; Varma, Sreejith Jayasree; Talukdar, Pinaki

2012-03-11

A new chromenoquinoline-based fluorescent off-on thiol probe 2 is reported. In aqueous buffer solutions at physiological pH, the probe exhibited 223-fold enhancement in fluorescence intensity by a Michael addition of cysteine to the maleimide appended to a chromenoquinoline. Cell permeability and live cell imaging of thiols are also demonstrated. This journal is © The Royal Society of Chemistry 2012

Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

PubMed

Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

2017-06-01

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

Multimodality optical coherence tomography and fluorescence confocal scanning laser ophthalmoscopy for image-guided feedback of intraocular injections in mouse models

NASA Astrophysics Data System (ADS)

Benavides, Oscar R.; Terrones, Benjamin D.; Leeburg, Kelsey C.; Mehanathan, Sankarathi B.; Levine, Edward M.; Tao, Yuankai K.

2018-02-01

Rodent models are robust tools for understanding human retinal disease and function because of their similarities with human physiology and anatomy and availability of genetic mutants. Optical coherence tomography (OCT) has been well-established for ophthalmic imaging in rodents and enables depth-resolved visualization of structures and image-based surrogate biomarkers of disease. Similarly, fluorescence confocal scanning laser ophthalmoscopy (cSLO) has demonstrated utility for imaging endogenous and exogenous fluorescence and scattering contrast in the mouse retina. Complementary volumetric scattering and en face fluorescence contrast from OCT and cSLO, respectively, enables cellular-resolution longitudinal imaging of changes in ophthalmic structure and function. We present a non-contact multimodal OCT+cSLO small animal imaging system with extended working distance to the pupil, which enables imaging during and after intraocular injection. While injections are routinely performed in mice to develop novel models of ophthalmic diseases and screen novel therapeutics, the location and volume delivered is not precisely controlled and difficult to reproduce. Animals were imaged using a custom-built OCT engine and scan-head combined with a modified commercial cSLO scan-head. Post-injection imaging showed structural changes associated with retinal puncture, including the injection track, a retinal elevation, and detachment of the posterior hyaloid. When combined with imagesegmentation, we believe OCT can be used to precisely identify injection locations and quantify injection volumes. Fluorescence cSLO can provide complementary contrast for either fluorescently labeled compounds or transgenic cells for improved specificity. Our non-contact OCT+cSLO system is uniquely-suited for concurrent imaging with intraocular injections, which may be used for real-time image-guided injections.

Recording membrane potential changes through photoacoustic voltage sensitive dye

NASA Astrophysics Data System (ADS)

Zhang, Haichong K.; Kang, Jeeun; Yan, Ping; Abou, Diane S.; Le, Hanh N. D.; Thorek, Daniel L. J.; Kang, Jin U.; Gjedde, Albert; Rahmim, Arman; Wong, Dean F.; Loew, Leslie M.; Boctor, Emad M.

2017-03-01

Monitoring of the membrane potential is possible using voltage sensitive dyes (VSD), where fluorescence intensity changes in response to neuronal electrical activity. However, fluorescence imaging is limited by depth of penetration and high scattering losses, which leads to low sensitivity in vivo systems for external detection. In contrast, photoacoustic (PA) imaging, an emerging modality, is capable of deep tissue, noninvasive imaging by combining near infrared light excitation and ultrasound detection. In this work, we develop the theoretical concept whereby the voltage-dependent quenching of dye fluorescence leads to a reciprocal enhancement of PA intensity. Based on this concept, we synthesized a novel near infrared photoacoustic VSD (PA-VSD) whose PA intensity change is sensitive to membrane potential. In the polarized state, this cyanine-based probe enhances PA intensity while decreasing fluorescence output in a lipid vesicle membrane model. With a 3-9 μM VSD concentration, we measured a PA signal increase in the range of 5.3 % to 18.1 %, and observed a corresponding signal reduction in fluorescence emission of 30.0 % to 48.7 %. A theoretical model successfully accounts for how the experimental PA intensity change depends on fluorescence and absorbance properties of the dye. These results not only demonstrate the voltage sensing capability of the dye, but also indicate the necessity of considering both fluorescence and absorbance spectral sensitivities in order to optimize the characteristics of improved photoacoustic probes. Together, our results demonstrate photoacoustic sensing as a potential new modality for sub-second recording and external imaging of electrophysiological and neurochemical events in the brain.

Readily Available Fluorescent Probe for Carbon Monoxide Imaging in Living Cells.

PubMed

Feng, Weiyong; Liu, Dandan; Feng, Shumin; Feng, Guoqiang

2016-11-01

Carbon monoxide (CO) is an important gasotransmitter in living systems and its fluorescent detection is of particular interest. However, fluorescent detection of CO in living cells is still challenging due to lack of effective probes. In this paper, a readily available fluorescein-based fluorescent probe was developed for rapid detection of CO. This probe can be used to detect CO in almost wholly aqueous solution under mild conditions and shows high selectivity and sensitivity for CO with colorimetric and remarkable fluorescent turn-on signal changes. The detection limit of this probe for CO is as low as 37 nM with a linear range of 0-30 μM. More importantly, this probe (1 μM dose) can be conveniently used for fluorescent imaging CO in living cells.

A selective colorimetric and fluorescent sensor for Al3+ ion and its application to cellular imaging

NASA Astrophysics Data System (ADS)

Manjunath, Rangasamy; Hrishikesan, Elango; Kannan, Palaninathan

2015-04-01

A new rhodamine-based fluorescent turn-on chemosensor (L) for selective detection of Al3+ ion has been developed and characterized. The fluorescent chemosensor L was synthesized by the reaction of intermediate (4) with 2,5-bis (4-phenylacyl chloride)-1,3,4-oxadiazole (3). The chemosensor L displays an excellent selective and sensitive response to Al3+ ion over other metal ions, in which the spirocyclic (non-fluorescent) to ring opened amide (fluorescent) process was utilized and a 1:2 stoichiometry for L-Al3+ complex was formed with an association constant of 2.03 × 103 M-1. Furthermore, chemosensor L can be applied as a fluorescent probe for monitoring Al3+ in living cells by performing cell imaging studies.

3D tensor-based blind multispectral image decomposition for tumor demarcation

NASA Astrophysics Data System (ADS)

Kopriva, Ivica; Peršin, Antun

2010-03-01

Blind decomposition of multi-spectral fluorescent image for tumor demarcation is formulated exploiting tensorial structure of the image. First contribution of the paper is identification of the matrix of spectral responses and 3D tensor of spatial distributions of the materials present in the image from Tucker3 or PARAFAC models of 3D image tensor. Second contribution of the paper is clustering based estimation of the number of the materials present in the image as well as matrix of their spectral profiles. 3D tensor of the spatial distributions of the materials is recovered through 3-mode multiplication of the multi-spectral image tensor and inverse of the matrix of spectral profiles. Tensor representation of the multi-spectral image preserves its local spatial structure that is lost, due to vectorization process, when matrix factorization-based decomposition methods (such as non-negative matrix factorization and independent component analysis) are used. Superior performance of the tensor-based image decomposition over matrix factorization-based decompositions is demonstrated on experimental red-green-blue (RGB) image with known ground truth as well as on RGB fluorescent images of the skin tumor (basal cell carcinoma).

Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging.

PubMed

Cho, Hong-Jun; Lee, Sung-Jin; Park, Sung-Jun; Paik, Chang H; Lee, Sang-Myung; Kim, Sehoon; Lee, Yoon-Sik

2016-09-10

A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Pupil-segmentation-based adaptive optical correction of a high-numerical-aperture gradient refractive index lens for two-photon fluorescence endoscopy.

PubMed

Wang, Chen; Ji, Na

2012-06-01

The intrinsic aberrations of high-NA gradient refractive index (GRIN) lenses limit their image quality as well as field of view. Here we used a pupil-segmentation-based adaptive optical approach to correct the inherent aberrations in a two-photon fluorescence endoscope utilizing a 0.8 NA GRIN lens. By correcting the field-dependent aberrations, we recovered diffraction-limited performance across a large imaging field. The consequent improvements in imaging signal and resolution allowed us to detect fine structures that were otherwise invisible inside mouse brain slices.

Detecting crop population growth using chlorophyll fluorescence imaging.

PubMed

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

2017-12-10

For both field and greenhouse crops, it is challenging to evaluate their growth information on a large area over a long time. In this work, we developed a chlorophyll fluorescence imaging-based system for crop population growth information detection. Modular design was used to make the system provide high-intensity uniform illumination. This system can perform modulated chlorophyll fluorescence induction kinetics measurement and chlorophyll fluorescence parameter imaging over a large area of up to 45  cm×34  cm. The system can provide different lighting intensity by modulating the duty cycle of its control signal. Results of continuous monitoring of cucumbers in nitrogen deficiency show the system can reduce the judge error of crop physiological status and improve monitoring efficiency. Meanwhile, the system is promising in high throughput application scenarios.

Polypyrrole-based nanotheranostics for activatable fluorescence imaging and chemo/photothermal dual therapy of triple-negative breast cancer

NASA Astrophysics Data System (ADS)

Park, Dongjin; Ahn, Kyung-Ohk; Jeong, Kyung-Chae; Choi, Yongdoo

2016-05-01

Here, we fabricated polypyrrole nanoparticles (PPys) (termed HA10-PPy, HA20-PPy, and HA40-PPy) doped with different average molecular weight hyaluronic acids (HAs) (10, 20, and 40 kDa, respectively), and evaluated the effect of molecular weight of doped HA on photothermal induction, fluorescence quenching, and drug loading efficiencies. Doxorubicin-loaded HA-doped PPys (DOX@HA-PPys) could be used for imaging and therapy of triple-negative breast cancer (TNBC). Fluorescence turn-on, stimuli-responsive drug release, and photo-induced heating of DOX@HA-PPys enabled not only activatable fluorescence imaging but also subsequent chemo/photothermal dual therapy for TNBC. In particular, we illustrated the potential usefulness of the photothermal effect of the nanoparticles for overcoming chemoresistance in TNBC.

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

PubMed Central

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

2008-01-01

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

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

PubMed

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

2008-07-01

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

Super-resolution imaging based on the temperature-dependent electron-phonon collision frequency effect of metal thin films

NASA Astrophysics Data System (ADS)

Ding, Chenliang; Wei, Jingsong; Xiao, Mufei

2018-05-01

We herein propose a far-field super-resolution imaging with metal thin films based on the temperature-dependent electron-phonon collision frequency effect. In the proposed method, neither fluorescence labeling nor any special properties are required for the samples. The 100 nm lands and 200 nm grooves on the Blu-ray disk substrates were clearly resolved and imaged through a laser scanning microscope of wavelength 405 nm. The spot size was approximately 0.80 μm , and the imaging resolution of 1/8 of the laser spot size was experimentally obtained. This work can be applied to the far-field super-resolution imaging of samples with neither fluorescence labeling nor any special properties.

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

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

Superresolution Imaging using Single-Molecule Localization

PubMed Central

Patterson, George; Davidson, Michael; Manley, Suliana; Lippincott-Schwartz, Jennifer

2013-01-01

Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (∼10–20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales. PMID:20055680

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

PubMed Central

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-01

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

PubMed

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

PubMed

Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

Construction of dual-functional polymer nanomaterials with near-infrared fluorescence imaging and polymer prodrug by RAFT-mediated aqueous dispersion polymerization.

PubMed

Tian, Chun; Niu, Jinyun; Wei, Xuerui; Xu, Yujie; Zhang, Lifen; Cheng, Zhenping; Zhu, Xiulin

2018-05-31

The performance of functional polymer nanomaterials is a vigorously discussed topic in polymer science. We devoted ourselves to investigating polymer nanomaterials based on near-infrared (NIR) fluorescence imaging and polymer prodrug in this study. Aza-boron dipyrromethene (BODIPY) is an important organic dye, having characteristics such as environmental resistance, light resistance, high molar extinction coefficient, and fluorescence quantum yield. We incorporated it into our target monomer, which can be polymerized without changing its parent structure in a polar solvent and copolymerized with water-soluble monomer to improve the solubility of the dye in an aqueous solution. At the same time, the hydrophobic drug camptothecin (CPT) was designed as a prodrug monomer, and the polymeric nanoparticles (NPs) with NIR fluorescence imaging and prodrug were synthesized in situ in reversible addition-fragmentation chain transfer (RAFT)-mediated aqueous dispersion polymerization. The dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed the final uniform size of the dual-functional polymeric NPs morphology. The dual-functional polymeric NPs had a strong absorption and emission signal in the NIR region (>650 nm) based on the fluorescence tests. In consideration of the long-term biological toxicity, confocal laser scanning microscopy (CLSM) results indicated that the dual-functional NPs with controlled drug content exhibited effective capability of killing HeLa cells. In addition, in vivo imaging of the dual-functional NPs was observed in real time, and the fluorescent signals clearly demonstrated the dynamic process of prodrug transfer.

Synthesis and validation of novel cholesterol-based fluorescent lipids designed to observe the cellular trafficking of cationic liposomes.

PubMed

Kim, Bieong-Kil; Seu, Young-Bae; Choi, Jong-Soo; Park, Jong-Won; Doh, Kyung-Oh

2015-09-15

Cholesterol-based fluorescent lipids with ether linker were synthesized using NBD (Chol-E-NBD) or Rhodamine B (Chol-E-Rh), and the usefulnesses as fluorescent probes for tracing cholesterol-based liposomes were validated. The fluorescent intensities of liposomes containing these modified lipids were measured and observed under a microscope. Neither compound interfered with the expression of GFP plasmid, and live cell images were obtained without interferences. Changes in the fluorescent intensity of liposomes containing Chol-E-NBD were followed by flow cytometry for up to 24h. These fluorescent lipids could be useful probes for trafficking of cationic liposome-mediated gene delivery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Imaging systems and algorithms to analyze biological samples in real-time using mobile phone microscopy.

PubMed

Shanmugam, Akshaya; Usmani, Mohammad; Mayberry, Addison; Perkins, David L; Holcomb, Daniel E

2018-01-01

Miniaturized imaging devices have pushed the boundaries of point-of-care imaging, but existing mobile-phone-based imaging systems do not exploit the full potential of smart phones. This work demonstrates the use of simple imaging configurations to deliver superior image quality and the ability to handle a wide range of biological samples. Results presented in this work are from analysis of fluorescent beads under fluorescence imaging, as well as helminth eggs and freshwater mussel larvae under white light imaging. To demonstrate versatility of the systems, real time analysis and post-processing results of the sample count and sample size are presented in both still images and videos of flowing samples.

Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope.

PubMed

Qiu, Zhen; Liu, Zhongyao; Duan, Xiyu; Khondee, Supang; Joshi, Bishnu; Mandella, Michael J; Oldham, Kenn; Kurabayashi, Katsuo; Wang, Thomas D

2013-02-01

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0-400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument.

Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope

PubMed Central

Qiu, Zhen; Liu, Zhongyao; Duan, Xiyu; Khondee, Supang; Joshi, Bishnu; Mandella, Michael J.; Oldham, Kenn; Kurabayashi, Katsuo; Wang, Thomas D.

2013-01-01

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0–400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument. PMID:23412564

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.

