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Results for “Fluorescence imaging”

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At least 19 recordsLinked to original sources

PubMed1990

Tissue-localizing properties of some photosensitizers studied by in vivo fluorescence imaging.

Using fluorescence imaging, the tissue-localizing properties of five photosensitizers were studied in vivo in tumours in 'sandwich' observation chambers and in tumours growing on thigh muscle. The preliminary results indicate that of the three photodynamically active dyes tested (haematoporphyrin derivative, Photofrin II and aluminium phthalocyanine tetrasulphonate), the phthalocyanine possesses the best tumour-localizing properties. This makes it possible to combine tumour fluorescence detection and photodynamic therapy with reduced skin photosensitivity. The two photodynamically inactive dyes tested (uroporphyrin I and acridine red) may be useful for application in fluorescence imaging to localize superficial tumours without inducing skin photosensitivity. In particular, acridine red has remarkable tumour-localizing properties, but is rather toxic.

Animals↗

PubMed1988

Cancer detection by quantitative fluorescence image analysis.

Quantitative fluorescence image analysis is a rapidly evolving biophysical cytochemical technology with the potential for multiple clinical and basic research applications. We report the application of this technique for bladder cancer detection and discuss its potential usefulness as an adjunct to methods used currently by urologists for the diagnosis and management of bladder cancer. Quantitative fluorescence image analysis is a cytological method that incorporates 2 diagnostic techniques, quantitation of nuclear deoxyribonucleic acid and morphometric analysis, in a single semiautomated system to facilitate the identification of rare events, that is individual cancer cells. When compared to routine cytopathology for detection of bladder cancer in symptomatic patients, quantitative fluorescence image analysis demonstrated greater sensitivity (76 versus 33 per cent) for the detection of low grade transitional cell carcinoma. The specificity of quantitative fluorescence image analysis in a small control group was 94 per cent and with the manual method for quantitation of absolute nuclear fluorescence intensity in the screening of high risk asymptomatic subjects the specificity was 96.7 per cent. The more familiar flow cytometry is another fluorescence technique for measurement of nuclear deoxyribonucleic acid. However, rather than identifying individual cancer cells, flow cytometry identifies cellular pattern distributions, that is the ratio of normal to abnormal cells. Numerous studies by others have shown that flow cytometry is a sensitive method to monitor patients with diagnosed urological disease. Based upon results in separate quantitative fluorescence image analysis and flow cytometry studies, it appears that these 2 fluorescence techniques may be complementary tools for urological screening, diagnosis and management, and that they also may be useful separately or in combination to elucidate the oncogenic process, determine the biological potential of tumors and monitor the results of chemopreventive, immunological and chemotherapeutic regimens. To our knowledge there has been no study in which quantitative fluorescence image analysis and flow cytometry were compared directly to assess the relative strengths and weaknesses for urinary tract cytology. Such a study could provide important information for urologists.

Cytodiagnosis↗

PubMed1991

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.

3T3 Cells↗

PubMed1992

Practical applications in molecular biology of sensitive fluorescence detection by a laser-excited fluorescence image analyzer.

A new kind of fluorescence image analyzer was developed for a variety of uses, especially in molecular biology. Compounds labeled with fluorescent groups on a gel or nitrocellulose membrane are excited with 532 nm of light from a green laser. The fluorescence emitted passes through light-collecting fibers to a photomultiplier. Imaging data converted from the emitted light are analyzed by a microcomputer and stored on a magnetic optical disk. Dideoxy DNA sequencing was done with the same amount of DNA used for autoradiography, and the sequencing ladders obtained from gel scanning were automatically converted to sequence data by the analyzer. When an agarose gel was analyzed after electrophoresis, DNA stained with ethidium bromide was detected by the analyzer with higher sensitivity rather than by the conventional photographic method. Nylon and nitrocellulose membranes could be read by the analyzer, so blot hybridization experiments can be done without radioisotopes. High-quality computer storage of the imaging data from gel electrophoresis and hybridized membranes, including pulsed-field gels, make it possible to quantify image intensity and to construct many kinds of databases.

