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Results for “Fluorescence imaging”

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PubMed2005

Spectral- and frequency-encoded fluorescence imaging.

A method for obtaining fluorescence images with a high number of resolvable points by using spectral and frequency encoding is presented. Broadband excitation light is encoded with a wavelength-dependent frequency modulation and dispersed onto the sample with a grating to simultaneously illuminate an entire image line. The Fourier transform of the frequency-encoded fluorescence emission provides one line of the image. Mechanical scanning along a direction orthogonal to the wavelength-encoded axis allows creation of the two-dimensional fluorescent image. This method is applicable for developing submillimeter diameter endoscopes. The principles of the technique are validated by imaging indocyanine green fluorescence in microfluidic channels.

Endoscopes↗

PubMed1996

Multiparameter fluorescence imaging microscopy: reagents and instruments.

Fluorescence imaging microscopes and fluorescent reagents have both evolved greatly in the past 10 years. Sensitive imaging cameras developed over the last decade allow detection of fluorescence signals from even a single fluorescent tag on a protein. At the same time, numerous antibody markers for growth control proteins and DNA probes for chromosomal alterations have been discovered and these can be labeled with the new multicolor fluorescent dyes for detection by microscopy. Image analysis software automatically localizes and quantifies of as many as 5-6 of these different color fluorescent markers in the individual cells of a preparation. The results is that powerful methods for multiparameter analysis of molecular structures and dynamic processes are now accessible to the pathologist.

DNA Probes↗

PubMed2002

Spectral imaging fluorescence microscopy.

The spectral resolution of fluorescence microscope images in living cells is achieved by using a confocal laser scanning microscope equipped with grating optics. This capability of temporal and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP).

Fluorescein-5-isothiocyanate↗

PubMed2000

A comparison of Cunyite and Fosterite NIR tunable laser tissue welding using native collagen fluorescence imaging.

OBJECTIVE: To evaluate the technique of native collagen fluorescence imaging for assessing the extent of welded areas for tissues exposed to different near-infrared (NIR) laser wavelengths. BACKGROUND: Native fluorescence imaging may be used to identify the distribution of collagen and elastin in tissues. Our past work demonstrated that different welding strengths were obtained under the same laser power conditions using different NIR wavelengths. The role of collagen in tissue welding experiments is not well understood. METHODS: Two new NIR tunable lasers were used to weld canine skin. The welded areas on the surface and in cross sections were analyzed by measuring the spatial distribution of native collagen fluorescence at 380 nm excited by 340 nm radiation. RESULTS: The results show that native collagen fluorescence imaging is a useful technique for analyzing the extent of tissue welds produced under a range of laser exposures. Fluorescence imaging reveals the depth of laser interaction with the tissue as well as evaluating collateral damage to the tissue surface. The welded volume obtained in skin using Cunyite laser exposure at 1,430 nm is deeper than that produced with Forsterite laser exposure at 1,250 nm. The post welded tensile strength for the same power density is greater for the Cunyite lasers. Ablated tissue on the surface is more prevalent with Forsterite laser welding at 1,250 nm than with Cunyite at 1,430 nm. CONCLUSION: Native collagen fluorescence can distinguish between tissue welds that have been produced by different NIR wavelengths. Tissue welding using 1,430 nm radiation is more effective than that using 1,250 nm.

Animals↗

PubMed1997

In vivo fluorescence imaging for tissue diagnostics.

Non-invasive fluorescence imaging has the potential to provide in vivo diagnostic information for many clinical specialties. Techniques have been developed over the years for simple ocular observations following UV excitation to sophisticated spectroscopic imaging using advanced equipment. Much of the impetus for research on fluorescence imaging for tissue diagnostics has come from parallel developments in photodynamic therapy of malignant lesions with fluorescent photosensitizers. However, the fluorescence of endogenous molecules (tissue autofluorescence) also plays an important role in most applications. In this paper, the possibilities of imaging tissues using fluorescence spectroscopy as a mean of tissue characterization are discussed. The various imaging techniques for extracting diagnostic information suggested in the literature are reviewed. The development of exogenous fluorophores for this purpose is also presented. Finally, the present status of clinical evaluation and future directions are discussed.

Diagnostic Imaging↗

PubMed2001

An affordable, portable fluorescence imaging device for skin lesion detection using a dual wavelength approach for image contrast enhancement and aminolaevulinic acid-induced protoporphyrin IX. Part I. Design, spectral and spatial characteristics.