Design and characterization of an optimized simultaneous color and near-infrared fluorescence rigid endoscopic imaging system

NASA Astrophysics Data System (ADS)

Venugopal, Vivek; Park, Minho; Ashitate, Yoshitomo; Neacsu, Florin; Kettenring, Frank; Frangioni, John V.; Gangadharan, Sidhu P.; Gioux, Sylvain

2013-12-01

We report the design, characterization, and validation of an optimized simultaneous color and near-infrared (NIR) fluorescence rigid endoscopic imaging system for minimally invasive surgery. This system is optimized for illumination and collection of NIR wavelengths allowing the simultaneous acquisition of both color and NIR fluorescence at frame rates higher than 6.8 fps with high sensitivity. The system employs a custom 10-mm diameter rigid endoscope optimized for NIR transmission. A dual-channel light source compatible with the constraints of an endoscope was built and includes a plasma source for white light illumination and NIR laser diodes for fluorescence excitation. A prism-based 2-CCD camera was customized for simultaneous color and NIR detection with a highly efficient filtration scheme for fluorescence imaging of both 700- and 800-nm emission dyes. The performance characterization studies indicate that the endoscope can efficiently detect fluorescence signal from both indocyanine green and methylene blue in dimethyl sulfoxide at the concentrations of 100 to 185 nM depending on the background optical properties. Finally, we performed the validation of this imaging system in vivo during a minimally invasive procedure for thoracic sentinel lymph node mapping in a porcine model.

Listening to membrane potential: photoacoustic voltage-sensitive dye recording.

PubMed

Zhang, Haichong K; Yan, Ping; Kang, Jeeun; Abou, Diane S; Le, Hanh N D; Jha, Abhinav K; Thorek, Daniel L J; Kang, Jin U; Rahmim, Arman; Wong, Dean F; Boctor, Emad M; Loew, Leslie M

2017-04-01

Voltage-sensitive dyes (VSDs) are designed to monitor membrane potential by detecting fluorescence changes in response to neuronal or muscle electrical activity. However, fluorescence imaging is limited by depth of penetration and high scattering losses, which leads to low sensitivity in vivo systems for external detection. By contrast, photoacoustic (PA) imaging, an emerging modality, is capable of deep tissue, noninvasive imaging by combining near-infrared light excitation and ultrasound detection. Here, we show that voltage-dependent quenching of dye fluorescence leads to a reciprocal enhancement of PA intensity. We synthesized a near-infrared photoacoustic VSD (PA-VSD), whose PA intensity change is sensitive to membrane potential. In the polarized state, this cyanine-based probe enhances PA intensity while decreasing fluorescence output in a lipid vesicle membrane model. A theoretical model accounts for how the experimental PA intensity change depends on fluorescence and absorbance properties of the dye. These results not only demonstrate PA voltage sensing but also emphasize the interplay of both fluorescence and absorbance properties in the design of optimized PA probes. Together, our results demonstrate PA sensing as a potential new modality for recording and external imaging of electrophysiological and neurochemical events in the brain.

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

Listening to membrane potential: photoacoustic voltage-sensitive dye recording

NASA Astrophysics Data System (ADS)

Zhang, Haichong K.; Yan, Ping; Kang, Jeeun; Abou, Diane S.; Le, Hanh N. D.; Jha, Abhinav K.; Thorek, Daniel L. J.; Kang, Jin U.; Rahmim, Arman; Wong, Dean F.; Boctor, Emad M.; Loew, Leslie M.

2017-04-01

Voltage-sensitive dyes (VSDs) are designed to monitor membrane potential by detecting fluorescence changes in response to neuronal or muscle electrical activity. However, fluorescence imaging is limited by depth of penetration and high scattering losses, which leads to low sensitivity in vivo systems for external detection. By contrast, photoacoustic (PA) imaging, an emerging modality, is capable of deep tissue, noninvasive imaging by combining near-infrared light excitation and ultrasound detection. Here, we show that voltage-dependent quenching of dye fluorescence leads to a reciprocal enhancement of PA intensity. We synthesized a near-infrared photoacoustic VSD (PA-VSD), whose PA intensity change is sensitive to membrane potential. In the polarized state, this cyanine-based probe enhances PA intensity while decreasing fluorescence output in a lipid vesicle membrane model. A theoretical model accounts for how the experimental PA intensity change depends on fluorescence and absorbance properties of the dye. These results not only demonstrate PA voltage sensing but also emphasize the interplay of both fluorescence and absorbance properties in the design of optimized PA probes. Together, our results demonstrate PA sensing as a potential new modality for recording and external imaging of electrophysiological and neurochemical events in the brain.

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

NASA Astrophysics Data System (ADS)

Pandey, Vikas; Gupta, Shalini; Elangovan, Ravikrishnan

2018-01-01

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

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

Wavefront sensorless adaptive optics versus sensor-based adaptive optics for in vivo fluorescence retinal imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wahl, Daniel J.; Zhang, Pengfei; Jian, Yifan; Bonora, Stefano; Sarunic, Marinko V.; Zawadzki, Robert J.

2017-02-01

Adaptive optics (AO) is essential for achieving diffraction limited resolution in large numerical aperture (NA) in-vivo retinal imaging in small animals. Cellular-resolution in-vivo imaging of fluorescently labeled cells is highly desirable for studying pathophysiology in animal models of retina diseases in pre-clinical vision research. Currently, wavefront sensor-based (WFS-based) AO is widely used for retinal imaging and has demonstrated great success. However, the performance can be limited by several factors including common path errors, wavefront reconstruction errors and an ill-defined reference plane on the retina. Wavefront sensorless (WFS-less) AO has the advantage of avoiding these issues at the cost of algorithmic execution time. We have investigated WFS-less AO on a fluorescence scanning laser ophthalmoscopy (fSLO) system that was originally designed for WFS-based AO. The WFS-based AO uses a Shack-Hartmann WFS and a continuous surface deformable mirror in a closed-loop control system to measure and correct for aberrations induced by the mouse eye. The WFS-less AO performs an open-loop modal optimization with an image quality metric. After WFS-less AO aberration correction, the WFS was used as a control of the closed-loop WFS-less AO operation. We can easily switch between WFS-based and WFS-less control of the deformable mirror multiple times within an imaging session for the same mouse. This allows for a direct comparison between these two types of AO correction for fSLO. Our results demonstrate volumetric AO-fSLO imaging of mouse retinal cells labeled with GFP. Most significantly, we have analyzed and compared the aberration correction results for WFS-based and WFS-less AO imaging.

1-Million droplet array with wide-field fluorescence imaging for digital PCR.

PubMed

Hatch, Andrew C; Fisher, Jeffrey S; Tovar, Armando R; Hsieh, Albert T; Lin, Robert; Pentoney, Stephen L; Yang, David L; Lee, Abraham P

2011-11-21

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

Real-time monitoring of endogenous cysteine levels in living cells using a CD-based ratiometric fluorescent nanoprobe.

PubMed

Wang, Hong; Zhang, Peisheng; Tian, Yong; Zhang, Yuan; Yang, Heping; Chen, Shu; Zeng, Rongjin; Long, Yunfei; Chen, Jian

2018-04-30

A simple and readily available fluorescent probe is needed for the real-time monitoring of endogenous cysteine (Cys) levels in living cells, as such a probe could be used to study the role of Cys in related diseases. Herein, we report the first fluorescent probe based on carbon dots (CDs-FITA) for the selective and ratiometric imaging of endogenous Cys in live cells. In this ratiometric fluorescent probe, a fluorescein derivative (FITA) that recognizes Cys is covalently linked to the surfaces of carbon dots (CDs); employing CDs greatly improves the water solubility of the probe. Acrylate on FITA is selectively cleaved by Cys in aqueous solution under mild conditions, leading to a dramatic increase in the fluorescence from fluorescein. The probe therefore allows the highly selective ratiometric fluorescent detection of Cys even in the presence of various interferents. The as-prepared CDs-FITA showed excellent performance when applied to detect Cys in blood serum. In addition, due to its negligible cytotoxicity, the CDs-FITA can also be utilized for the real-time monitoring of endogenous cysteine (Cys) levels in living cells. Graphical abstract Illustration of the CD-based probe for Cys imaging in living cells.

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

Optical imaging of tumor microenvironment

PubMed Central

Wu, Yihan; Zhang, Wenjie; Li, Jinbo; Zhang, Yan

2013-01-01

Tumor microenvironment plays important roles in tumor development and metastasis. Features of the tumor microenvironment that are significantly different from normal tissues include acidity, hypoxia, overexpressed proteases and so on. Therefore, these features can serve as not only biomarkers for tumor diagnosis but also theraputic targets for tumor treatment. Imaging modalities such as optical, positron emission tomography (PET) and magnetic resonance imaging (MRI) have been intensively applied to investigate tumor microenvironment. Various imaging probes targeting pH, hypoxia and proteases in tumor microenvironment were thus well developed. In this review, we will focus on recent examples on fluorescent probes for optical imaging of tumor microenvironment. Construction of these fluorescent probes were based on characteristic feature of pH, hypoxia and proteases in tumor microenvironment. Strategies for development of these fluorescent probes and applications of these probes in optical imaging of tumor cells or tissues will be discussed in this review paper. PMID:23342297

Hyperspectral fluorescence imaging coupled with multivariate image analysis techniques for contaminant screening of leafy greens

NASA Astrophysics Data System (ADS)

Everard, Colm D.; Kim, Moon S.; Lee, Hoyoung

2014-05-01

The production of contaminant free fresh fruit and vegetables is needed to reduce foodborne illnesses and related costs. Leafy greens grown in the field can be susceptible to fecal matter contamination from uncontrolled livestock and wild animals entering the field. Pathogenic bacteria can be transferred via fecal matter and several outbreaks of E.coli O157:H7 have been associated with the consumption of leafy greens. This study examines the use of hyperspectral fluorescence imaging coupled with multivariate image analysis to detect fecal contamination on Spinach leaves (Spinacia oleracea). Hyperspectral fluorescence images from 464 to 800 nm were captured; ultraviolet excitation was supplied by two LED-based line light sources at 370 nm. Key wavelengths and algorithms useful for a contaminant screening optical imaging device were identified and developed, respectively. A non-invasive screening device has the potential to reduce the harmful consequences of foodborne illnesses.

Fluorescent Microscopy Enhancement Using Imaging

NASA Astrophysics Data System (ADS)

Conrad, Morgan P.; Reck tenwald, Diether J.; Woodhouse, Bryan S.

1986-06-01

To enhance our capabilities for observing fluorescent stains in biological systems, we are developing a low cost imaging system based around an IBM AT microcomputer and a commercial image capture board compatible with a standard RS-170 format video camera. The image is digitized in real time with 256 grey levels, while being displayed and also stored in memory. The software allows for interactive processing of the data, such as histogram equalization or pseudocolor enhancement of the display. The entire image, or a quadrant thereof, can be averaged over time to improve the signal to noise ratio. Images may be stored to disk for later use or comparison. The camera may be selected for better response in the UV or near IR. Combined with signal averaging, this increases the sensitivity relative to that of the human eye, while still allowing for the fluorescence distribution on either the surface or internal cytoskeletal structure to be observed.

Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

NASA Astrophysics Data System (ADS)

Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

2015-03-01

Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

Lagrangian 3D tracking of fluorescent microscopic objects in motion

NASA Astrophysics Data System (ADS)

Darnige, T.; Figueroa-Morales, N.; Bohec, P.; Lindner, A.; Clément, E.

2017-05-01

We describe the development of a tracking device, mounted on an epi-fluorescent inverted microscope, suited to obtain time resolved 3D Lagrangian tracks of fluorescent passive or active micro-objects in microfluidic devices. The system is based on real-time image processing, determining the displacement of a x, y mechanical stage to keep the chosen object at a fixed position in the observation frame. The z displacement is based on the refocusing of the fluorescent object determining the displacement of a piezo mover keeping the moving object in focus. Track coordinates of the object with respect to the microfluidic device as well as images of the object are obtained at a frequency of several tenths of Hertz. This device is particularly well adapted to obtain trajectories of motile micro-organisms in microfluidic devices with or without flow.

Lagrangian 3D tracking of fluorescent microscopic objects in motion.

PubMed

Darnige, T; Figueroa-Morales, N; Bohec, P; Lindner, A; Clément, E

2017-05-01

We describe the development of a tracking device, mounted on an epi-fluorescent inverted microscope, suited to obtain time resolved 3D Lagrangian tracks of fluorescent passive or active micro-objects in microfluidic devices. The system is based on real-time image processing, determining the displacement of a x, y mechanical stage to keep the chosen object at a fixed position in the observation frame. The z displacement is based on the refocusing of the fluorescent object determining the displacement of a piezo mover keeping the moving object in focus. Track coordinates of the object with respect to the microfluidic device as well as images of the object are obtained at a frequency of several tenths of Hertz. This device is particularly well adapted to obtain trajectories of motile micro-organisms in microfluidic devices with or without flow.

Cd(II)-terpyridine-based complex as a ratiometric fluorescent probe for pyrophosphate detection in solution and as an imaging agent in living cells.

PubMed

Jiao, Shu-Yan; Li, Kun; Zhang, Wei; Liu, Yan-Hong; Huang, Zeng; Yu, Xiao-Qi

2015-01-21

The terpyridine anthracene ligand was synthesized and characterized. is a ratiometric fluorescent probe for Cd(2+) with a recognition mechanism based on intramolecular charge transfer (ICT). An complex was isolated, and its structure was established using single-crystal XRD. The complex was able to serve as a novel reversible chemosensing ensemble to allow ratiometric response to pyrophosphate (PPi) in aqueous media. Moreover, the fluorescence imaging in living cells from these two emission channels suggested that was a ratiometric probe for Cd(2+), and the in situ generated complex was also a ratiometric ensemble for PPi detection in living cells.

Molecular Imaging for Breast Cancer Using Magnetic Resonance-Guided Positron Emission Mammography and Excitation-Resolved Near-Infrared Fluorescence Imaging

NASA Astrophysics Data System (ADS)

Cho, Jaedu

The aim of this work is to develop novel breast-specific molecular imaging techniques for management of breast cancer. In this dissertation, we describe two novel molecular imaging approaches for breast cancer management. In Part I, we introduce our multimodal molecular imaging approach for breast cancer therapy monitoring using magnetic resonance imaging and positron emission mammography (MR/PEM). We have focused on the therapy monitoring technique for aggressive cancer molecular subtypes, which is challenging due to time constraint. Breast cancer therapy planning relies on a fast and accurate monitoring of functional and anatomical change. We demonstrate a proof-of-concept of sequential dual-modal magnetic resonance and positron emission mammography (MR/PEM) for the cancer therapy monitoring. We have developed dedicated breast coils with breast compression mechanism equipped with MR-compatible PEM detector heads. We have designed a fiducial marker that allows straightforward image registration of data obtained from MRI and PEM. We propose an optimal multimodal imaging procedure for MR/PEM. In Part II, we have focused on the development of a novel intraoperative near-infrared fluorescence imaging system (NIRF) for image-guided breast cancer surgery. Conventional spectrally-resolved NIRF systems are unable to resolve various NIR fluorescence dyes for the following reasons. First, the fluorescence spectra of viable NIR fluorescence dyes are heavily overlapping. Second, conventional emission-resolved NIRF suffers from a trade-off between the fluence rate and the spectral resolution. Third, the multiple scattering in tissue degrades not only the spatial information but also the spectral contents by the red-shift. We develop a wavelength-swept laser-based NIRF system that can resolve the excitation shift of various NIR fluorescence dyes without substantial loss of the fluence rate. A linear ratiometric model is employed to measure the relative shift of the excitation spectrum of a fluorescence dye.

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.

[Development of a Surgical Navigation System with Beam Split and Fusion of the Visible and Near-Infrared Fluorescence].

