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PubMed2004

Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering.

Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).

Erythrocytes↗

PubMed2003

High-throughput screening of selectivity of melt polymerization catalysts using fluorescence spectroscopy and two-wavelength fluorescence imaging.

A new general approach for rapid assessment of polymerization catalysts is introduced. Native fluorescence emission of solid polymers is measured directly in combinatorial 96-microreactor arrays and polymers produced in a laboratory-scale validation reactor. Fluorescence features collected with a CCD-based spectrofluorometer are correlated with chemical properties of interest such as polymer molecular weight, amount of branching, and catalyst selectivity. The approach is illustrated by screening of selectivity of melt polymerization catalysts used in synthesis of an aromatic bisphenol A polycarbonate. Selectivity of catalysts correlated with the ratio of fluorescence intensities at 400 and 500 nm at 340-nm excitation. The relative standard deviation (RSD) in spectroscopic serial measurements was 1-12.5%. This spread included instrument variability (< or = 1% RSD) and sample inhomogeneity. Parallel quantitative screening of catalyst selectivity in combinatorial 96-microreactor arrays was performed as a two-wavelength ratiometric fluorescence imaging through 400- and 500-nm interference filters and showed a good correlation (R2 = 0.994) with serial screening. Our approach is an attractive alternative to traditional separation-based techniques if speed and nondestructive nature of analysis are critical and when the high cross-linking or solvent resistance of polymers complicates traditional analysis.

Journal Article↗

PubMed1984

Imaging fluorescence bronchoscopy for localizing early bronchial cancer and carcinoma in situ.

A system of imaging fluorescence bronchoscopy instrumentation and methods has been devised that has succeeded in localizing very small (1 X 2 mm) areas of bronchial mucosal cancer, in individuals with radiologically occult lung cancer (positive sputum cytology for malignant cells and a negative chest X-ray). These areas were located solely by their fluorescence, and were visibly normal on white-light examination. The detection of lung cancer in individuals with radiologically occult lung cancer depends upon adequate methods of sputum collection and processing. Proving that fluorescing areas show "true positive" fluorescence depends upon accurate brush and forceps biopsies, providing adequate cytological and biopsy material. The entire system of the diagnosis and localization of early or pre-invasive lung cancer (while still confined to a bronchus) rests on skilled cytopathology methods and interpretation, not only skilled fluorescence bronchoscopic examination and adequate instrumentation.

Aged↗

PubMed1997

Temporal and spatial monitoring of exocytosis with native fluorescence imaging microscopy.

Exocytosis of rat peritoneal mast cells (RPMCs) was monitored temporally and spatially using native fluorescence imaging microscopy with 305-nm laser excitation. Real time chemical images of the relative amounts of serotonin and protein released from each cell are obtained. Individual cells released different amounts of material and the time delay of the release event after stimulation by polymyxin varied from cell to cell. Release consisted of a main burst of activity followed by slow sustained secretion over many seconds. The images show that different regions of a given cell behave asynchronously in releasing material into the surrounding medium. On rare occasions, highly localized fluorescence bursts can be seen in the vicinity of the cell. Presumably, these are due to delayed release of fluorescent mediators from single granules, following detachment of the latter from the cell. These quantitative fluorescence measurements allow one to follow the time-course of the physiologically important parameter---the amount of material that is secreted into the body fluid on stimulation.

Animals↗

PubMed2000

Fluorescence imaging during photodynamic therapy of experimental tumors in mice sensitized with disulfonated aluminum phthalocyanine.

A fluorescence imaging system was used to monitor the emission of disulfonated aluminum phthalocyanine (AlS2Pc) during the photodynamic therapy (PDT) of murine tumors. Cells of the MS-2 fibrosarcoma were injected in mice in two compartments in order to cause the development of tumors in different host tissues. Two drug doses and two uptake times were considered. Moreover, the fluorescence of the AlS2Pc was excited using two wavelengths on the opposite sides of the absorption peak to detect a possible change in the absorption spectrum of the sensitizer induced by the PDT. In the tumors, the treatment induces a variation of the fluorescence intensity: in some mice a mild photobleaching takes place, in others a fluorescence enhancement occurs. Which effect predominates depends on the experimental conditions, even though a large spread of data was found amongst mice of the same group. In all mice, independently of the drug dose, uptake time or tumor compartment, a marked increase in the fluorescence signal takes place at the borders of the irradiated area. To quantify this effect we evaluated the ratio between the fluorescence intensities in the peritumoral area and in the tumor itself. This ratio increases monotonically during the PDT, showing a different behavior with the two excitation wavelengths. This indicates that the AlS2Pc absorption spectrum shifts toward shorter wavelengths as a result of the irradiation.