Steady-state fluorescence imaging can be used in conjunction with selective exogenous or endogenous fluorescent compounds for the diagnosis of skin lesions, for example cancerous lesions. Depending on the excitation and emission properties of the fluorescent compound used, various excitation and/or emission wavelengths can be chosen in order to allow fluorescence imaging. Unwanted background signals like autofluorescence and scattering can decrease the image quality and hence the diagnosis potential of this imaging method. A method involving two excitation and/or emission wavelengths was used in order to suppress the unwanted background signal and allow contrast enhanced fluorescence imaging. A fluorescence imaging device prototype was assembled using both the two wavelength excitation method and the two wavelength emission method. Additionally, a white light source was included to allow the collection of images as seen with the naked eye. The prototype was designed to be affordable and portable and was laid out towards the diagnosis of skin lesions using aminolaevulinic acid-induced protoporphyrin IX (PpIX). This paper describes the excitation and detection characteristics of a fluorescence imaging device prototype. This includes spectral and spatial characteristics of the various light sources included in the device as well as specifications of the image detector used. Furthermore, the image analysis procedure used for the dual wavelength excitation/emission is described.

Aminolevulinic Acid↗

PubMed2003

Measurements of associations of cell-surface receptors by single-particle fluorescence imaging.

SPFI (single-particle fluorescence imaging) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. The images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional Gaussian function. The spot intensities depend on whether they arise from one or more particles; this provides the basis for determining self-association of cell-surface receptors. We have used this approach to determine dimerization of MHC class II molecules and its disruption by interface peptides. We have also exploited the positional information obtained from SPFI to detect co-localization of cell-surface molecules. This involves labelling two different molecules with different coloured fluorophores and determining their positions separately by dual wavelength imaging. The images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. The technique provides quantification of the extent of co-localization and can detect whether co-localized molecules occur singly or in clusters. We have obtained preliminary data for co-localization of lipopolysaccharide and CD14 on intact cells. We also show that HLA-DR (human leukocyte antigen-DR) and CD74 are partially co-localized and that interaction between these molecules involves the peptide-binding groove of HLA-DR.

Animals↗

PubMed2006

Bispectral fluorescence imaging of aggressive basal cell carcinoma combined with histopathological mapping: a preliminary study indicating a possible adjunct to Mohs micrographic surgery.

BACKGROUND: Fluorescence imaging is an attractive diagnostic technique for skin tumour demarcation with potential to move to clinical use. Bispectral fluorescence imaging combines skin autofluorescence with delta-aminolaevulinic acid-induced fluorescence. To evaluate the technique, fluorescence data must be compared with the histopathological extent of the tumour, which is the purpose of the current study. OBJECTIVES: To investigate the agreement between bispectral fluorescence images and the histopathological tumour boundary of ill-defined basal cell carcinomas (BCCs). After fluorescence imaging the tumours were removed using Mohs micrographic surgery (MMS) to obtain histopathological maps of the tumour boundaries. METHODS: Twelve patients with aggressive BCC of mean diameter 16 mm (range 5-32) in the face were included in the study. The patients were subjected to bispectral fluorescence imaging within the 2 months prior to MMS. The fluorescence images and histopathological maps were aligned using image warping. RESULTS: Five patients (42%) showed good agreement with the histopathological mapping and the remaining seven patients (58%) showed partial agreement. Bispectral investigation combining autofluorescence with protoporphyrin IX (PpIX) fluorescence generally yielded better agreement with the histopathological boundaries of the tumours compared with using only the PpIX fluorescence. CONCLUSIONS: In this preliminary study the fluorescence has been compared with the histopathological tumour boundaries. The result implies that the technique can be applied as a useful tool for indicating tumour boundary of aggressive BCCs. Further refinement is needed to be able to indicate the exact tumour border.

Adult↗

PubMed1994

Using dyes and filters in a fluorescent imaging system.

The FluorImager fluorescence imaging system uses monochromatic 488-nm laser light to excite fluorochromes. It contains a built-in 515-nm long-pass filter that rejects excitation laser light, but allows emission light with wavelengths longer than 515 nm to pass through. A fluorochrome appropriate for use in the system is excitable by 488-nm light and emits at least some of its fluorescence at wavelengths longer than 515 nm. Two types of optical filters, long-pass and band-pass, are used in the system. Long-pass filters reject shorter wavelengths and transmit longer wavelengths. The number in the filter name denotes the cutoff wavelength (midpoint of the transition between rejected and transmitted light) for the filter. Band-pass filters transmit a band of wavelengths and reject both shorter and longer wavelengths. The numbers in the filter name denote the center wavelength of the passed band and the width of the band at half maximum transmission. Generally, when scanning for a single fluorochrome, only the built-in 515-nm long-pass filter is needed. An interchangeable filter can be added to decrease the contribution from a broad-spectrum background signal and to attenuate strong fluorochrome signals.