PubMed

Yang, Xiaofeng; Wu, Wei; Wang, Guoan

2015-04-01

This paper presents a surgical optical navigation system with non-invasive, real-time, and positioning characteristics for open surgical procedure. The design was based on the principle of near-infrared fluorescence molecular imaging. The in vivo fluorescence excitation technology, multi-channel spectral camera technology and image fusion software technology were used. Visible and near-infrared light ring LED excitation source, multi-channel band pass filters, spectral camera 2 CCD optical sensor technology and computer systems were integrated, and, as a result, a new surgical optical navigation system was successfully developed. When the near-infrared fluorescence was injected, the system could display anatomical images of the tissue surface and near-infrared fluorescent functional images of surgical field simultaneously. The system can identify the lymphatic vessels, lymph node, tumor edge which doctor cannot find out with naked eye intra-operatively. Our research will guide effectively the surgeon to remove the tumor tissue to improve significantly the success rate of surgery. The technologies have obtained a national patent, with patent No. ZI. 2011 1 0292374. 1.

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

NASA Astrophysics Data System (ADS)

Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

2018-05-01

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

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.

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.

Low-frequency wide-field fluorescence lifetime imaging using a high-power near-infrared light-emitting diode light source

PubMed Central

Gioux, Sylvain; Lomnes, Stephen J.; Choi, Hak Soo; Frangioni, John V.

2010-01-01

Fluorescence lifetime imaging (FLi) could potentially improve exogenous near-infrared (NIR) fluorescence imaging, because it offers the capability of discriminating a signal of interest from background, provides real-time monitoring of a chemical environment, and permits the use of several different fluorescent dyes having the same emission wavelength. We present a high-power, LED-based, NIR light source for the clinical translation of wide-field (larger than 5 cm in diameter) FLi at frequencies up to 35 MHz. Lifetime imaging of indocyanine green (ICG), IRDye 800-CW, and 3,3′-diethylthiatricarbocyanine iodide (DTTCI) was performed over a large field of view (10 cm by 7.5 cm) using the LED light source. For comparison, a laser diode light source was employed as a gold standard. Experiments were performed both on the bench by diluting the fluorescent dyes in various chemical environments in Eppendorf tubes, and in vivo by injecting the fluorescent dyes mixed in Matrigel subcutaneously into CD-1 mice. Last, measured fluorescence lifetimes obtained using the LED and the laser diode sources were compared with those obtained using a state-of-the-art time-domain imaging system and with those previously described in the literature. On average, lifetime values obtained using the LED and the laser diode light sources were consistent, exhibiting a mean difference of 3% from the expected values and a coefficient of variation of 12%. Taken together, our study offers an alternative to laser diodes for clinical translation of FLi and explores the use of relatively low frequency modulation for in vivo imaging. PMID:20459250

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

Super-resolution of fluorescence-free plasmonic nanoparticles using enhanced dark-field illumination based on wavelength-modulation

DOE PAGES

Zhang, Peng; Lee, Seungah; Yu, Hyunung; ...

2015-06-15

Super-resolution imaging of fluorescence-free plasmonic nanoparticles (NPs) was achieved using enhanced dark-field (EDF) illumination based on wavelength-modulation. Indistinguishable adjacent EDF images of 103-nm gold nanoparticles (GNPs), 40-nm gold nanorods (GNRs), and 80-nm silver nanoparticles (SNPs) were modulated at their wavelengths of specific localized surface plasmon scattering. The coordinates (x, y) of each NP were resolved by fitting their point spread functions with a two-dimensional Gaussian. The measured localization precisions of GNPs, GNRs, and SNPs were 2.5 nm, 5.0 nm, and 2.9 nm, respectively. From the resolved coordinates of NPs and the corresponding localization precisions, super-resolution images were reconstructed. Depending onmore » the spontaneous polarization of GNR scattering, the orientation angle of GNRs in two-dimensions was resolved and provided more elaborate localization information. This novel fluorescence-free super-resolution method was applied to live HeLa cells to resolve NPs and provided remarkable subdiffraction limit images.« less

Quantitative multiphoton imaging

NASA Astrophysics Data System (ADS)

König, Karsten; Weinigel, Martin; Breunig, Hans Georg; Uchugonova, Aisada

2014-02-01

Certified clinical multiphoton tomographs for label-free multidimensional high-resolution in vivo imaging have been introduced to the market several years ago. Novel tomographs include a flexible 360° scan head attached to a mechanooptical arm for autofluorescence and SHG imaging as well as a CARS module. Non-fluorescent lipids and water, mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged in vivo with submicron resolution in human skin. Sensitive and rapid detectors allow single photon counting and the construction of 3D maps where the number of detected photons per voxel is depicted. Intratissue concentration profiles from endogenous as well exogenous substances can be generated when the number of detected photons can be correlated with the number of molecules with respect to binding and scattering behavior. Furthermore, the skin ageing index SAAID based on the ratio elastin/collagen as well as the epidermis depth based on the onset of SHG generation can be determined.

Intracellular distribution and stability of a luminescent rhenium(I) tricarbonyl tetrazolato complex using epifluorescence microscopy in conjunction with X-ray fluorescence imaging

DOE PAGES

Wedding, Jason L.; Harris, Hugh H.; Bader, Christie A.; ...

2016-11-23

Optical fluorescence microscopy was used in conjunction with X-ray fluorescence microscopy to monitor the stability and intracellular distribution of the luminescent rhenium(I) complex fac-[Re(CO) 3(phen)L], where phen = 1,10-phenathroline and L = 5-(4-iodophenyl)tetrazolato, in 22Rv1 cells. The rhenium complex showed no signs of ancillary ligand dissociation, a conclusion based on data obtained via X-ray fluorescence imaging aligning iodine and rhenium distributions. A diffuse reticular localisation was detected for the complex, in the nuclear/perinuclear region of cells, by either optical or X-ray fluorescence techniques. Furthermore, X-ray fluorescence also showed that the Re-I complex disrupted the homeostasis of some biologically relevant elements,more » such as chlorine, potassium and zinc.« less

Preparation of a Nile Red-Pd-based fluorescent CO probe and its imaging applications in vitro and in vivo.

PubMed

Liu, Keyin; Kong, Xiuqi; Ma, Yanyan; Lin, Weiying

2018-05-01

Carbon monoxide (CO) is a key gaseous signaling molecule in living cells and organisms. This protocol illustrates the synthesis of a highly sensitive Nile Red (NR)-Pd-based fluorescent probe, NR-PdA, and its applications for detecting endogenous CO in tissue culture cells, ex vivo organs, and zebrafish embryos. In the NR-PdA synthesis process, 3-diethylamine phenol reacts with sodium nitrite in the acidic condition to afford 5-(diethylamino)-2-nitrosophenol hydrochloride (compound 1), which is further treated with 1-naphthalenol at a high temperature to provide the NR dye via a cyclization reaction. Finally, NR is reacted with palladium acetate to obtain the desired Pd-based fluorescent probe NR-PdA. NR-PdA possesses excellent two-photon excitation and near-IR emission properties, high stability, low background fluorescence, and a low detection limit. In addition to the chemical synthesis procedures, we provide step-by-step procedures for imaging endogenous CO in RAW 264.7 cells, mouse organs ex vivo, and live zebrafish embryos. The synthesis process for the probe requires ∼4 d, and the biological imaging experiments take ∼14 d.

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

First-in-human intraoperative near-infrared fluorescence imaging of glioblastoma using cetuximab-IRDye800.

PubMed

Miller, Sarah E; Tummers, Willemieke S; Teraphongphom, Nutte; van den Berg, Nynke S; Hasan, Alifia; Ertsey, Robert D; Nagpal, Seema; Recht, Lawrence D; Plowey, Edward D; Vogel, Hannes; Harsh, Griffith R; Grant, Gerald A; Li, Gordon H; Rosenthal, Eben L

2018-04-06

Maximizing extent of surgical resection with the least morbidity remains critical for survival in glioblastoma patients, and we hypothesize that it can be improved by enhancements in intraoperative tumor detection. In a clinical study, we determined if therapeutic antibodies could be repurposed for intraoperative imaging during resection. Fluorescently labeled cetuximab-IRDye800 was systemically administered to three patients 2 days prior to surgery. Near-infrared fluorescence imaging of tumor and histologically negative peri-tumoral tissue was performed intraoperatively and ex vivo. Fluorescence was measured as mean fluorescence intensity (MFI), and tumor-to-background ratios (TBRs) were calculated by comparing MFIs of tumor and histologically uninvolved tissue. The mean TBR was significantly higher in tumor tissue of contrast-enhancing (CE) tumors on preoperative imaging (4.0 ± 0.5) compared to non-CE tumors (1.2 ± 0.3; p = 0.02). The TBR was higher at a 100 mg dose than at 50 mg (4.3 vs. 3.6). The smallest detectable tumor volume in a closed-field setting was 70 mg with 50 mg of dye and 10 mg with 100 mg. On sections of paraffin embedded tissues, fluorescence positively correlated with histological evidence of tumor. Sensitivity and specificity of tumor fluorescence for viable tumor detection was calculated and fluorescence was found to be highly sensitive (73.0% for 50 mg dose, 98.2% for 100 mg dose) and specific (66.3% for 50 mg dose, 69.8% for 100 mg dose) for viable tumor tissue in CE tumors while normal peri-tumoral tissue showed minimal fluorescence. This first-in-human study demonstrates the feasibility and safety of antibody based imaging for CE glioblastomas.

Imaging of Fluoride Ion in Living Cells and Tissues with a Two-Photon Ratiometric Fluorescence Probe

PubMed Central

Zhu, Xinyue; Wang, Jianxi; Zhang, Jianjian; Chen, Zhenjie; Zhang, Haixia; Zhang, Xiaoyu

2015-01-01

A reaction-based two-photon (TP) ratiometric fluorescence probe Z2 has been developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z2 probe was designed designed to utilize an ICT mechanism between n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane (TBDPS) as a response group. Upon addition of fluoride ion, the Si-O bond in the Z2 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z2 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy (TPM). PMID:25594597

Near Infrared Fluorescence Imaging in Nano-Therapeutics and Photo-Thermal Evaluation

PubMed Central

Vats, Mukti; Mishra, Sumit Kumar; Baghini, Mahdieh Shojaei; Chauhan, Deepak S.; Srivastava, Rohit; De, Abhijit

2017-01-01

The unresolved and paramount challenge in bio-imaging and targeted therapy is to clearly define and demarcate the physical margins of tumor tissue. The ability to outline the healthy vital tissues to be carefully navigated with transection while an intraoperative surgery procedure is performed sets up a necessary and under-researched goal. To achieve the aforementioned objectives, there is a need to optimize design considerations in order to not only obtain an effective imaging agent but to also achieve attributes like favorable water solubility, biocompatibility, high molecular brightness, and a tissue specific targeting approach. The emergence of near infra-red fluorescence (NIRF) light for tissue scale imaging owes to the provision of highly specific images of the target organ. The special characteristics of near infra-red window such as minimal auto-fluorescence, low light scattering, and absorption of biomolecules in tissue converge to form an attractive modality for cancer imaging. Imparting molecular fluorescence as an exogenous contrast agent is the most beneficial attribute of NIRF light as a clinical imaging technology. Additionally, many such agents also display therapeutic potentials as photo-thermal agents, thus meeting the dual purpose of imaging and therapy. Here, we primarily discuss molecular imaging and therapeutic potentials of two such classes of materials, i.e., inorganic NIR dyes and metallic gold nanoparticle based materials. PMID:28452928

Ratiometric Near-Infrared Fluorescent Probes Based On Through-Bond Energy Transfer and π-Conjugation Modulation between Tetraphenylethene and Hemicyanine Moieties for Sensitive Detection of pH Changes in Live Cells.

PubMed

Wang, Jianbo; Xia, Shuai; Bi, Jianheng; Fang, Mingxi; Mazi, Wafa; Zhang, Yibin; Conner, Nathan; Luo, Fen-Tair; Lu, H Peter; Liu, Haiying

2018-04-18

In this paper, we present three ratiometric near-infrared fluorescent probes (A-C) for accurate, ratiometric detection of intracellular pH changes in live cells. Probe A consists of a tetraphenylethene (TPE) donor and near-infrared hemicyanine acceptor in a through-bond energy transfer (TBET) strategy, while probes B and C are composed of TPE and hemicyanine moieties through single and double sp 2 carbon-carbon bond connections in a π-conjugation modulation strategy. The specific targeting of the probes to lysosomes in live cells was achieved by introducing morpholine residues to the hemicyanine moieties to form closed spirolactam ring structures. Probe A shows aggregation-induced emission (AIE) property at neutral or basic pH, while probes B and C lack AIE properties. At basic or neutral pH, the probes only show fluorescence of TPE moieties with closed spirolactam forms of hemicyanine moieties, and effectively avoid blind fluorescence imaging spots, an issue which typical intensity-based pH fluorescent probes encounter. Three probes show ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with TPE fluorescence decreases and hemicyanine fluorescence increases, because acidic pH makes the spirolactam rings open to enhance π-conjugation of hemicyanine moieties. However, probe A shows much more sensitive ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with remarkable ratio increase of TPE fluorescence to hemicyanine fluorescence up to 238-fold than probes B and C because of its high efficiency of energy transfer from TPE donor to the hemicyanine acceptor in the TBET strategy. The probe offers dual Stokes shifts with a large pseudo-Stokes shift of 361 nm and well-defined dual emissions, and allows for colocalization of the imaging readouts of visible and near-infrared fluorescence channels to achieve more precisely double-checked ratiometric fluorescence imaging. These platforms could be employed to develop a variety of novel ratiometric fluorescent probes for accurate detection of different analytes in applications of chemical and biological sensing, imaging, and diagnostics by introducing appropriate sensing ligands to hemicyanine moieties to form on-off spirolactam switches.

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.

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.

Luminescent probes for optical in vivo imaging

NASA Astrophysics Data System (ADS)

Texier, Isabelle; Josserand, Veronique; Garanger, Elisabeth; Razkin, Jesus; Jin, Zhaohui; Dumy, Pascal; Favrot, Marie; Boturyn, Didier; Coll, Jean-Luc

2005-04-01

Going along with instrumental development for small animal fluorescence in vivo imaging, we are developing molecular fluorescent probes, especially for tumor targeting. Several criteria have to be taken into account for the optimization of the luminescent label. It should be adapted to the in vivo imaging optical conditions : red-shifted absorption and emission, limited overlap between absorption and emission for a good signal filtering, optimized luminescence quantum yield, limited photo-bleaching. Moreover, the whole probe should fulfill the biological requirements for in vivo labeling : adapted blood-time circulation, biological conditions compatibility, low toxicity. We here demonstrate the ability of the imaging fluorescence set-up developed in LETI to image the bio-distribution of molecular probes on short times after injection. Targeting with Cy5 labeled holo-transferrin of subcutaneous TS/Apc (angiogenic murine breast carcinoma model) or IGROV1 (human ovarian cancer) tumors was achieved. Differences in the kinetics of the protein uptake by the tumors were evidenced. IGROV1 internal metastatic nodes implanted in the peritoneal cavity could be detected in nude mice. However, targeted metastatic nodes in lung cancer could only be imaged after dissection of the mouse. These results validate our fluorescence imaging set-up and the use of Cy5 as a luminescent label. New fluorescent probes based on this dye and a molecular delivery template (the RAFT molecule) can thus be envisioned.