Base Sequence↗

PubMed1987

Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope.

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.

Animals↗

PubMed1991

Fluorescent image cytometry: from qualitative to quantitative measurements.

Image analysis is being increasingly used in biology and medicine; however, in order to obtain truly quantitative data and thus avoid errors in interpretation, a certain number of precautions must be taken when the image is digitized, well before any attempt is made to analyse or interpret the data. This is particularly true for image microfluorometry. In this article we will examine an image analysis system for fluorescent images composed of a mercury lamp, a microscope, a high sensitivity video camera and an image analyser and evaluate the principal sources of random and non-random errors, various constraints, and their relative importance. A signal correction protocol is proposed to minimize non-random errors during digitalization. A few examples are given to illustrate its efficiency.

Analog-Digital Conversion↗

PubMed1991

[Spatial fluorescence imaging of atherosclerotic plaque: contrast enhancement by 2 wavelength laser stimulation, digital image processing and dye marking].

A new video-enhanced fluorescence imaging technique has been used for the first time for in vitro differentiation of human atherosclerotic plaques vs normal arterial wall. Laser-induced superficial tissue fluorescence of specimens from human aorta was documented after alternating excitation with violet (405 nm +/- 5 nm) and blue (470 nm +/- 10 nm) krypton-ion laser light. Subsequent digital subtraction of the corresponding fluorescence images allowed to differentiate areas of atherosclerosis from normal intima. Fluorescence intensity was correlated with the morphological aspect of samples and histology of the plaque. Dihematoporphyrin-ether/ester (DHE) incubation enhanced fluorescence contrast of plaques in comparison to normal artery vessel wall. Depending on the concentration of the incubation solution (10, 20, and 40 micrograms DHE/ml NaCl solution), fluorescence increased. Fluorescence intensity was highest in fatty plaque areas, while calcific lesions showed no substantial DHE uptake.

Angioplasty, Laser↗

PubMed1988

Simultaneous Nomarski and fluorescence imaging during video microscopy of cells.

A video microscope designed to allow low light level fluorescence imaging of cells during simultaneous high-resolution differential interference contrast (DIC) imaging, without the fluorescence light losses of 60-90% normally associated with this contrast-enhancement technique, is described. Transmitted light for DIC imaging, filtered at greater than 620 nm, passes through standard DIC optical components, (1/4 lambda-plate, polarizer, and Wollaston prism) before illuminating the cells. Transmitted light and fluorescence emission pass through a second Wollaston prism but not through the analyzer, which is repositioned more distally in the optical path. Prisms designed to reflect light out a side port of the microscope to a video camera have been replaced with a dichroic mirror. This mirror reflects fluorescence emission out the side port to a low light-sensitive video camera. The spectrally distinct transmitted light continues through the dichroic mirror to an overhead camera through a polarizer (analyzer), which completes the DIC optical path. The fluorescence and DIC images can be viewed simultaneously on side-by-side video monitors, examined sequentially by an image-processing computer, or examined simultaneously using a video splitter/inserter. The ability to image cells with high resolution simultaneously with low light level fluorescence imaging should find wide applicability whenever it is necessary or desirable to correlate fluorescence intensity or distribution with specific cell structure or function.

Animals↗

PubMedDate not supplied

Digital background subtraction for fluorescence imaging.

A method of enhancing contrast in fluorescence imaging has been devised, based on real-time digital subtraction of a background video image from a signal-plus-background video image. Color filters are used to differentiate signal from background. The technique has been applied to detection of small tumors labeled with the tumor specific fluorescent drug hematoporphyrin derivative.

Biophysical Phenomena↗

PubMed1990

Analysis of cholesterol ester accumulation in macrophages by the use of digital imaging fluorescence microscopy.