Animals↗

PubMed1994

Fluorescence image analysis of plasma cells in monoclonal gammopathies.

The DNA content of plasma cells was estimated by fluorescence image analysis in monoclonal gammopathies. Image analysis provides the possibility of measuring the bone marrow plasma cell population separately from the other myeloid cells. Plasma cells were labeled, identified and then relocated for propidium iodide intensity measurements. Patients with DNA malignancy grade > 0.20 or with DNA index > 1.15 had a poorer survival rate than did patients with a low DNA malignancy grade or DNA index. DNA malignancy grade is based on the variance of DNA values around the normal diploid peak, as proposed by Böcking. Of all the biologic and clinical parameters investigated, the serum beta 2 microglobulin level was the most important prognostic factor for survival in the multivariate regression analysis, and the DNA entropy index provided the most significant additional information.

Aged↗

PubMed1999

Signal to noise analysis of multiple color fluorescence imaging microscopy.

BACKGROUND: Various approaches that were recently developed demonstrate the ability to simultaneously detect all human (or other species) chromosomes by using combinatorial labeling and fluorescence in situ hybridization (FISH). With the growing interest in this field, it is important to develop tools for optimizing and estimating the accuracy of different experimental methods. METHODS: We have analyzed the principles of multiple color fluorescence imaging microscopy. First, formalism based on the physical principles of fluorescence microscopy and noise analysis is introduced. Next, a signal to noise (S/N) analysis is performed and summarized in a simple accuracy criterion. The analysis assumes shot noise to be the dominant source of noise. RESULTS: The accuracy criterion was used to calculate the S/N of multicolor FISH (M-FISH), spectral karyotyping, ratio imaging, and a method based on using a set of broad band filters. Spectral karyotyping is tested on various types of samples and shows accurate classifications. We have also tested classification accuracy as a function of total measurement time. CONCLUSIONS: The accuracy criterion that we have developed can be used for optimizing and analyzing different multiple color fluorescence microscopy methods. The assumption that shot noise is dominant in these measurements is supported by our measurements.

Female↗

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 II. In vivo testing.

A fluorescence imaging prototype for skin lesion detection and diagnosis using aminolaevulinic acid (ALA) induced protoporphyrin IX (PpIX) was tested in vivo and in the clinic. The prototype was designed as an affordable, portable device to allow contrast enhanced imaging of skin lesions using either the dual excitation wavelength method or the dual emission wavelength method or both. In this study the prototype was tested first on an animal model. Topical application of ALA on defined spots on mouse skin gave PpIX fluorescence after about 3 hours of application. After successful in vivo testing the instrument was tested on basal cell carcinoma patients before ALA-PpIX photodynamic therapy. The patients were topically applied with ALA. After three hours the device was tested (immediately before treatment). The prototype showed good results in terms of contrast enhancement (elimination of unwanted background signals, e.g. autofluorescence) using either contrast enhancement method, both methods achieving similar results. The results achieved in this study, combined with the affordable design of the device, seem to allow cost-effective, contrast-enhanced imaging of skin lesions before or during photodynamic therapy using ALA induced PpIX.

Aminolevulinic Acid↗

PubMed2001

Novel surface and multicolor charge coupled device-based fluorescent imaging system for DNA microarrays.

We report a novel support, concomitant attachment chemistry, and a fluorescent imaging system for DNA microarrays. The support consists of soda lime glass coated with a layer of chromium, which eliminates any autofluorescence from the underlying glass substrate and reduces nonspecific probe binding. Attachment of DNA fragments exceeding 300 nucleotides in length is achieved without chemical modifications of either the chrome surface or the DNA itself. The charge coupled device (CCD)-based imaging system employs a 175 W xenon arc lamp as the light source, allowing the use of many different fluorophors. A 14 mm x 9 mm sample area is imaged with a single exposure, which takes between 5 and 20 s for each color plane in typical genomic comparative genomic hybridization type assays. The spatial resolution is limited only by the pixel size of the CCD chip (9 microm). The oblique illumination geometry combined with effective background reduction afforded by the chromium surface enables the system to achieve a detection limit of <5 x 10(7) fluorophors/cm(2) with 10 s integration. In a model system with arrayed lambda DNA targets a dose response was observed over four orders of magnitude in response to hybridizations with increasing amounts of the fluorescent labeled lambda probe.

Escherichia coli↗

PubMed1995

Fluorescence image cytometry for measurement of nuclear DNA content in surgical pathology.