Amine-Reactive Fluorene Probes: Synthesis, Optical Characterization, Bioconjugation, and Two-Photon Fluorescence Imaging

PubMed Central

2008-01-01

With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially “lighting up” after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated. PMID:19090700

Identification of tissular origin of particles based on autofluorescence multispectral image analysis at the macroscopic scale

NASA Astrophysics Data System (ADS)

Corcel, Mathias; Devaux, Marie-Françoise; Guillon, Fabienne; Barron, Cécile

2017-06-01

Powders produced from plant materials are heterogeneous in relation to native plant heterogeneity, and during grinding, dissociation often occurred at the tissue scale. The tissue composition of powdery samples could be modified through dry fractionation diagrams and impact their end-uses properties. If tissue identification is often made on native plant structure, this characterization is not straightforward in destructured samples such powders. Taking advantage of the autofluorescence properties of cell wall components, multispectral image acquisition is envisioned to identify the tissular origin of particles. Images were acquired on maize stem sections and ground tissues isolated from the same stem by hand dissection. The variability in fluorescence intensity profiles was analysed using principal component analysis. The correspondence between fluorescence profiles and the different tissues observed in maize sections was assessed based on histology or known compositional heterogeneity. Similar variability was encountered in fluorescence profiles extracted from powder leading to the potential ability to predict tissular origin based on this autofluorescence multispectral signal.

Fast and accurate image recognition algorithms for fresh produce food safety sensing

NASA Astrophysics Data System (ADS)

Yang, Chun-Chieh; Kim, Moon S.; Chao, Kuanglin; Kang, Sukwon; Lefcourt, Alan M.

2011-06-01

This research developed and evaluated the multispectral algorithms derived from hyperspectral line-scan fluorescence imaging under violet LED excitation for detection of fecal contamination on Golden Delicious apples. The algorithms utilized the fluorescence intensities at four wavebands, 680 nm, 684 nm, 720 nm, and 780 nm, for computation of simple functions for effective detection of contamination spots created on the apple surfaces using four concentrations of aqueous fecal dilutions. The algorithms detected more than 99% of the fecal spots. The effective detection of feces showed that a simple multispectral fluorescence imaging algorithm based on violet LED excitation may be appropriate to detect fecal contamination on fast-speed apple processing lines.

Fast globally optimal segmentation of cells in fluorescence microscopy images.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2011-01-01

Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

Hydrophobic-carbon-dot-based dual-emission micelle for ratiometric fluorescence biosensing and imaging of Cu2+ in liver cells.

PubMed

Lu, Linlin; Feng, Chongchong; Xu, Jie; Wang, Fengyang; Yu, Haijun; Xu, Zhiai; Zhang, Wen

2017-06-15

Copper is closely related to liver damage, therefore, it is essential to develop a simple and sensitive strategy to detect copper ions (Cu 2+ ) in liver cells. A hydrophobic carbon dots (HCDs)-based dual-emission fluorescent probe for Cu 2+ was prepared by encapsulating HCDs in micelles formed by self-assembly of amphiphilic polymer DSPE-PEG and tetrakis (4-carboxyphenyl) porphyrin (TCPP)-modified DSPE-PEG. The obtained probe showed characteristic fluorescence emissions of HCDs and TCPP with large emission shift of 170nm with single-wavelength excitation. In the presence of Cu 2+ , the fluorescence of TCPP was quenched and that of HCDs remained unchanged, displaying ratiometric fluorescence response to Cu 2+ . The developed probe exhibited high sensitivity (detection limit down to 36nM) and selectivity to Cu 2+ over other substances, and the probe was used to image the changes of Cu 2+ level in liver cells successfully. Copyright © 2017 Elsevier B.V. All rights reserved.

Ratiometric sensing of fluoride and acetate anions based on a BODIPY-azaindole platform and its application to living cell imaging.

PubMed

Mahapatra, Ajit Kumar; Maji, Rajkishor; Maiti, Kalipada; Adhikari, Susanta Sekhar; Das Mukhopadhyay, Chitrangada; Mandal, Debasish

2014-01-07

A new BODIPY-azaindole based fluorescent sensor 1 was designed and synthesized as a new colorimetric and ratiometric fluorescent chemosensor for fluoride. The binding and sensing abilities of sensor 1 towards various anions were studied by absorption, emission and (1)H NMR titration spectroscopies. The spectral responses of 1 to fluoride in acetonitrile-water were studied: an approximately 69 nm red shift in absorption and ratiometric fluorescent response was observed. The striking light yellow to deep brown color change in ambient light and green to blue emission color change are thought to be due to the deprotonation of the indole moiety of the azaindole fluorophore. From the changes in the absorption, fluorescence, and (1)H NMR titration spectra, proton-transfer mechanisms were deduced. Density function theory and time-dependent density function theory calculations were conducted to rationalize the optical response of the sensor. Results were supported by confocal fluorescence imaging and MTT assay of live cells.

A fluorescent probe for the efficient discrimination of Cys, Hcy and GSH based on different cascade reactions.

PubMed

Li, Ying; Liu, Weimin; Zhang, Panpan; Zhang, Hongyan; Wu, Jiasheng; Ge, Jiechao; Wang, Pengfei

2017-04-15

A fluorescent probe (1) for distinguishing amongst biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), is developed based on different cascade reactions. The key design feature of fluorescent probe 1 is the integration of two potential reaction groups for the thiol and amino groups of biothiols in one molecule. By reacting with the halogen atom and α, β-unsaturated malonitrile in probe 1, Cys, Hcy and GSH can generate a total of three main products with distinct photophysical properties. Probe 1 shows a strong fluorescence turn-on response to Cys with blue-green emission by using an excitation wavelength of 390nm. At an excitation wavelength of 500nm, probe 1 responds to GSH over Cys and Hcy and emits strong orange fluorescence. The discrimination of biothiols can be demonstrated by cell imaging experiments, indicating that probe 1 can be a useful tool for the selective imaging of Cys and GSH in living cells. Copyright © 2016 Elsevier B.V. All rights reserved.

One-step synthesis of solid state luminescent carbon-based silica nanohybrids for imaging of latent fingerprints

NASA Astrophysics Data System (ADS)

Li, Feng; Li, Hongren; Cui, Tianfang

2017-11-01

Fluorescent carbon-based nanomaterials(CNs) with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. Despite the successes in preparing strongly fluorescent CNs, preserving the luminescence in solid materials is still challenging because of the serious emission quenching of CNs in solid state materials. In this work, fluorescent carbon and silica nanohybrids (SiCNHs) were synthesized via a simple one-step hydrothermal approach by carbonizing sodium citrate and (3-aminopropyl)triethoxysilane(APTES), and hydrolysis of tetraethyl orthosilicate(TEOS). The resultant SiCNs were characterized through X-ray diffraction (XRD), transmission electron microscopy (TEM), FT-IR, X-ray photoelectron spectroscopy (XPS), and photoluminescence (PL) spectroscopy. The SiCNs exhibited strong fluorescence in both aqueous and solid states. The luminescent solid state SiCNs power were successfully used as a fluorescent labeling material for enhanced imaging of latent fingerprints(LFPs) on single background colour and multi-coloured surfaces substrates in forensic science for individual identification.

Design of a smartphone-camera-based fluorescence imaging system for the detection of oral cancer

NASA Astrophysics Data System (ADS)

Uthoff, Ross

Shown is the design of the Smartphone Oral Cancer Detection System (SOCeeDS). The SOCeeDS attaches to a smartphone and utilizes its embedded imaging optics and sensors to capture images of the oral cavity to detect oral cancer. Violet illumination sources excite the oral tissues to induce fluorescence. Images are captured with the smartphone's onboard camera. Areas where the tissues of the oral cavity are darkened signify an absence of fluorescence signal, indicating breakdown in tissue structure brought by precancerous or cancerous conditions. With this data the patient can seek further testing and diagnosis as needed. Proliferation of this device will allow communities with limited access to healthcare professionals a tool to detect cancer in its early stages, increasing the likelihood of cancer reversal.

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

PubMed Central

Tichauer, Kenneth M.; Holt, Robert W.; Samkoe, Kimberley S.; El-Ghussein, Fadi; Gunn, Jason R.; Jermyn, Michael; Dehghani, Hamid; Leblond, Frederic; Pogue, Brian W.

2012-01-01

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3. Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model. The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging. Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17. PMID:22847515

Computed tomography-guided time-domain diffuse fluorescence tomography in small animals for localization of cancer biomarkers.

PubMed

Tichauer, Kenneth M; Holt, Robert W; Samkoe, Kimberley S; El-Ghussein, Fadi; Gunn, Jason R; Jermyn, Michael; Dehghani, Hamid; Leblond, Frederic; Pogue, Brian W

2012-07-17

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals. Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity, while our group and a few others have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model. The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control, laser referencing, and co-registration with a small animal computed tomography (microCT) system. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging. Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat.

In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope

NASA Astrophysics Data System (ADS)

Flusberg, Benjamin A.; Jung, Juergen C.; Cocker, Eric D.; Anderson, Erik P.; Schnitzer, Mark J.

2005-09-01

We introduce a compact two-photon fluorescence microendoscope based on a compound gradient refractive index endoscope probe, a DC micromotor for remote adjustment of the image plane, and a flexible photonic bandgap fiber for near distortion-free delivery of ultrashort excitation pulses. The imaging head has a mass of only 3.9 g and provides micrometer-scale resolution. We used portable two-photon microendoscopy to visualize hippocampal blood vessels in the brains of live mice.

Full color emitting fluorescent carbon material as reversible pH sensor with multicolor live cell imaging.

PubMed

Sharma, Vinay; Kaur, Navpreet; Tiwari, Pranav; Mobin, Shaikh M

2018-05-01

Carbon-based nano materials are developed as a cytocompatible alternative to semiconducting quantum dots for bioimaging and fluorescence-based sensing. The green alternatives for the synthesis of carbon materials are imminent. The present study demonstrates microwave based one step quick synthesis of fluorescent carbon material (FCM) having three variants: (i) un-doped fluorescent carbon material (UFCM) (ii) nitrogen doped FCM (N@FCM), and (iii) nitrogen & phosphorus co-doped FCM (N-P@FCM) using sugarcane extract as a carbon source. The N doping was performed using ethylenediamine and phosphoric acid was used for P doping. The heteroatom doped FCM were synthesized due to insolubility of UFCM in water. Unlike, UFCM, the N@FCM and N-P@FCM were found to be highly soluble in water. The N-P@FCM shows highest quantum yield among the three. The N-P@FCM was explored for alkaline pH sensing and it shows a quenching of fluorescence in the pH range 09-14. The sensing behaviour shows reversibility and high selectivity. Further, the sensor was also investigated for their biocompatibility and hence employed as a promising multicolour probe for cancer cell imaging. The generality in cell imaging was investigated by flow cytometry. The hetero-atom doped green carbon-dots may open new avenues for sensing and selective cellular targeting. Copyright © 2018 Elsevier B.V. All rights reserved.

Structural effects of naphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in live zebrafish

NASA Astrophysics Data System (ADS)

Choi, Seon-Ae; Park, Chul Soon; Kwon, Oh Seok; Giong, Hoi-Khoanh; Lee, Jeong-Soo; Ha, Tai Hwan; Lee, Chang-Soo

2016-05-01

Hydrogen sulfide (H2S) is an important biological messenger, but few biologically-compatible methods are available for its detection in aqueous solution. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe (L1), which is a highly versatile building unit that absorbs and emits at long wavelengths and is selective for hydrogen sulfide over cysteine, glutathione, and other reactive sulfur, nitrogen, and oxygen species in aqueous solution. We describe turn-on fluorescent probes based on azide group reduction on the fluorogenic ‘naphthalene’ moiety to fluorescent amines and intracellular hydrogen sulfide detection without the use of an organic solvent. L1 and L2 were synthetically modified to functional groups with comparable solubility on the N-imide site, showing a marked change in turn-on fluorescent intensity in response to hydrogen sulfide in both PBS buffer and living cells. The probes were readily employed to assess intracellular hydrogen sulfide level changes by imaging endogenous hydrogen sulfide signal in RAW264.7 cells incubated with L1 and L2. Expanding the use of L1 to complex and heterogeneous biological settings, we successfully visualized hydrogen sulfide detection in the yolk, brain and spinal cord of living zebrafish embryos, thereby providing a powerful approach for live imaging for investigating chemical signaling in complex multicellular systems.

Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2012-10-01

In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging.

PubMed

Poëa-Guyon, Sandrine; Pasquier, Hélène; Mérola, Fabienne; Morel, Nicolas; Erard, Marie

2013-05-01

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

General Staining and Segmentation Procedures for High Content Imaging and Analysis.

PubMed

Chambers, Kevin M; Mandavilli, Bhaskar S; Dolman, Nick J; Janes, Michael S

2018-01-01

Automated quantitative fluorescence microscopy, also known as high content imaging (HCI), is a rapidly growing analytical approach in cell biology. Because automated image analysis relies heavily on robust demarcation of cells and subcellular regions, reliable methods for labeling cells is a critical component of the HCI workflow. Labeling of cells for image segmentation is typically performed with fluorescent probes that bind DNA for nuclear-based cell demarcation or with those which react with proteins for image analysis based on whole cell staining. These reagents, along with instrument and software settings, play an important role in the successful segmentation of cells in a population for automated and quantitative image analysis. In this chapter, we describe standard procedures for labeling and image segmentation in both live and fixed cell samples. The chapter will also provide troubleshooting guidelines for some of the common problems associated with these aspects of HCI.

Multispectral open-air intraoperative fluorescence imaging.

PubMed

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

2017-08-01

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

Time-resolved wide-field optically sectioned fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dupuis, Guillaume; Benabdallah, Nadia; Chopinaud, Aurélien; Mayet, Céline; Lévêque-Fort, Sandrine

2013-02-01

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.

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.

Highly sensitive detection of human papillomavirus type 16 DNA using time-resolved fluorescence microscopy and long lifetime probes

NASA Astrophysics Data System (ADS)

Wang, Xue F.; Periasamy, Ammasi; Wodnicki, Pawel; Siadat-Pajouh, M.; Herman, Brian

1995-04-01

We have been interested in the role of Human Papillomavirus (HPV) in cervical cancer and its diagnosis; to that end we have been developing microscopic imaging and fluorescent in situ hybridization (FISH) techniques to genotype and quantitate the amount of HPV present at a single cell level in cervical PAP smears. However, we have found that low levels of HPV DNA are difficult to detect accurately because theoretically obtainable sensitivity is never achieved due to nonspecific autofluorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components in the microscopic system. In addition, the absorption stains used for PAP smears are intensely autofluorescent. Autofluorescence is a rapidly decaying process with lifetimes in the range of 1-100 nsec, whereas phosphorescence and delayed fluorescence have lifetimes in the range of 1 microsecond(s) ec-10 msec. The ability to discriminate between specific fluorescence and autofluorescence in the time-domain has improved the sensitivity of diagnostic test such that they perform comparably to, or even more sensitive than radioisotopic assays. We have developed a novel time-resolved fluorescence microscope to improve the sensitivity of detection of specific molecules of interest in slide based specimens. This time-resolved fluorescence microscope is based on our recently developed fluorescence lifetime imaging microscopy (FILM) in conjunction with the use of long lifetime fluorescent labels. By using fluorescence in situ hybridization and the long lifetime probe (europium), we have demonstrated the utility of this technique for detection of HPV DNA in cervicovaginal cells. Our results indicate that the use of time-resolved fluorescence microscopy and long lifetime probes increases the sensitivity of detection by removing autofluorescence and will thus lead to improved early diagnosis of cervical cancer. Since the highly sensitive detection of DNA in clinical samples using fluorescence in situ hybridization image is useful for the diagnosis of many other type of diseases, the system we have developed should find numerous applications for the diagnosis of disease states.

Imaging systems and algorithms to analyze biological samples in real-time using mobile phone microscopy

PubMed Central

Mayberry, Addison; Perkins, David L.; Holcomb, Daniel E.