Low density lipoprotein (LDL) induced accumulation of cholesterol esters was analyzed by the digital imaging fluorescence microscopy (DIFM) in murine tumor macrophages. To analyze cholesterol ester accumulation, P388D1 macrophages were incubated with increasing quantities of unmodified or acetylated human LDL, washed, and live stained with a lipophylic fluorescent dye Nile Red. The increase in fluorescence intensity was quantitatively determined by the interactive laser cytometer (ACAS 470) and compared with the accumulation of cellular cholesterol esters determined by the gas liquid chromatography. Correlation between the two methods was highly significant (r greater than 0.9, P less than 0.001). A good agreement between the two methods was also found in terms of sensitivity and reproducibility. With the use of 589 nm narrowband interference filter in the light path of emitted light the intensity of fluorescence correlated well with cellular cholesterol ester content even in the presence of relatively high concentrations of triglycerides. Therefore, digital imaging fluorescence microscopy appears to be a reliable method for quantification of cholesterol ester accumulation at the single cell level offering new possibilities of studying interactions between cells and cholesterol ester rich lipoproteins.

Animals↗

PubMed1990

Quantitative fluorescence image analysis in bladder cancer screening.

Quantitative fluorescence image analysis (QFIA) cytology combines image analysis to measure DNA with visual cytology for bladder cancer detection. QFIA sensitivity is 76% to 81% and 95% to 100% for low- and high-grade tumors respectively, with 94% specificity in asymptomatic controls. QFIA screening of 504 persons within a beta-naphthylamine exposed cohort found DNA hyperploidy correlated with the duration of carcinogenic exposure and smoking history; marker prevalence was 23% for exposed workers who smoked and 2% for nonexposed nonsmokers. In prospective animal studies, QFIA was useful in monitoring carcinogenesis and chemoprevention with retinoids. Current QFIA research focuses on measurements of oncogenes, growth factors and their receptors, cytoskeleton, and tumor-associated antigens to improve sensitivity and specificity to low-grade tumors and to identify premalignant conditions. Profiles of biochemical and immunological markers on single cells may further assist in the study of high-risk cohorts and individual risk assessment.

Biomarkers, Tumor↗

PubMed1992

Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.

Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased. We modified a standard epifluorescence microscope for time-resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD-camera was used for detection and the images were digitally processed. A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix. The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehyde-fixed, wax-embedded specimens.

Alkaline Phosphatase↗

PubMed1991

Carcinogen-induced alterations in rat liver DNA adduct formation determined by computerized fluorescent image analysis.

These studies employed continuous feeding of a carcinogenic level of N-2-acetylaminofluorene to male rats for 28 days. Under these conditions normal hepatocytes are known to be inhibited from proliferation, whereas xenobiotic-resistant putative preneoplastic hepatocytes with altered liver enzyme phenotypic expression appear to have a growth advantage. A novel technique using computerized fluorescent image analysis of triple-stained frozen liver sections was developed and used to visualize three different molecular markers in individual hepatic cells. Proliferating liver cells were identified by anti-5-bromodeoxyuridine immunostaining in livers of rats injected with 5-bromodeoxyuridine 1 hour before sacrifice. Anti-cytokeratin immunostaining was used to identify bile ducts and putative oval cells. Characterization of DNA adduct formation was achieved with an antiserum specific for N-(deoxyguanosine-8-yl)-2-aminofluorene, the major DNA adduct of 2-acetylaminofluorene. The image analysis demonstrated low but distinct DNA adduct concentrations in putative oval cells identified by anti-cytokeratin staining and in scattered, replicating liver cells recognized by anti-5-bromodeoxyuridine. Adducts were not detected in replicating foci consisting of 3 to 11 nuclei. It is possible that proliferating liver cells that have low N-2-acetylaminofluorene-DNA adduct levels may clonally expand to become foci protected from further adduct accumulation and preneoplastic liver lesions. Thus, the computerized fluorescent image analysis demonstrated here may provide a novel procedure for identification of carcinogen-induced liver cell alterations.