A study was made on various methodological aspects of fluorescence image cytometry (FICM) for measurement of nuclear DNA content by using CCD cameras attached to an epifluorescence microscope. Cell nuclei of paraffin-embedded specimens from mouse tissues and human prostate carcinomas were isolated and stained with 4'-6-diamidino-2-phenylindole (DAPI). We found that fluorescence fading, lamp stability, and the homogeneity of the illumination can easily be controlled. A camera with a signal-to-noise ratio of 53 dB gave a slightly more precise measurement than did a 46-dB camera. The linearity of the analysis results was very good. The coefficient of variation of mouse kidney standard cells in the DNA histograms was about 5% and 7.4% in histograms of prostate carcinoma biopsies. Stained cell nuclei can be stored for long periods at -20 degrees C without impairment of quality. Comparative measurements of ploidy by FICM and flow cytometry confirmed the accuracy of the FICM analyses. Thus, FICM appears to be an easy method for quantifying the DNA content of visually inspected cell nuclei in surgical pathology.

Animals↗

PubMed1999

Analysis of Apparent Noncompetitive Responses to Competitive H(1)-Histamine Receptor Antagonists in Fluorescent Imaging Plate Reader-Based Calcium Assays.

We have examined the utility of high throughput fluorescent imaging plate reader (FLIPR)-based calcium assays for pharmacological characterization of G-protein coupled receptors (GPCRs) using recombinant and native human H1-histamine receptors (H(1)-HR), expressed in HEK293 and HeLa S3 cells, respectively, as model systems. For stably transfected HEK293 cell lines, the potency of histamine for elevating intracellular calcium increased (pD(2), 7.13 and 7.86) with increased H(1)-HR density (about 0.8 and 14 pmol/mg protein, respectively), though histamine binding affinities were similar. The classic H(1)-HR competitive antagonists diphenhydramine and chlorpheniramine appeared noncompetitive by causing depressions of the maximal histamine responses along with rightward shifts of histamine concentration-response curves, thus precluding Schild analysis. Applying the generalized Cheng-Prusoff equation to antagonist concentration-response curves for inhibition of fixed histamine concentrations yielded apparent pK(b) values that were consistent among recombinant and native receptors at different expression levels. These pK(b) values for diphenhydramine and chlorpheniramine (e.g., 7.83 and 8.77, respectively) were in good agreement with binding pK(i) values (e.g., 7.98 and 8.52, respectively). Apparent antagonist affinities determined from FLIPR calcium and competition binding assays were also consistent for the competitive antagonists mepyramine, tripelennamine, and promethazine. In phosphoinositide hydrolysis assays, chlorpheniramine exhibited insurmountable inhibition of histamine calcium responses, although to a lesser extent than that observed in calcium assays; pK(b) values were similar. These results demonstrate that competitive antagonist potencies can be attained from FLIPR-derived data by application of the generalized Cheng-Prusoff equation, despite apparent noncompetitive antagonism under these assay conditions. Apparent noncompetitive antagonist effects may in part be attributable to a lack of equilibrium of histamine and antagonists with H(1)-HR within the short duration of rapid transient effects of histamine on intracellular calcium.

Journal Article↗

PubMedDate not supplied

A digitized fluorescence imaging study of intracellular Ca2+, pH, and mitochondrial function in primary cultures of rabbit corneal epithelial cells exposed to sodium dodecyl sulfate.

Primary cultures of rabbit corneal epithelial cells have been developed as an in vitro system to predict irritancy potential and delayed cytotoxicity of surfactants in our laboratory. The objective of this study was to evaluate the effects of the surfactant sodium dodecyl sulfate (SDS), a common ingredient in consumer products, on intracellular Ca2+, pH, and mitochondrial function in this culture system. Ca2+ and pH were measured in single living corneal epithelial cells by ratio imaging of fura-2 and 2,'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein fluorescence, respectively. Mitochondrial function was examined by probing mitochondrial membrane potential with the fluorescent dye rhodamine 123 and by measuring the ratio of ATP to ADP with an HPLC method. Cell viability was determined by fluorescence imaging of propidium iodide in single cells and LDH leakage assay in populations of cells. SDS (40 micrograms/ml) increased intracellular Ca2+ from 180 +/- 28nM to 453 +/- 86 nM within 2 min, and induced intracellular acidification (pHi dropped 0.3 units in 15 min). Treatment of the cultures with SDS also resulted in dissipation of the mitochondrial membrane potential and decrease of intracellular ATP/ADP. SDS-induced Ca2+ elevation and intracellular acidification preceded the loss of cell viability observed 20 min after exposure. However, SDS-induced cell injury does not appear to be triggered by extracellular Ca(2+)-influx, as absence of extracellular Ca2+ did not attenuate SDS-induced cytotoxicity while it completely blocked ionomycin-induced cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1993

Analysis of the interleukin 6 receptor on normal human keratinocytes by digital imaging fluorescence microscopy.