2018-01-01

Miniaturized imaging devices have pushed the boundaries of point-of-care imaging, but existing mobile-phone-based imaging systems do not exploit the full potential of smart phones. This work demonstrates the use of simple imaging configurations to deliver superior image quality and the ability to handle a wide range of biological samples. Results presented in this work are from analysis of fluorescent beads under fluorescence imaging, as well as helminth eggs and freshwater mussel larvae under white light imaging. To demonstrate versatility of the systems, real time analysis and post-processing results of the sample count and sample size are presented in both still images and videos of flowing samples. PMID:29509786

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.

In situ fluorescence activation of DNA-silver nanoclusters as a label-free and general strategy for cell nucleus imaging.

PubMed

Li, Duo; Qiao, Zhenzhen; Yu, Yanru; Tang, Jinlu; He, Xiaoxiao; Shi, Hui; Ye, Xiaosheng; Lei, Yanli; Wang, Kemin

2018-01-25

A facile, general and turn-on nucleus imaging strategy was first developed based on in situ fluorescence activation of C-rich dark silver nanoclusters by G-rich telomeres. After a simple incubation without washing, nanoclusters could selectively stain the nucleus with intense red luminescence, which was confirmed using fixed/living cells and several cell lines.

A miniaturised image based fluorescence detection system for point-of-care-testing of cocaine abuse

NASA Astrophysics Data System (ADS)

Walczak, Rafał; Krüger, Jan; Moynihan, Shane

2015-08-01

In this paper, we describe a miniaturised image-based fluorescence detection system and demonstrate its viability as a highly sensitive tool for point-of-care-analysis of drugs of abuse in human sweat with a focus on monitor individuals for drugs of abuse. Investigations of miniaturised and low power optoelectronic configurations and methodologies for real-time image analysis were successfully carried out. The miniaturised fluorescence detection system was validated against a reference detection system under controlled laboratory conditions by analysing spiked sweat samples in dip stick and then strip with sample pad. As a result of the validation studies, a 1 ng mL-1 limit of detection of cocaine in sweat and full agreement of test results with the reference detection system can be reported. Results of the investigations open the way towards a detection system that integrates a hand-held fluorescence reader and a wearable skinpatch, and which can collect and in situ analyse sweat for the presence of cocaine at any point for up to tenths hours.

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.

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.

Temporal Effects on Internal Fluorescence Emissions Associated with Aflatoxin Contamination from Corn Kernel Cross-Sections Inoculated with Toxigenic and Atoxigenic Aspergillus flavus.

PubMed

Hruska, Zuzana; Yao, Haibo; Kincaid, Russell; Brown, Robert L; Bhatnagar, Deepak; Cleveland, Thomas E

2017-01-01

Non-invasive, easy to use and cost-effective technology offers a valuable alternative for rapid detection of carcinogenic fungal metabolites, namely aflatoxins, in commodities. One relatively recent development in this area is the use of spectral technology. Fluorescence hyperspectral imaging, in particular, offers a potential rapid and non-invasive method for detecting the presence of aflatoxins in maize infected with the toxigenic fungus Aspergillus flavus . Earlier studies have shown that whole maize kernels contaminated with aflatoxins exhibit different spectral signatures from uncontaminated kernels based on the external fluorescence emission of the whole kernels. Here, the effect of time on the internal fluorescence spectral emissions from cross-sections of kernels infected with toxigenic and atoxigenic A. flavus , were examined in order to elucidate the interaction between the fluorescence signals emitted by some aflatoxin contaminated maize kernels and the fungal invasion resulting in the production of aflatoxins. First, the difference in internal fluorescence emissions between cross-sections of kernels incubated in toxigenic and atoxigenic inoculum was assessed. Kernels were inoculated with each strain for 5, 7, and 9 days before cross-sectioning and imaging. There were 270 kernels (540 halves) imaged, including controls. Second, in a different set of kernels (15 kernels/group; 135 total), the germ of each kernel was separated from the endosperm to determine the major areas of aflatoxin accumulation and progression over nine growth days. Kernels were inoculated with toxigenic and atoxigenic fungal strains for 5, 7, and 9 days before the endosperm and germ were separated, followed by fluorescence hyperspectral imaging and chemical aflatoxin determination. A marked difference in fluorescence intensity was shown between the toxigenic and atoxigenic strains on day nine post-inoculation, which may be a useful indicator of the location of aflatoxin contamination. This finding suggests that both, the fluorescence peak shift and intensity as well as timing, may be essential in distinguishing toxigenic and atoxigenic fungi based on spectral features. Results also reveal a possible preferential difference in the internal colonization of maize kernels between the toxigenic and atoxigenic strains of A. flavus suggesting a potential window for differentiating the strains based on fluorescence spectra at specific time points.

Temporal Effects on Internal Fluorescence Emissions Associated with Aflatoxin Contamination from Corn Kernel Cross-Sections Inoculated with Toxigenic and Atoxigenic Aspergillus flavus

PubMed Central

Hruska, Zuzana; Yao, Haibo; Kincaid, Russell; Brown, Robert L.; Bhatnagar, Deepak; Cleveland, Thomas E.

2017-01-01

Non-invasive, easy to use and cost-effective technology offers a valuable alternative for rapid detection of carcinogenic fungal metabolites, namely aflatoxins, in commodities. One relatively recent development in this area is the use of spectral technology. Fluorescence hyperspectral imaging, in particular, offers a potential rapid and non-invasive method for detecting the presence of aflatoxins in maize infected with the toxigenic fungus Aspergillus flavus. Earlier studies have shown that whole maize kernels contaminated with aflatoxins exhibit different spectral signatures from uncontaminated kernels based on the external fluorescence emission of the whole kernels. Here, the effect of time on the internal fluorescence spectral emissions from cross-sections of kernels infected with toxigenic and atoxigenic A. flavus, were examined in order to elucidate the interaction between the fluorescence signals emitted by some aflatoxin contaminated maize kernels and the fungal invasion resulting in the production of aflatoxins. First, the difference in internal fluorescence emissions between cross-sections of kernels incubated in toxigenic and atoxigenic inoculum was assessed. Kernels were inoculated with each strain for 5, 7, and 9 days before cross-sectioning and imaging. There were 270 kernels (540 halves) imaged, including controls. Second, in a different set of kernels (15 kernels/group; 135 total), the germ of each kernel was separated from the endosperm to determine the major areas of aflatoxin accumulation and progression over nine growth days. Kernels were inoculated with toxigenic and atoxigenic fungal strains for 5, 7, and 9 days before the endosperm and germ were separated, followed by fluorescence hyperspectral imaging and chemical aflatoxin determination. A marked difference in fluorescence intensity was shown between the toxigenic and atoxigenic strains on day nine post-inoculation, which may be a useful indicator of the location of aflatoxin contamination. This finding suggests that both, the fluorescence peak shift and intensity as well as timing, may be essential in distinguishing toxigenic and atoxigenic fungi based on spectral features. Results also reveal a possible preferential difference in the internal colonization of maize kernels between the toxigenic and atoxigenic strains of A. flavus suggesting a potential window for differentiating the strains based on fluorescence spectra at specific time points. PMID:28966606

Sentinel lymph nodes and lymphatic vessels: noninvasive dual-modality in vivo mapping by using indocyanine green in rats--volumetric spectroscopic photoacoustic imaging and planar fluorescence imaging.

PubMed

Kim, Chulhong; Song, Kwang Hyun; Gao, Feng; Wang, Lihong V

2010-05-01

To noninvasively map sentinel lymph nodes (SLNs) and lymphatic vessels in rats in vivo by using dual-modality nonionizing imaging-volumetric spectroscopic photoacoustic imaging, which measures optical absorption, and planar fluorescence imaging, which measures fluorescent emission-of indocyanine green (ICG). Institutional animal care and use committee approval was obtained. Healthy Sprague-Dawley rats weighing 250-420 g (age range, 60-120 days) were imaged by using volumetric photoacoustic imaging (n = 5) and planar fluorescence imaging (n = 3) before and after injection of 1 mmol/L ICG. Student paired t tests based on a logarithmic scale were performed to evaluate the change in photoacoustic signal enhancement of SLNs and lymphatic vessels before and after ICG injection. The spatial resolutions of both imaging systems were compared at various imaging depths (2-8 mm) by layering additional biologic tissues on top of the rats in vivo. Spectroscopic photoacoustic imaging was applied to identify ICG-dyed SLNs. In all five rats examined with photoacoustic imaging, SLNs were clearly visible, with a mean signal enhancement of 5.9 arbitrary units (AU) + or - 1.8 (standard error of the mean) (P < .002) at 0.2 hour after injection, while lymphatic vessels were seen in four of the five rats, with a signal enhancement of 4.3 AU + or - 0.6 (P = .001). In all three rats examined with fluorescence imaging, SLNs and lymphatic vessels were seen. The average full width at half maximum (FWHM) of the SLNs in the photoacoustic images at three imaging depths (2, 6, and 8 mm) was 2.0 mm + or - 0.2 (standard deviation), comparable to the size of a dissected lymph node as measured with a caliper. However, the FWHM of the SLNs in fluorescence images widened from 8 to 22 mm as the imaging depth increased, owing to strong light scattering. SLNs were identified spectroscopically in photoacoustic images. These two modalities, when used together with ICG, have the potential to help map SLNs in axillary staging and to help evaluate tumor metastasis in patients with breast cancer.

S- Cis Diene Conformation: A New Bathochromic Shift Strategy for Near-Infrared Fluorescence Switchable Dye and the Imaging Applications.

PubMed

Chen, Hsiang-Jung; Chew, Chee Ying; Chang, En-Hao; Tu, Yu-Wei; Wei, Li-Yu; Wu, Bo-Han; Chen, Chien-Hung; Yang, Ya-Ting; Huang, Su-Chin; Chen, Jen-Kun; Chen, I-Chia; Tan, Kui-Thong

2018-04-18

In this paper, we present a novel charge-free fluorescence-switchable near-infrared (IR) dye based on merocyanine for target specific imaging. In contrast to the typical bathochromic shift approach by extending π-conjugation, the bathochromic shift of our merocyanine dye to the near-IR region is due to an unusual S- cis diene conformer. This is the first example where a fluorescent dye adopts the stable S- cis conformation. In addition to the novel bathochromic shift mechanism, the dye exhibits fluorescence-switchable properties in response to polarity and viscosity. By incorporating a protein-specific ligand to the dye, the probes (for SNAP-tag and hCAII proteins) exhibited dramatic fluorescence increase (up to 300-fold) upon binding with its target protein. The large fluorescence enhancement, near-IR absorption/emission, and charge-free scaffold enabled no-wash and site-specific imaging of target proteins in living cells and in vivo with minimum background fluorescence. We believe that our unconventional approach for a near-IR dye with the S- cis diene conformation can lead to new strategies for the design of near-IR dyes.

Rotational multispectral fluorescence lifetime imaging and intravascular ultrasound: bimodal system for intravascular applications

PubMed Central

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

2014-01-01

Abstract. We report the development and validation of a hybrid intravascular diagnostic system combining multispectral fluorescence lifetime imaging (FLIm) and intravascular ultrasound (IVUS) for cardiovascular imaging applications. A prototype FLIm system based on fluorescence pulse sampling technique providing information on artery biochemical composition was integrated with a commercial IVUS system providing information on artery morphology. A customized 3-Fr bimodal catheter combining a rotational side-view fiberoptic and a 40-MHz IVUS transducer was constructed for sequential helical scanning (rotation and pullback) of tubular structures. Validation of this bimodal approach was conducted in pig heart coronary arteries. Spatial resolution, fluorescence detection efficiency, pulse broadening effect, and lifetime measurement variability of the FLIm system were systematically evaluated. Current results show that this system is capable of temporarily resolving the fluorescence emission simultaneously in multiple spectral channels in a single pullback sequence. Accurate measurements of fluorescence decay characteristics from arterial segments can be obtained rapidly (e.g., 20 mm in 5 s), and accurate co-registration of fluorescence and ultrasound features can be achieved. The current finding demonstrates the compatibility of FLIm instrumentation with in vivo clinical investigations and its potential to complement conventional IVUS during catheterization procedures. PMID:24898604

Elemental mapping in a contemporary miniature by full-field X-ray fluorescence imaging with gaseous detector vs. scanning X-ray fluorescence imaging with polycapillary optics

NASA Astrophysics Data System (ADS)

Silva, A. L. M.; Cirino, S.; Carvalho, M. L.; Manso, M.; Pessanha, S.; Azevedo, C. D. R.; Carramate, L. F. N. D.; Santos, J. P.; Guerra, M.; Veloso, J. F. C. A.

2017-03-01

Energy dispersive X-ray imaging can be used in several research fields and industrial applications. Elemental mapping through energy dispersive X-ray imaging technique has become a promising method to obtain positional distribution of specific elements in a non-destructive way. To obtain the elemental distribution of a sample it is necessary to use instruments capable of providing a precise positioning together with a good energy resolution. Polycapillary beams together with silicon drift chamber detectors are used in several commercial systems and are considered state-of-the-art spectrometers, however they are usually very costly. A new concept of large energy dispersive X-ray imaging systems based on gaseous radiation detectors emerged in the last years enabling a promising 2D elemental detection at a very reduced price. The main goal of this work is to analyze a contemporary Indian miniature with both X-ray fluorescence imaging systems, the one based on a gaseous detector 2D-THCOBRA and the state-of-the-art spectrometer M4 Tornado, from Bruker. The performance of both systems is compared and evaluated in the context of the sample's analysis.

Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos.

PubMed

Karnowski, Karol; Ajduk, Anna; Wieloch, Bartosz; Tamborski, Szymon; Krawiec, Krzysztof; Wojtkowski, Maciej; Szkulmowski, Maciej

2017-06-23

Imaging of living cells based on traditional fluorescence and confocal laser scanning microscopy has delivered an enormous amount of information critical for understanding biological processes in single cells. However, the requirement for a high numerical aperture and fluorescent markers still limits researchers' ability to visualize the cellular architecture without causing short- and long-term photodamage. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of fluorescence imaging techniques and provides unique access to fundamental aspects of early embryonic development, without the requirement for sample pre-processing or labeling. In the present paper, we utilized the internal motion of cytoplasm, as well as custom scanning and signal processing protocols, to effectively reduce the speckle noise typical for standard OCM and enable high-resolution intracellular time-lapse imaging. To test our imaging system we used mouse and pig oocytes and embryos and visualized them through fertilization and the first embryonic division, as well as at selected stages of oogenesis and preimplantation development. Because all morphological and morphokinetic properties recorded by OCM are believed to be biomarkers of oocyte/embryo quality, OCM may represent a new chapter in imaging-based preimplantation embryo diagnostics.

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

Identification of the optimal therapeutic antibody for fluorescent imaging of cutaneous squamous cell carcinoma

PubMed Central

Day, Kristine E.; Beck, Lauren N.; Heath, C. Hope; Huang, Conway C.; Zinn, Kurt R.; Rosenthal, Eben L.