Calcium↗

PubMed2006

Quantitative DNA imaging in breast tumor cells by a Hadamard transform fluorescence imaging microscope.

This paper presents a novel Hadamard transform (HT) fluorescence imaging microscope by combining multiplexed imaging technique with a conventional upright fluorescence microscope for single-cell imaging and quantitative cellular analysis. The HT imaging microscope can provide 511 x 512-pixel single-cell image with high sensitivity within 21 s. In this study, the high potential value of the microscope in biomedical analysis has been demonstrated by using it to evaluate the malignancy degree of thirty cases of human breast tumors based on the measurements of cellular DNA contents, with conclusions highly accordant with pathological diagnosis. The results show that the HT microscope has the ability to analyze very small specimens and the capability of detecting very high ploidy cells, which are advantages over flow cytometry. The microscope was also successfully applied to cellular morphological analysis, and it was demonstrated that a significant linear relationship exists between tumor nuclear DNA contents and the nuclear area, and malignant and benign tumors are significantly different in both DNA contents and nuclear area. The reliability of the HT microscope in cellular DNA measurements was also investigated.

Breast Neoplasms↗

PubMed1994

A model for dendritic Ca2+ accumulation in hippocampal pyramidal neurons based on fluorescence imaging measurements.

1. High-speed fluorescence imaging was used to measure intracellular Ca2+ concentration ([Ca2+]i) changes in hippocampal neurons injected with the Ca(2+)-sensitive indicator fura-2 during intrasomatic and synaptic stimulation. The results of these experiments were used to construct a biophysical model of [Ca2+]i dynamics in hippocampal neurons. 2. A compartmental model of a pyramidal neuron was constructed incorporating published passive membrane properties of these cells, three types of voltage-gated Ca2+ channels characterized from adult hippocampal neurons, voltage-gated Na+ and K+ currents, and mechanisms for Ca2+ buffering and extrusion. 3. In hippocampal pyramidal neurons imaging of Na+ entry during electrical activity suggests that Na+ channels, at least in sufficient density to sustain action potentials, are localized in the soma and the proximal part of the apical dendritic tree. The model, which incorporates this distribution, demonstrates that action potentials attenuate steeply in passive distal dendritic compartments or distal dendritic compartments containing Ca2+ and K+ channels. This attenuation was affected by intracellular resistivity but not membrane resistivity. 4. Consistent with fluorescence imaging experiments, a non-uniform distribution of Ca2+ accumulation was generated by Ca2+ entry through voltage-gated Ca2+ channels opened by decrementally propagating Na+ action potentials. Consequently, the largest increases in [C2+]i were produced in the proximal dendrites. Distal voltage-gated Ca2+ currents were activated by broad, almost isopotential action potentials produced by reducing the overall density of K+ channels. 5. Simulations of subthreshold synaptic stimulation produced dendritic Ca2+ entry by the activation of voltage-gated Ca2+ channels. In the model these Ca2+ signals were localized near the site of synaptic input because of the attenuation of synaptic potentials with distance from the site of origin and the steep voltage-dependence of Ca2+ channel activation. 6. These simulations support the hypotheses generated from experimental evidence regarding the differential distribution of voltage-gated Ca2+ and Na+ channels in hippocampal neurons and the resulting voltage-gated Ca2+ accumulation from action and synaptic potentials.

Animals↗

PubMed1993

Nile red labeling of single living cells for contour delineation to quantify and evaluate the distribution of rhodamine 123 with fluorescence image cytometry.

Simultaneous study of intracellular quantification and distribution of fluorescent probes is difficult when cell staining is not homogeneous. This occurs after mitochondrial staining with rhodamine 123 (R123). Classical techniques for evaluation of intracellular R123 fluorescence, such as flow cytometry, are based on measurement of the global fluorescence intensity but do not take into account parameters that reflecting cellular distribution of the probe. For simultaneously studying intracellular quantification and distribution of R123 with fluorescence image analysis, we delineated a mask of the cell, generated from a fluorescent image of the plasma membrane stained by nile red (NR). After a preliminary study of the fluorescence characteristics of R123 and NR to avoid artifacts and optimize conditions of staining, quantification and distribution of intracellular R123 studies were performed by superimposition of the mask on the R123 fluorescence image. This protocol was applied to leukemic cells and allowed estimation of individual cell parameters such as mean fluorescence intensity and standard deviation, the latter providing information of the cellular distribution of R123. Moreover, it permitted demonstration of the redistribution of R123 in the whole cell when coincubated in the presence of nigericin.