The interleukin 6 receptor (IL 6 receptor) on normal human keratinocytes was analyzed by digital imaging fluorescence microscopy (DIFM). Non-fixed keratinocytes were incubated with monoclonal anti-IL 6 receptor antibody (MT18). The fluorescence intensity was quantitatively determined by an interactive laser cytometer (ACAS 470). IL 6 receptor-positive cells showed high density staining with the specific antibody in the center of the elevated cell surface. Therefore, DIFM appears to be an effective method for study of the distribution of IL 6 receptors at the single living cell level.

Cells, Cultured↗

PubMed2002

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Cell Division↗

PubMed2006

A novel fluorescence imaging technique combining deconvolution microscopy and spectral analysis for quantitative detection of opportunistic pathogens.

A novel fluorescence imaging technique based on deconvolution microscopy and spectral analysis is presented here as an alternative to confocal laser scanning microscopy. It allowed rapid, specific and simultaneous identification of five major opportunistic pathogens, relevant for public health, in suspension and provided quantitative results.

Bacteria↗

PubMed2005

Validation of a fluorescent imaging plate reader membrane potential assay for high-throughput screening of glycine transporter modulators.

A fluorescent imaging plate reader (FLIPR) membrane potential (V(m)) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT(2)) in a stable rGlyT(2)-HEK cell line. Data show that glycine activation of rGlyT(2) consistently results in a concentration-dependent V(m) response on the FLIPR that is blocked by the potent and selective GlyT(2) antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [(3)H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT(2) physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT(2) inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based V(m) assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT(2).

Amino Acid Transport Systems, Neutral↗

PubMed2000

Time-resolved fluorescence imaging for specific and quantitative immunodetection of human kallikrein 2 and prostate-specific antigen in prostatic tissue sections.

OBJECTIVES: To design protocols for specific and quantitative immunohistochemical detection of human kallikrein 2 (hK2) using lanthanide chelate-labeled monoclonal antibodies (Mabs) and time-resolved fluorescence imaging. METHODS: Anti-prostate-specific antigen (PSA) Mabs were tested in microtiterplate assays for their ability to prevent PSA from cross-reacting with the anti-hK2 Mab 6H10. Europium-labeled 6H10 and terbium-labeled anti-PSA Mab 2E9, selected as the best blocker antibody, were used for dual-label immunodetection in routinely fixed benign (n = 7) and malignant (n = 5) prostate specimens. The amounts of IgG bound in tissue were calculated from drops containing known Mab concentrations. RESULTS: The use of anti-PSA Mab 2E9 for blocking diminished the cross-reaction from 5% to 0.3%. In the analyzed tissues, there was considerable variation in staining intensity for both proteins; PSA signals varied from 0.1 to 36.6 times that of hK2, with on average 10-fold more bound anti-PSA Mab than anti-hK2 Mab. In malignant tissue, the amounts of bound IgGs were lower and more variable than in benign tissue using both the anti-PSA Mab and the anti-hK2 Mab. The variation in signal intensities for PSA and hK2 correlated significantly in benign tissue (P >0.05), but not in benign hyperplastic and malignant specimens (P <0.05). CONCLUSIONS: Quantification of two lanthanide chelate-labeled antibodies bound in the same tissue section enabled comparison of PSA and hK2 content in individual cells. The average cellular content of hK2 relative to that of PSA was consistent with previous mRNA studies. The time-resolved fluorescence imaging-based quantification method has universal applicability in fixed tissue specimens.

Antibodies, Monoclonal↗

PubMedDate not supplied

Near-infrared fluorescence imaging of tumor integrin alpha v beta 3 expression with Cy7-labeled RGD multimers.

PURPOSE: Cell adhesion molecule integrin alpha v beta 3 is an excellent target for tumor interventions because of its unique expression on the surface of several types of solid tumor cells and on almost all sprouting tumor vasculatures. Here, we describe the development of near-infrared (NIR) fluorochrome Cy7-labeled RGD peptides for tumor integrin targeting. PROCEDURES: Mono-, di-, and tetrameric RGD peptides were synthesized and conjugated with Cy7. The integrin specificity of these fluorescent probes was tested in vitro for receptor binding assay and fluorescence microscopy and in vivo for subcutaneous U87MG tumor targeting. RESULTS: The tetrameric RGD peptide probe with the highest integrin affinity showed the highest tumor activity accumulation and strongest tumor-to-normal tissue contrast. This uptake is integrin-specific as the signal accumulated in the tumor can be effectively blocked by unconjugated RGD peptide antagonist of integrin alpha v beta 3. CONCLUSIONS: Noninvasive NIR fluorescence imaging is able to detect and semiquantify tumor integrin expression based upon the highly potent tetrameric RGD peptide probe.

Animals↗