2013-01-01

Intraoperative, real-time fluorescence imaging may significantly improve tumor visualization and resection and postoperatively, in pathological assessment. To this end, we sought to determine the optimal FDA approved therapeutic monoclonal antibody for optical imaging of human cutaneous squamous cell carcinoma (cSCC). A near-infrared (NIR) fluorescent probe (IRDye800) was covalently linked to bevacizumab, panitumumab or tocilizumab and injected systemically into immunodeficient mice bearing either cutaneous tumor cell lines (SCC13) or cutaneous human tumor explants. Tumors were then imaged and resected under fluorescent guidance with the SPY, an FDA-approved intraoperative imaging system, and the Pearl Impulse small animal imaging system. All fluorescently labeled antibodies delineated normal tissue from tumor in SCC13 xenografts based on tumor-to-background (TBR) ratios. The conjugated antibodies produced TBRs of 1.2–2 using SPY and 1.6–3.6 using Pearl; in comparison, isotype control antibody IgG-IRDye produced TBRs of 1.0 (SPY) and 0.98 (Pearl). Comparison between antibodies revealed them to be roughly equivalent for imaging purposes with both the SPY and Pearl (p = 0.89 SPY, p = 0.99 Pearl; one way ANOVA). Human tumor explants were also imaged and tumor detection was highest with panitumumab-IRDye800 when using the SPY (TBR 3.0) and Pearl (TBR 4.0). These data suggest that FDA approved antibodies may be clinically used for intraoperative detection of cSCC. PMID:23298904

Magnetic resonance-coupled fluorescence tomography scanner for molecular imaging of tissue

NASA Astrophysics Data System (ADS)

Davis, Scott C.; Pogue, Brian W.; Springett, Roger; Leussler, Christoph; Mazurkewitz, Peter; Tuttle, Stephen B.; Gibbs-Strauss, Summer L.; Jiang, Shudong S.; Dehghani, Hamid; Paulsen, Keith D.

2008-06-01

A multichannel spectrally resolved optical tomography system to image molecular targets in small animals from within a clinical MRI is described. Long source/detector fibers operate in contact mode and couple light from the tissue surface in the magnet bore to 16 spectrometers, each containing two optical gratings optimized for the near infrared wavelength range. High sensitivity, cooled charge coupled devices connected to each spectrograph provide detection of the spectrally resolved signal, with exposure times that are automated for acquisition at each fiber. The design allows spectral fitting of the remission light, thereby separating the fluorescence signal from the nonspecific background, which improves the accuracy and sensitivity when imaging low fluorophore concentrations. Images of fluorescence yield are recovered using a nonlinear reconstruction approach based on the diffusion approximation of photon propagation in tissue. The tissue morphology derived from the MR images serves as an imaging template to guide the optical reconstruction algorithm. Sensitivity studies show that recovered values of indocyanine green fluorescence yield are linear to concentrations of 1nM in a 70mm diameter homogeneous phantom, and detection is feasible to near 10pM. Phantom data also demonstrate imaging capabilities of imperfect fluorophore uptake in tissue volumes of clinically relevant sizes. A unique rodent MR coil provides optical fiber access for simultaneous optical and MR data acquisition of small animals. A pilot murine study using an orthotopic glioma tumor model demonstrates optical-MRI imaging of an epidermal growth factor receptor targeted fluorescent probe in vivo.

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.

Spatially-controlled illumination with rescan confocal microscopy enhances image quality, resolution and reduces photodamage

NASA Astrophysics Data System (ADS)

Krishnaswami, Venkataraman; De Luca, Giulia M. R.; Breedijk, Ronald M. P.; Van Noorden, Cornelis J. F.; Manders, Erik M. M.; Hoebe, Ron A.

2017-02-01

Fluorescence microscopy is an important tool in biomedical imaging. An inherent trade-off lies between image quality and photodamage. Recently, we have introduced rescan confocal microscopy (RCM) that improves the lateral resolution of a confocal microscope down to 170 nm. Previously, we have demonstrated that with controlled-light exposure microscopy, spatial control of illumination reduces photodamage without compromising image quality. Here, we show that the combination of these two techniques leads to high resolution imaging with reduced photodamage without compromising image quality. Implementation of spatially-controlled illumination was carried out in RCM using a line scanning-based approach. Illumination is spatially-controlled for every line during imaging with the help of a prediction algorithm that estimates the spatial profile of the fluorescent specimen. The estimation is based on the information available from previously acquired line images. As a proof-of-principle, we show images of N1E-115 neuroblastoma cells, obtained by this new setup with reduced illumination dose, improved resolution and without compromising image quality.

Methodological challenges of optical tweezers-based X-ray fluorescence imaging of biological model organisms at synchrotron facilities.

PubMed

Vergucht, Eva; Brans, Toon; Beunis, Filip; Garrevoet, Jan; Bauters, Stephen; De Rijcke, Maarten; Deruytter, David; Janssen, Colin; Riekel, Christian; Burghammer, Manfred; Vincze, Laszlo

2015-07-01

Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.

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.

Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

PubMed

Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

2009-01-01

Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

13-fold resolution gain through turbid layer via translated unknown speckle illumination

PubMed Central

Guo, Kaikai; Zhang, Zibang; Jiang, Shaowei; Liao, Jun; Zhong, Jingang; Eldar, Yonina C.; Zheng, Guoan

2017-01-01

Fluorescence imaging through a turbid layer holds great promise for various biophotonics applications. Conventional wavefront shaping techniques aim to create and scan a focus spot through the turbid layer. Finding the correct input wavefront without direct access to the target plane remains a critical challenge. In this paper, we explore a new strategy for imaging through turbid layer with a large field of view. In our setup, a fluorescence sample is sandwiched between two turbid layers. Instead of generating one focus spot via wavefront shaping, we use an unshaped beam to illuminate the turbid layer and generate an unknown speckle pattern at the target plane over a wide field of view. By tilting the input wavefront, we raster scan the unknown speckle pattern via the memory effect and capture the corresponding low-resolution fluorescence images through the turbid layer. Different from the wavefront-shaping-based single-spot scanning, the proposed approach employs many spots (i.e., speckles) in parallel for extending the field of view. Based on all captured images, we jointly recover the fluorescence object, the unknown optical transfer function of the turbid layer, the translated step size, and the unknown speckle pattern. Without direct access to the object plane or knowledge of the turbid layer, we demonstrate a 13-fold resolution gain through the turbid layer using the reported strategy. We also demonstrate the use of this technique to improve the resolution of a low numerical aperture objective lens allowing to obtain both large field of view and high resolution at the same time. The reported method provides insight for developing new fluorescence imaging platforms and may find applications in deep-tissue imaging. PMID:29359102

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.

Improved axial point spread function in a two-frequency laser scanning confocal fluorescence microscope

NASA Astrophysics Data System (ADS)

Wu, Jheng-Syong; Chung, Yung-Chin; Chien, Jun-Jei; Chou, Chien

2018-01-01

A two-frequency laser scanning confocal fluorescence microscope (TF-LSCFM) based on intensity modulated fluorescence signal detection was proposed. The specimen-induced spherical aberration and scattering effect were suppressed intrinsically, and high image contrast was presented due to heterodyne interference. An improved axial point spread function in a TF-LSCFM compared with a conventional laser scanning confocal fluorescence microscope was demonstrated and discussed.

[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.

Red fluorescent zinc oxide nanoparticle: A novel platform for cancer targeting

DOE PAGES

Hong, Hao; Wang, Fei; Zhang, Yin; ...

2015-01-21

Multifunctional zinc oxide (ZnO) nanoparticles (NPs) with well-integrated multimodality imaging capacities have generated increasing research interest in the past decade. However, limited progress has been made in developing ZnO NP-based multimodality tumor-imaging agents. In this paper, we developed novel red fluorescent ZnO NPs and described the successful conjugation of 64Cu ( t 1/2 = 12.7 h) and TRC105, a chimeric monoclonal antibody against CD105, to these ZnO NPs via well-developed surface engineering procedures. The produced dual-modality ZnO NPs were readily applicable for positron emission tomography (PET) imaging and fluorescence imaging of the tumor vasculature. Their pharmacokinetics and tumor-targeting efficacy/specificity inmore » mice bearing murine breast 4T1 tumor were thoroughly investigated. In conclusion, ZnO NPs with dual-modality imaging properties can serve as an attractive candidate for future cancer theranostics.« less

Fast optically sectioned fluorescence HiLo endomicroscopy.

PubMed

Ford, Tim N; Lim, Daryl; Mertz, Jerome

2012-02-01

We describe a nonscanning, fiber bundle endomicroscope that performs optically sectioned fluorescence imaging with fast frame rates and real-time processing. Our sectioning technique is based on HiLo imaging, wherein two widefield images are acquired under uniform and structured illumination and numerically processed to reject out-of-focus background. This work is an improvement upon an earlier demonstration of widefield optical sectioning through a flexible fiber bundle. The improved device features lateral and axial resolutions of 2.6 and 17 μm, respectively, a net frame rate of 9.5 Hz obtained by real-time image processing with a graphics processing unit (GPU) and significantly reduced motion artifacts obtained by the use of a double-shutter camera. We demonstrate the performance of our system with optically sectioned images and videos of a fluorescently labeled chorioallantoic membrane (CAM) in the developing G. gallus embryo. HiLo endomicroscopy is a candidate technique for low-cost, high-speed clinical optical biopsies.

Fast optically sectioned fluorescence HiLo endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.; Lim, Daryl; Mertz, Jerome

2012-02-01

We describe a nonscanning, fiber bundle endomicroscope that performs optically sectioned fluorescence imaging with fast frame rates and real-time processing. Our sectioning technique is based on HiLo imaging, wherein two widefield images are acquired under uniform and structured illumination and numerically processed to reject out-of-focus background. This work is an improvement upon an earlier demonstration of widefield optical sectioning through a flexible fiber bundle. The improved device features lateral and axial resolutions of 2.6 and 17 μm, respectively, a net frame rate of 9.5 Hz obtained by real-time image processing with a graphics processing unit (GPU) and significantly reduced motion artifacts obtained by the use of a double-shutter camera. We demonstrate the performance of our system with optically sectioned images and videos of a fluorescently labeled chorioallantoic membrane (CAM) in the developing G. gallus embryo. HiLo endomicroscopy is a candidate technique for low-cost, high-speed clinical optical biopsies.

A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Fassel, T. A.; Schultz, E.; Greaser, M. L.; Cassens, R. G.

1996-01-01

A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

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

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.

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.

Three-photon tissue imaging using moxifloxacin.

PubMed

Lee, Seunghun; Lee, Jun Ho; Wang, Taejun; Jang, Won Hyuk; Yoon, Yeoreum; Kim, Bumju; Jun, Yong Woong; Kim, Myoung Joon; Kim, Ki Hean

2018-06-20

Moxifloxacin is an antibiotic used in clinics and has recently been used as a clinically compatible cell-labeling agent for two-photon (2P) imaging. Although 2P imaging with moxifloxacin labeling visualized cells inside tissues using enhanced fluorescence, the imaging depth was quite limited because of the relatively short excitation wavelength (<800 nm) used. In this study, the feasibility of three-photon (3P) excitation of moxifloxacin using a longer excitation wavelength and moxifloxacin-based 3P imaging were tested to increase the imaging depth. Moxifloxacin fluorescence via 3P excitation was detected at a >1000 nm excitation wavelength. After obtaining the excitation and emission spectra of moxifloxacin, moxifloxacin-based 3P imaging was applied to ex vivo mouse bladder and ex vivo mouse small intestine tissues and compared with moxifloxacin-based 2P imaging by switching the excitation wavelength of a Ti:sapphire oscillator between near 1030 and 780 nm. Both moxifloxacin-based 2P and 3P imaging visualized cellular structures in the tissues via moxifloxacin labeling, but the image contrast was better with 3P imaging than with 2P imaging at the same imaging depths. The imaging speed and imaging depth of moxifloxacin-based 3P imaging using a Ti:sapphire oscillator were limited by insufficient excitation power. Therefore, we constructed a new system for moxifloxacin-based 3P imaging using a high-energy Yb fiber laser at 1030 nm and used it for in vivo deep tissue imaging of a mouse small intestine. Moxifloxacin-based 3P imaging could be useful for clinical applications with enhanced imaging depth.

Fluorescence detection of oral squamous cell carcinoma using Hyperflav

NASA Astrophysics Data System (ADS)

Melnik, Ivan S.; Dets, Sergiy M.; Rawicz, Andrew H.; Zhang, Lewei

2000-05-01

A novel hypericin-based drug HyperflavTM has been evaluated for light-induced fluorescence detection of oral cancer. Squamous cell carcinoma was induced with carcinogenic agent in right pouches of forty hamsters (20/20 males/females). Solution of HyperflavTM was sprinkled into stomach with a single dose 0.2 - 4 mg of pure hypericin per kg b.w. and 4 - 8 hours before fluorescence analysis. In two animal groups with cancer symptoms the autofluorescence and hypericin-induced fluorescence were taken under 442 nm excitation. The buccal mucosa and adjacent areas were measured fiberoptically in-vivo and in-vitro using orange/green ratio (610/540). The in-vivo fluorescence imaging of malignant areas was conducted to assist the biopsy guidance and to compare with white-light images. Histological and morphological analyses were performed from biopsies. Oral squamous cell carcinoma in its early stage demonstrated specific higher 610/540 ratio for 37 tested hamsters. Advanced state involved another higher fluorescence maximum around 640 nm that in our opinion caused by strong porphyrin-induced native fluorescence. Such deformation of fluorescence spectra may lead to inadequate perception of diseased tissue area. To avoid this problem the autofluorescence spectra & images were added. HyperflavTM application is promising for demarcation of early oral cancer when combined with autofluorescence measurements.

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins[S

PubMed Central

Gaebler, Anne; Penno, Anke; Kuerschner, Lars; Thiele, Christoph

2016-01-01

The demand to study the cellular localization of specific lipids has led to recent advances in lipid probes and microscopy. Alkyne lipids bear a small, noninterfering tag and can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne lipid imaging crucially depends on appropriate azide reporters and labeling protocols that allow for an efficient click reaction and therefore a sensitive detection. We synthesized several azide reporters with different spacer components and tested their suitability for alkyne lipid imaging in fixed cells. The implementation of a copper-chelating picolyl moiety into fluorescent or biotin-based azide reagents strongly increased the sensitivity of the imaging routine. We demonstrate the applicability and evaluate the performance of this approach using different lipid classes and experimental setups. As azide picolyl reporters allow for reduced copper catalyst concentrations, they also enable coimaging of alkyne lipids with multiple fluorescent proteins including enhanced green fluorescent protein. Alternatively, and as we also show, microscopy of alkyne lipids can be combined with protein detection by immunocytochemistry. In summary, we present a robust, sensitive, and highly versatile protocol for the labeling of alkyne lipids with azide-coupled reporters for fluorescence microscopy that can be combined with different protein detection and imaging techniques. PMID:27565170

Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

PubMed Central

Ai, Hui-wang; Olenych, Scott G; Wong, Peter; Davidson, Michael W; Campbell, Robert E

2008-01-01

Background In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. Results Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His). Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP). To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. Conclusion Based on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mTFP1. With respect to live cell imaging applications, the mTFP1-derived mWasabi should be particularly useful in two-color imaging in conjunction with a Sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue FP donor. In all fusions attempted, both mTFP1 and mWasabi give patterns of fluorescent localization indistinguishable from that of well-established avGFP variants. PMID:18325109

Latest results of 5-ALA-based fluorescence diagnosis and other medical disciplines

NASA Astrophysics Data System (ADS)

Baumgartner, Reinhold

1999-02-01

Preclinical and clinical studies on 5-aminolevulinic acid (5- ALA) induced Protoporphyrin IX (PPIX) are performed in various departments now following promising clinical results for the detection of bladder cancer in urology. This paper provides an overview on the progress of 5-ALA assisted fluorescence diagnosis in urology, pulmonology, neurosurgery, gynecology and ENT coordinated by the Laser Research Laboratory of the Ludwig-Maximilians-University in Munich. 5-ALA can be applied either topically or systematically to induce an intracellular accumulation of fluorescing PPIX. With appropriate dosage of 5-ALA, malignant tissue can be stained selectively, and irradiation with violet light excites a bright red fluorescence of the tumor visible with naked eyes. Optical properties of the tissue tend to hamper the precise identification and demarcation of suspect areas in fluorescence images. Multicolor remission and fluorescence imaging, therefore, should improve tumor localization in future.