Animals↗

PubMed2006

In vivo spectral fluorescence imaging of submillimeter peritoneal cancer implants using a lectin-targeted optical agent.

Intraperitoneal metastases commonly recur after surgery because small tumor foci escape detection within the complex anatomy of the peritoneal cavity and mesentery. Accurate localization of peritoneal implants during surgery could improve the resection of ovarian cancer and other malignancies, but few practical techniques to enhance detectability are currently available. Here, we describe a targeted molecular imaging method that employs fluorescently labeled avidin to detect submillimeter peritoneal implants of ovarian cancer in mice. After binding to surface lectins on the tumor, fluorescein-conjugated avidin enabled the spectral fluorescence imaging of disseminated peritoneal implants. High spatial resolution and high tumor-to-background ratio allowed the visualization of implants as small as 0.3 mm (with 100% sensitivity and specificity; n = 150) and the identification of even smaller lesions ex vivo. These results suggest that targeted molecular imaging with a fluorescence-labeled lectin-ligand system is a promising technique for the detection of disseminated submillimeter foci of cancer.

Animals↗

PubMed2006

Monitoring dynamic systems with multiparameter fluorescence imaging.

A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.

Algorithms↗

PubMed2006

Fluorescent image analysis of lipid hydroperoxides in fish muscle with 3-perylene diphenylphosphine.

A fluorescent image analysis method was developed to evaluate lipid hydroperoxide formation in fish muscle. The lipid hydroperoxides generated in white and dark fish muscles during storage at 5-6 degrees C oxidized 3-perylene diphenylphosphine located in the tissue to yield the fluorescent derivative, 3-perylene diphenylphosphine oxide (3-PeDPPO). 3-PeDPPO thus obtained was determined by digital fluorescent image analysis. The 3-PeDPPO fluorescence intensity of white and dark muscle increased during low-temperature storage (0-24 h) and was clearly correlated with total lipid hydroperoxide levels in muscle extracts, which were determined by using HPLC based on a triphenylphosphine oxidation method (R2 = 0.954). These results suggest that 3-PeDPPO fluorescence, coupled with fluorescent image analysis, is a novel tool for direct determination of lipid hydroperoxides in fish muscle without a need for extraction of lipid.

Animals↗

PubMed1998

Detection of dimers of dimers of human leukocyte antigen (HLA)-DR on the surface of living cells by single-particle fluorescence imaging.

The technique of single-particle fluorescence imaging was used to investigate the oligomeric state of MHC class II molecules on the surface of living cells. Cells transfected with human leukocyte antigen (HLA)-DR A and B genes were labeled at saturation with a univalent probe consisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of single and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers. The proportion of double particles was lower at 37 degrees C than at 22 degrees C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependent equilibrium. These results are discussed in the context of a possible role for HLA-DR dimers of dimers in T cell receptor-MHC interactions. The technique is validated by demonstrating that fluorescence imaging can distinguish between dimers and tetramers of human erythrocyte spectrin deposited from solution onto a solid substrate. The methodology will have broad applicability to investigation of the oligomeric state of immunological and other membrane-bound receptors in living cells.

Cell Line↗

PubMed2003

Multispectral laser-induced fluorescence imaging system for large biological samples.

A laser-induced fluorescence imaging system developed to capture multispectral fluorescence emission images simultaneously from a relatively large target object is described. With an expanded, 355-nm Nd:YAG laser as the excitation source, the system captures fluorescence emission images in the blue, green, red, and far-red regions of the spectrum centered at 450, 550, 678, and 730 nm, respectively, from a 30-cm-diameter target area in ambient light. Images of apples and of pork meat artificially contaminated with diluted animal feces have demonstrated the versatility of fluorescence imaging techniques for potential applications in food safety inspection. Regions of contamination, including sites that were not readily visible to the human eye, could easily be identified from the images.

Animals↗

PubMed2005

Use of fluorescence imaging to investigate the structure and function of intestinal M cells.

Fluorescence imaging technology can be applied to many aspects of cell biology ranging from the analysis of specific markers in cells and tissues to the biological actions and distribution of fluorescent proteins or particles in living cells. In this review, we examine the role of fluorescence imaging, in conjunction with other microscopical techniques, to study sites of uptake of material across the gastrointestinal epithelium. We will focus primarily on intestinal M cells, specialised antigen-sampling cells in the epithelium of the gut-associated lymphoid tissue (GALT), including Peyer's patches. In addition to their importance as sites for uptake of inert material, and hence their potential as a route of delivery of vaccines, etc., M cells are also a major site of infection by a range of microbial pathogens. The application of new fluorescence imaging technologies has expanded our knowledge on the structure, development and function of these fascinating cells.

Animals↗