Hyperspectral small animal fluorescence imaging: spectral selection imaging

NASA Astrophysics Data System (ADS)

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

2008-02-01

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

Fluorescence enhancement of photoswitchable metal ion sensors

NASA Astrophysics Data System (ADS)

Sylvia, Georgina; Heng, Sabrina; Abell, Andrew D.

2016-12-01

Spiropyran-based fluorescence sensors are an ideal target for intracellular metal ion sensing, due to their biocompatibility, red emission frequency and photo-controlled reversible analyte binding for continuous signal monitoring. However, increasing the brightness of spiropyran-based sensors would extend their sensing capability for live-cell imaging. In this work we look to enhance the fluorescence of spiropyran-based sensors, by incorporating an additional fluorophore into the sensor design. We report a 5-membered monoazacrown bearing spiropyran with metal ion specificity, modified to incorporate the pyrene fluorophore. The effect of N-indole pyrene modification on the behavior of the spiropyran molecule is explored, with absorbance and fluorescence emission characterization. This first generation sensor provides an insight into fluorescence-enhancement of spiropyran molecules.

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

1,3-Bis(2-chloroethyl)-1-nitrosourea-loaded bovine serum albumin nanoparticles with dual magnetic resonance-fluorescence imaging for tracking of chemotherapeutic agents.

PubMed

Wei, Kuo-Chen; Lin, Feng-Wei; Huang, Chiung-Yin; Ma, Chen-Chi M; Chen, Ju-Yu; Feng, Li-Ying; Yang, Hung-Wei

To date, knowing how to identify the location of chemotherapeutic agents in the human body after injection is still a challenge. Therefore, it is urgent to develop a drug delivery system with molecular imaging tracking ability to accurately understand the distribution, location, and concentration of a drug in living organisms. In this study, we developed bovine serum albumin (BSA)-based nanoparticles (NPs) with dual magnetic resonance (MR) and fluorescence imaging modalities (fluorescein isothiocyanate [FITC]-BSA-Gd/1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU] NPs) to deliver BCNU for inhibition of brain tumor cells (MBR 261-2). These BSA-based NPs are water dispersible, stable, and biocompatible as confirmed by XTT cell viability assay. In vitro phantoms and in vivo MR and fluorescence imaging experiments show that the developed FITC-BSA-Gd/BCNU NPs enable dual MR and fluorescence imaging for monitoring cellular uptake and distribution in tumors. The T1 relaxivity (R1) of FITC-BSA-Gd/BCNU NPs was 3.25 mM(-1) s(-1), which was similar to that of the commercial T1 contrast agent (R1 =3.36 mM(-1) s(-1)). The results indicate that this multifunctional drug delivery system has potential bioimaging tracking of chemotherapeutic agents ability in vitro and in vivo for cancer therapy.

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

Electrosprayed synthesis of red-blood-cell-like particles with dual modality for magnetic resonance and fluorescence imaging.

PubMed

Hayashi, Koichiro; Ono, Kenji; Suzuki, Hiromi; Sawada, Makoto; Moriya, Makoto; Sakamoto, Wataru; Yogo, Toshinobu

2010-11-05

Red blood cells (RBCs) are able to avoid filtration in the spleen to prolong their half-time in the body because of their flexibility and unique shape, or a concave disk with diameter of some 10 μm. In addition, they can flow through capillary blood vessels, which are smaller than the diameter of RBCs, by morphing into a parachute-like shape. In this study, flexible RBC-like polymer particles are synthesized by electrospraying based on electrospinning. Furthermore, magnetite nanoparticles and fluorescent dye are encapsulated in the particles via in situ hydrolysis of an iron-organic compound in the presence of celluloses. The superparamagnetic behavior of the particles is confirmed by low-temperature magnetic measurements. The particles exhibited not only a dark contrast in magnetic resonance imaging (MRI), but also effective fluorescence. The RBC-like particles with flexibility are demonstrated to have a dual-modality for MRI and fluorescence imaging.

Software-based measurement of thin filament lengths: an open-source GUI for Distributed Deconvolution analysis of fluorescence images

PubMed Central

Gokhin, David S.; Fowler, Velia M.

2016-01-01

The periodically arranged thin filaments within the striated myofibrils of skeletal and cardiac muscle have precisely regulated lengths, which can change in response to developmental adaptations, pathophysiological states, and genetic perturbations. We have developed a user-friendly, open-source ImageJ plugin that provides a graphical user interface (GUI) for super-resolution measurement of thin filament lengths by applying Distributed Deconvolution (DDecon) analysis to periodic line scans collected from fluorescence images. In the workflow presented here, we demonstrate thin filament length measurement using a phalloidin-stained cryosection of mouse skeletal muscle. The DDecon plugin is also capable of measuring distances of any periodically localized fluorescent signal from the Z- or M-line, as well as distances between successive Z- or M-lines, providing a broadly applicable tool for quantitative analysis of muscle cytoarchitecture. These functionalities can also be used to analyze periodic fluorescence signals in nonmuscle cells. PMID:27644080

Strategies to overcome photobleaching in algorithm-based adaptive optics for nonlinear in-vivo imaging.

PubMed

Caroline Müllenbroich, M; McGhee, Ewan J; Wright, Amanda J; Anderson, Kurt I; Mathieson, Keith

2014-01-01

We have developed a nonlinear adaptive optics microscope utilizing a deformable membrane mirror (DMM) and demonstrated its use in compensating for system- and sample-induced aberrations. The optimum shape of the DMM was determined with a random search algorithm optimizing on either two photon fluorescence or second harmonic signals as merit factors. We present here several strategies to overcome photobleaching issues associated with lengthy optimization routines by adapting the search algorithm and the experimental methodology. Optimizations were performed on extrinsic fluorescent dyes, fluorescent beads loaded into organotypic tissue cultures and the intrinsic second harmonic signal of these cultures. We validate the approach of using these preoptimized mirror shapes to compile a robust look-up table that can be applied for imaging over several days and through a variety of tissues. In this way, the photon exposure to the fluorescent cells under investigation is limited to imaging. Using our look-up table approach, we show signal intensity improvement factors ranging from 1.7 to 4.1 in organotypic tissue cultures and freshly excised mouse tissue. Imaging zebrafish in vivo, we demonstrate signal improvement by a factor of 2. This methodology is easily reproducible and could be applied to many photon starved experiments, for example fluorescent life time imaging, or when photobleaching is a concern.

Rapid and sensitive detection of early esophageal squamous cell carcinoma with fluorescence probe targeting dipeptidylpeptidase IV

PubMed Central

Onoyama, Haruna; Kamiya, Mako; Kuriki, Yugo; Komatsu, Toru; Abe, Hiroyuki; Tsuji, Yosuke; Yagi, Koichi; Yamagata, Yukinori; Aikou, Susumu; Nishida, Masato; Mori, Kazuhiko; Yamashita, Hiroharu; Fujishiro, Mitsuhiro; Nomura, Sachiyo; Shimizu, Nobuyuki; Fukayama, Masashi; Koike, Kazuhiko; Urano, Yasuteru; Seto, Yasuyuki

2016-01-01

Early detection of esophageal squamous cell carcinoma (ESCC) is an important prognosticator, but is difficult to achieve by conventional endoscopy. Conventional lugol chromoendoscopy and equipment-based image-enhanced endoscopy, such as narrow-band imaging (NBI), have various practical limitations. Since fluorescence-based visualization is considered a promising approach, we aimed to develop an activatable fluorescence probe to visualize ESCCs. First, based on the fact that various aminopeptidase activities are elevated in cancer, we screened freshly resected specimens from patients with a series of aminopeptidase-activatable fluorescence probes. The results indicated that dipeptidylpeptidase IV (DPP-IV) is specifically activated in ESCCs, and would be a suitable molecular target for detection of esophageal cancer. Therefore, we designed, synthesized and characterized a series of DPP-IV-activatable fluorescence probes. When the selected probe was topically sprayed onto endoscopic submucosal dissection (ESD) or surgical specimens, tumors were visualized within 5 min, and when the probe was sprayed on biopsy samples, the sensitivity, specificity and accuracy reached 96.9%, 85.7% and 90.5%. We believe that DPP-IV-targeted activatable fluorescence probes are practically translatable as convenient tools for clinical application to enable rapid and accurate diagnosis of early esophageal cancer during endoscopic or surgical procedures. PMID:27245876

Utilizing fluorescence hyperspectral imaging to differentiate corn inoculated with toxigenic and atoxigenic fungal strains

NASA Astrophysics Data System (ADS)

Yao, Haibo; Hruska, Zuzana; Kincaid, Russell; Brown, Robert L.; Bhatnagar, Deepak; Cleveland, Thomas E.

2012-05-01

Naturally occurring Aspergillus flavus strains can be either toxigenic or atoxigenic, indicating their ability to produce aflatoxin or not, under specific conditions. Corn contaminated with toxigenic strains of A. flavus can result in great losses to the agricultural industry and pose threats to public health. Past research showed that fluorescence hyperspectral imaging could be a potential tool for rapid and non-invasive detection of aflatoxin contaminated corn. The objective of the current study was to assess, with the use of a hyperspectral sensor, the difference in fluorescence emission between corn kernels inoculated with toxigenic and atoxigenic inoculums of A. flavus. Corn ears were inoculated with AF13, a toxigenic strain of A. flavus, and AF38, an atoxigenic strain of A. flavus, at dough stage of development and harvested 8 weeks after inoculation. After harvest, single corn kernels were divided into three groups prior to imaging: control, adjacent, and glowing. Both sides of the kernel, germplasm and endosperm, were imaged separately using a fluorescence hyperspectral imaging system. It was found that the classification accuracies of the three manually designated groups were not promising. However, the separation of corn kernels based on different fungal inoculums yielded better results. The best result was achieved with the germplasm side of the corn kernels. Results are expected to enhance the potential of fluorescence hyperspectral imaging for the detection of aflatoxin contaminated corn.

Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo

NASA Astrophysics Data System (ADS)

Rudkouskaya, Alena; Sinsuebphon, Nattawut; Intes, Xavier; Mazurkiewicz, Joseph E.; Barroso, Margarida

2017-02-01

To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

Monte Carlo Simulation for Polychromatic X-Ray Fluorescence Computed Tomography with Sheet-Beam Geometry

PubMed Central

Jiang, Shanghai

2017-01-01

X-ray fluorescence computed tomography (XFCT) based on sheet beam can save a huge amount of time to obtain a whole set of projections using synchrotron. However, it is clearly unpractical for most biomedical research laboratories. In this paper, polychromatic X-ray fluorescence computed tomography with sheet-beam geometry is tested by Monte Carlo simulation. First, two phantoms (A and B) filled with PMMA are used to simulate imaging process through GEANT 4. Phantom A contains several GNP-loaded regions with the same size (10 mm) in height and diameter but different Au weight concentration ranging from 0.3% to 1.8%. Phantom B contains twelve GNP-loaded regions with the same Au weight concentration (1.6%) but different diameter ranging from 1 mm to 9 mm. Second, discretized presentation of imaging model is established to reconstruct more accurate XFCT images. Third, XFCT images of phantoms A and B are reconstructed by filter back-projection (FBP) and maximum likelihood expectation maximization (MLEM) with and without correction, respectively. Contrast-to-noise ratio (CNR) is calculated to evaluate all the reconstructed images. Our results show that it is feasible for sheet-beam XFCT system based on polychromatic X-ray source and the discretized imaging model can be used to reconstruct more accurate images. PMID:28567054

A new probe using hybrid virus-dye nanoparticles for near-infrared fluorescence tomography

NASA Astrophysics Data System (ADS)

Wu, Changfeng; Barnhill, Hannah; Liang, Xiaoping; Wang, Qian; Jiang, Huabei

2005-11-01

A fluorescent probe based on bionanoparticle cowpea mosaic virus has been developed for near-infrared fluorescence tomography. A unique advantage of this probe is that over 30 dye molecules can be loaded onto each viral nanoparticle with an average diameter of 30 nm, making high local dye concentration (∼1.8 mM) possible without significant fluorescence quenching. This ability of high loading of local dye concentration would increase the signal-to-noise ratio considerably, thus sensitivity for detection. We demonstrate successful tomographic fluorescence imaging of a target containing the virus-dye nanoparticles embedded in a tissue-like phantom. Tomographic fluorescence data were obtained through a multi-channel frequency-domain system and the spatial maps of fluorescence quantum yield were recovered with a finite-element-based reconstruction algorithm.

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.

Bacterial cell identification in differential interference contrast microscopy images.

PubMed

Obara, Boguslaw; Roberts, Mark A J; Armitage, Judith P; Grau, Vicente

2013-04-23

Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

DOE PAGES

Venkatraman, S.; Doktycz, M. J.; Qi, H.; ...

2006-01-01

The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

Multimodality Imaging of Gene Transfer with a Receptor-Based Reporter Gene

PubMed Central

Chen, Ron; Parry, Jesse J.; Akers, Walter J.; Berezin, Mikhail Y.; El Naqa, Issam M.; Achilefu, Samuel; Edwards, W. Barry; Rogers, Buck E.

2010-01-01

Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Non-invasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor–enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging. Methods An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of 111In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice. Results The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of 111In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2. Although fluorescence was observed, there were discrepancies between in vivo imaging and ex vivo imaging as well as between nuclear imaging and fluorescent imaging. Conclusion These studies showed that the SSTR2-EGFP fusion construct can be used for in vivo nuclear and optical imaging of gene transfer. PMID:20720053

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.

Generation of HIV-1 based bi-cistronic lentiviral vectors for stable gene expression and live cell imaging.

PubMed

Sehgal, Lalit; Budnar, Srikanth; Bhatt, Khyati; Sansare, Sneha; Mukhopadhaya, Amitabha; Kalraiya, Rajiv D; Dalal, Sorab N

2012-10-01

The study of protein-protein interactions, protein localization, protein organization into higher order structures and organelle dynamics in live cells, has greatly enhanced the understanding of various cellular processes. Live cell imaging experiments employ plasmid or viral vectors to express the protein/proteins of interest fused to a fluorescent protein. Unlike plasmid vectors, lentiviral vectors can be introduced into both dividing and non dividing cells, can be pseudotyped to infect a broad or narrow range of cells, and can be used to generate transgenic animals. However, the currently available lentiviral vectors are limited by the choice of fluorescent protein tag, choice of restriction enzyme sites in the Multiple Cloning Sites (MCS) and promoter choice for gene expression. In this report, HIV-1 based bi-cistronic lentiviral vectors have been generated that drive the expression of multiple fluorescent tags (EGFP, mCherry, ECFP, EYFP and dsRed), using two different promoters. The presence of a unique MCS with multiple restriction sites allows the generation of fusion proteins with the fluorescent tag of choice, allowing analysis of multiple fusion proteins in live cell imaging experiments. These novel lentiviral vectors are improved delivery vehicles for gene transfer applications and are important tools for live cell imaging in vivo.

Fluorescence-based microendoscopes for breast cancer ductoscopy

NASA Astrophysics Data System (ADS)

Zeylikovich, Iosif; Tang, Guichen C.; Katz, A.; Budansky, Yury; Alfano, R. R.

2006-02-01

Recently microendoscopes are being developed as a tool to detection cancer or pre-cancerous lesions in the milk ducts of the human breast. The microendoscope can be inserted into the duct through the nipple. Integration of fluorescence spectroscopy into microendoscopy can provide an improved platform for real-time cancer detection followed by immediate intervention. Typically, the optical fibers employed by existing microendoscope systems transmit in the 450 to 900 nm range. A prototype system combining fluorescence spectroscopy with visible imaging by microendoscopy is described and preliminary measurements on ex vivo human breast tissues are presented. Image resolution and distortion are discussed.

Mobile Phone Ratiometric Imaging Enables Highly Sensitive Fluorescence Lateral Flow Immunoassays without External Optical Filters.

PubMed

Shah, Kamal G; Singh, Vidhi; Kauffman, Peter C; Abe, Koji; Yager, Paul

2018-05-14

Paper-based diagnostic tests based on the lateral flow immunoassay concept promise low-cost, point-of-care detection of infectious diseases, but such assays suffer from poor limits of detection. One factor that contributes to poor analytical performance is a reliance on low-contrast chromophoric optical labels such as gold nanoparticles. Previous attempts to improve the sensitivity of paper-based diagnostics include replacing chromophoric labels with enzymes, fluorophores, or phosphors at the expense of increased fluidic complexity or the need for device readers with costly optoelectronics. Several groups, including our own, have proposed mobile phones as suitable point-of-care readers due to their low cost, ease of use, and ubiquity. However, extant mobile phone fluorescence readers require costly optical filters and were typically validated with only one camera sensor module, which is inappropriate for potential point-of-care use. In response, we propose to couple low-cost ultraviolet light-emitting diodes with long Stokes-shift quantum dots to enable ratiometric mobile phone fluorescence measurements without optical filters. Ratiometric imaging with unmodified smartphone cameras improves the contrast and attenuates the impact of excitation intensity variability by 15×. Practical application was shown with a lateral flow immunoassay for influenza A with nucleoproteins spiked into simulated nasal matrix. Limits of detection of 1.5 and 2.6 fmol were attained on two mobile phones, which are comparable to a gel imager (1.9 fmol), 10× better than imaging gold nanoparticles on a scanner (18 fmol), and >2 orders of magnitude better than gold nanoparticle-labeled assays imaged with mobile phones. Use of the proposed filter-free mobile phone imaging scheme is a first step toward enabling a new generation of highly sensitive, point-of-care fluorescence assays.

Fully integrated optical coherence tomography, ultrasound, and indocyanine green based fluorescence tri-modality system for intravascular imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Li, Yan; Jing, Joseph C.; Qu, Yueqiao; Miao, Yusi; Ma, Teng; Yu, Mingyue; Zhou, Qifa; Chen, Zhongping

2017-02-01

The rupture of atherosclerotic plaques is the leading cause of acute coronary events, so accurate assessment of plaque is critical. A large lipid pool, thin fibrous cap, and inflammatory reaction are the crucial characteristics for identifying vulnerable plaques. In our study, a tri-modality imaging system for intravascular imaging was designed and implemented. The tri-modality imaging system with a 1-mm probe diameter is able to simultaneously acquire optical coherence tomography (OCT), intravascular ultrasound (IVUS), and fluorescence imaging. Moreover, for fluorescence imaging, we used the FDA-approved indocyanine green (ICG) dye as the contrast agent to target lipid-loaded macrophages. Firstly, IVUS is used as the first step for identifying plaque since IVUS enables the visualization of the layered structures of the artery wall. Due to low soft-tissue contrast, IVUS only provides initial identification of the lipid plaque. Then OCT is used for differentiating fibrosis and lipid pool based on its relatively higher soft tissue contrast and high sensitivity/specificity. Last, fluorescence imaging is used for identifying inflammatory reaction to further confirm whether the plaque is vulnerable or not. Ex vivo experiment of a male New Zealand white rabbit aorta was performed to validate the performance of our tri-modality system. H and E histology results of the rabbit aorta were also presented to check assessment accuracy. The miniature tri-modality probe, together with the use of ICG dye suggest that the system is of great potential for providing a more accurate assessment of vulnerable plaques in clinical applications.

Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos.

PubMed

Strobl, Frederic; Stelzer, Ernst H K

2014-06-01

Insect development has contributed significantly to our understanding of metazoan development. However, most information has been obtained by analyzing a single species, the fruit fly Drosophila melanogaster. Embryonic development of the red flour beetle Tribolium castaneum differs fundamentally from that of Drosophila in aspects such as short-germ development, embryonic leg development, extensive extra-embryonic membrane formation and non-involuted head development. Although Tribolium has become the second most important insect model organism, previous live imaging attempts have addressed only specific questions and no long-term live imaging data of Tribolium embryogenesis have been available. By combining light sheet-based fluorescence microscopy with a novel mounting method, we achieved complete, continuous and non-invasive fluorescence live imaging of Tribolium embryogenesis at high spatiotemporal resolution. The embryos survived the 2-day or longer imaging process, developed into adults and produced fertile progeny. Our data document all morphogenetic processes from the rearrangement of the uniform blastoderm to the onset of regular muscular movement in the same embryo and in four orientations, contributing significantly to the understanding of Tribolium development. Furthermore, we created a comprehensive chronological table of Tribolium embryogenesis, integrating most previous work and providing a reference for future studies. Based on our observations, we provide evidence that serosa window closure and serosa opening, although deferred by more than 1 day, are linked. All our long-term imaging datasets are available as a resource for the community. Tribolium is only the second insect species, after Drosophila, for which non-invasive long-term fluorescence live imaging has been achieved. © 2014. Published by The Company of Biologists Ltd.

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

NASA Astrophysics Data System (ADS)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

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.

Remote imaging laser-induced breakdown spectroscopy and laser-induced fluorescence spectroscopy using nanosecond pulses from a mobile lidar system.

PubMed

Grönlund, Rasmus; Lundqvist, Mats; Svanberg, Sune

2006-08-01

A mobile lidar system was used in remote imaging laser-induced breakdown spectroscopy (LIBS) and laser-induced fluorescence (LIF) experiments. Also, computer-controlled remote ablation of a chosen area was demonstrated, relevant to cleaning of cultural heritage items. Nanosecond frequency-tripled Nd:YAG laser pulses at 355 nm were employed in experiments with a stand-off distance of 60 meters using pulse energies of up to 170 mJ. By coaxial transmission and common folding of the transmission and reception optical paths using a large computer-controlled mirror, full elemental imaging capability was achieved on composite targets. Different spectral identification algorithms were compared in producing thematic data based on plasma or fluorescence light.

A Dual Reporter Iodinated Labeling Reagent for Cancer Positron Emission Tomography Imaging and Fluorescence-Guided Surgery

PubMed Central

2018-01-01

The combination of early diagnosis and complete surgical resection offers the greatest prospect of curative cancer treatment. An iodine-124/fluorescein-based dual-modality labeling reagent, 124I-Green, constitutes a generic tool for one-step installation of a positron emission tomography (PET) and a fluorescent reporter to any cancer-specific antibody. The resulting antibody conjugate would allow both cancer PET imaging and intraoperative fluorescence-guided surgery. 124I-Green was synthesized in excellent radiochemical yields of 92 ± 5% (n = 4) determined by HPLC with an improved one-pot three-component radioiodination reaction. The A5B7 carcinoembryonic antigen (CEA)-specific antibody was conjugated to 124I-Green. High tumor uptake of the dual-labeled A5B7 of 20.21 ± 2.70, 13.31 ± 0.73, and 10.64 ± 1.86%ID/g was observed in CEA-expressing SW1222 xenograft mouse model (n = 3) at 24, 48, and 72 h post intravenous injection, respectively. The xenografts were clearly visualized by both PET/CT and ex vivo fluorescence imaging. These encouraging results warrant the further translational development of 124I-Green for cancer PET imaging and fluorescence-guided surgery. PMID:29388770

Non-invasive thermal IR detection of breast tumor development in vivo

NASA Astrophysics Data System (ADS)

Case, Jason R.; Young, Madison A.; Dréau, D.; Trammell, Susan R.

2015-03-01

Lumpectomy coupled with radiation therapy and/or chemotherapy comprises the treatment of breast cancer for many patients. We are developing an enhanced thermal IR imaging technique that can be used in real-time to guide tissue excision during a lumpectomy. This novel enhanced thermal imaging method is a combination of IR imaging (8- 10 μm) and selective heating of blood (~0.5 °C) relative to surrounding water-rich tissue using LED sources at low powers. Post-acquisition processing of these images highlights temporal changes in temperature and is sensitive to the presence of vascular structures. In this study, fluorescent and enhanced thermal imaging modalities were used to estimate breast cancer tumor volumes as a function of time in 19 murine subjects over a 30-day study period. Tumor volumes calculated from fluorescent imaging follow an exponential growth curve for the first 22 days of the study. Cell necrosis affected the tumor volume estimates based on the fluorescent images after Day 22. The tumor volumes estimated from enhanced thermal imaging show exponential growth over the entire study period. A strong correlation was found between tumor volumes estimated using fluorescent imaging and the enhanced IR images, indicating that enhanced thermal imaging is capable monitoring tumor growth. Further, the enhanced IR images reveal a corona of bright emission along the edges of the tumor masses. This novel IR technique could be used to estimate tumor margins in real-time during surgical procedures.

Enhanced fluorescence of tetrasulfonated zinc phthalocyanine by graphene quantum dots and its application in molecular sensing/imaging.

PubMed

Wang, Jian; Zhang, Yanjun; Ye, Jiqing; Jiang, Zhou

2017-06-01

When excited at 435 nm, tetra-sulfonate zinc phthalocyanine (ZnPcS 4 ) emitted dual fluorescence at 495 and 702 nm. The abnormal fluorescence at 495 nm was experimentally studied and analyzed in detail for the first time. The abnormal fluorescence at 495 nm was deduced to originate from triplet-triplet (T-T) energy transfer of excited phthalocyanine ( 3 *ZnPcS 4 ). Furthermore, graphene quantum dots (GQDs) enhanced the 495 nm fluorescence quantum yield (Q) of ZnPcS 4 . The fluorescence properties of ZnPcS 4 -GQDs conjugate were retained in a cellular environment. Based on the fluorescence of ZnPcS 4 -GQDs conjugate, we designed and prepared an Apt29/thrombin/Apt15 sandwich thrombin sensor with high specificity and affinity. This cost-saving, simple operational sensing strategy can be extended to use in sensing/imaging of other biomolecules. Copyright © 2016 John Wiley & Sons, Ltd.

Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome.

PubMed

Piatkevich, Kiryl D; Subach, Fedor V; Verkhusha, Vladislav V

2013-01-01

The ability to modulate the fluorescence of optical probes can be used to enhance signal-to-noise ratios for imaging within highly autofluorescent environments, such as intact tissues and living organisms. Here, we report two bacteriophytochrome-based photoactivatable near-infrared fluorescent proteins, named PAiRFP1 and PAiRFP2. PAiRFPs utilize haem-derived biliverdin, ubiquitous in mammalian tissues, as the chromophore. Initially weakly fluorescent PAiRFPs undergo photoconversion into a highly fluorescent state with excitation/emission at 690/717 nm following a brief irradiation with far-red light. After photoactivation, PAiRFPs slowly revert back to initial state, enabling multiple photoactivation-relaxation cycles. Low-temperature optical spectroscopy reveals several intermediates involved in PAiRFP photocycles, which all differ from that of the bacteriophytochrome precursor. PAiRFPs can be photoactivated in a spatially selective manner in mouse tissues, and optical modulation of their fluorescence allows for substantial contrast enhancement, making PAiRFPs advantageous over permanently fluorescent probes for in vivo imaging conditions of high autofluorescence and low signal levels.

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.

Visual Analysis for Detection and Quantification of Pseudomonas cichorii Disease Severity in Tomato Plants.

PubMed

Rajendran, Dhinesh Kumar; Park, Eunsoo; Nagendran, Rajalingam; Hung, Nguyen Bao; Cho, Byoung-Kwan; Kim, Kyung-Hwan; Lee, Yong Hoon

2016-08-01

Pathogen infection in plants induces complex responses ranging from gene expression to metabolic processes in infected plants. In spite of many studies on biotic stress-related changes in host plants, little is known about the metabolic and phenotypic responses of the host plants to Pseudomonas cichorii infection based on image-based analysis. To investigate alterations in tomato plants according to disease severity, we inoculated plants with different cell densities of P. cichorii using dipping and syringe infiltration methods. High-dose inocula (≥ 10(6) cfu/ml) induced evident necrotic lesions within one day that corresponded to bacterial growth in the infected tissues. Among the chlorophyll fluorescence parameters analyzed, changes in quantum yield of PSII (ΦPSII) and non-photochemical quenching (NPQ) preceded the appearance of visible symptoms, but maximum quantum efficiency of PSII (Fv/Fm) was altered well after symptom development. Visible/near infrared and chlorophyll fluorescence hyperspectral images detected changes before symptom appearance at low-density inoculation. The results of this study indicate that the P. cichorii infection severity can be detected by chlorophyll fluorescence assay and hyperspectral images prior to the onset of visible symptoms, indicating the feasibility of early detection of diseases. However, to detect disease development by hyperspectral imaging, more detailed protocols and analyses are necessary. Taken together, change in chlorophyll fluorescence is a good parameter for early detection of P. cichorii infection in tomato plants. In addition, image-based visualization of infection severity before visual damage appearance will contribute to effective management of plant diseases.

Synthesis and biological imaging of cross-linked fluorescent polymeric nanoparticles with aggregation-induced emission characteristics based on the combination of RAFT polymerization and the Biginelli reaction.

PubMed

Dong, Jiande; Liu, Meiying; Jiang, Ruming; Huang, Hongye; Wan, Qing; Wen, Yuanqing; Tian, Jianwen; Dai, Yanfeng; Zhang, Xiaoyong; Wei, Yen

2018-05-17

Fluorescent probes have long been regarded as tools for imaging living organisms with advantages such as high sensitivity, good designability and multifunctional potential. Many fluorescent probes, especially the probes based on aggregation-induced emission (AIE) dyes, have received increasing attention since the AIE phenomenon was discovered. These AIE dye-based fluorescent probes could elegantly overcome the notorious quenching effect caused by aggregation of conventional organic dyes. However, it is still difficult to directly apply these AIE-active dyes for biomedical applications owing to their hydrophobic nature. Therefore, the development of novel and facile strategies to endow them with water dispersibility is of critical importance. In this work, we exploit an efficient and simple strategy to fabricate an AIE dye-based fluorescent copolymer through the combination of reversible addition-fragmentation chain transfer and the Biginelli reaction. Moreover, the copolymer can self-assemble to fluorescent polymeric nanoparticles (FPNs) in water solution. Hydrophilic poly(PEGMA-co-AEMA) was reacted with the AIE-active dye 4',4‴-(1,2-diphenylethene-1,2-diyl)bis([1,1'-biphenyl]-4-carbaldehyde (CHO-TPE-CHO) to form amphiphilic luminescent polymers using urea as the connection bridge. The successful synthesis of the final products (poly(PEGMA-co-AEMA-TPE) FPNs) was confirmed by various instruments. Furthermore, Transmission electron microscopy (TEM) images manifest that poly(PEGMA-co-AEMA-TPE) copolymers will self-assemble into spherical nanoparticles in aqueous environments with sizes between 100 nm and 200 nm. The cell uptake and bioimaging experiment confirm that poly(PEGMA-co-AEMA-TPE) FPNs have excellent biocompatibility and emit strong green fluorescence in a cellular environment. Thus, poly(PEGMA-co-AEMA-TPE) FPNs are excellent candidates for biomedical applications. Copyright © 2018. Published by Elsevier Inc.