2-Acetylaminofluorene↗

PubMed2025

Application of Perioperative Real-Time Fluorescence Imaging to Achieve High-Quality Debridement: A Randomized Control Trial.

OBJECTIVE: To investigate the effectiveness of real-time fluorescence imaging (RTFI)-assisted debridement in managing chronic wounds compared with standard surgical debridement. APPROACH: This study was a patient-blinded, randomized clinical trial conducted from February 17, 2021, to July 30, 2021, on patients with chronic wounds. Patients were randomized to an RTFI group (M group) or conventional group (C group). The primary outcomes were as follows: percentage of residual bacterial area (preoperative and postoperative), number of debridements, high-quality debridement ratio, operation duration, and wound healing duration. RESULTS: A total of 100 patients were enrolled in both groups. No significant difference in the percentage of preoperative residual bacterial area or high-quality debridement ratio was seen. The M group underwent debridement an average of 2.6 times and had a significantly longer duration of operation (33.5 ± 12.7 min) than the C group (29.9 ± 10.4 min; p = 0.031). The postoperative residual bacterial area was significantly lower in the M than in the C group (6.83% ± 1.39% vs. 30.0% ± 12.37%, respectively; p < 0.001). The M group required significantly fewer wound healing days (49.2 ± 25.3 vs. 63.0 ± 27.9, p < 0.001). Secondary outcomes also demonstrated statistically significant differences in total hospitalized days (17.5 ± 9.3 vs. 21.5 ± 12.5, p < 0.01), days of antibiotic use (15.5 ± 8.7 vs. 18.7 ± 6.7, p < 0.01), and reinfection rates (4 of 100 vs. 22 of 100, p < 0.001). INNOVATION: RTFI can detect signals from normal skin components and bacterial metabolites. Therefore, interpretation of RTFI results should be correlated with the clinical condition. RTFI is associated with high-quality debridement. This technique can also be applied in targeted biopsy and in training young staff to mature debridement procedures. CONCLUSION: RTFI in debridement is associated with favorable clinical outcomes and may have a positive influence on chronic wound healing.

Humans↗

PubMed1991

Quantitative fluorescence image analysis of deoxyribonucleic acid ploidy in urine from normal children.

Quantitative fluorescence image analysis incorporates the 2 diagnostic techniques of cytological analysis with quantitation of deoxyribonucleic acid (DNA). Exfoliated urinary cells are ideal for analysis by this method, which allows the identification of "rare event" abnormal cells. We evaluated the urine from 50 children who had undergone cystoscopy or were catheterized for other reasons. The urine was free of infection by urinalysis. Cytological analysis demonstrated normal or atypical cells in all patients. Of the patients 1 (2%) had greater than 2 of 500 cells analyzed with greater than 5C DNA and 4 (8%) had greater than 2 of 500 cells with greater than 5C double stranded nucleic acid. These data suggest that it may be "normal" for urine to contain "rare event" abnormal cells. The significance of this finding is unclear at present.

Adolescent↗

PubMed1987

DNA cytometry and cytology by quantitative fluorescence image analysis in symptomatic bladder cancer patients.

A semi-automated quantitative fluorescence image analysis (QFIA) technique was developed with the Leitz TAS-Plus to detect bladder cancer using hyperploidy in urinary cells. Absolute nuclear fluorescence intensity (ANFI) (emission at 540 nm with excitation at 436 nm) of individual acridine-orange-stained cells was quantitated using (1) QFIA and (2) simple filter microspectrofluorophotometry (SFM). Both methods employed an internal phosphor particle standard which, when once calibrated against the DNA content of normal cells, obviates the necessity of routinely calibrating against normal cells in each sample. Results of SFM and QFIA were compared with routine Papanicolaou (Pap) cytopathology, using histopathology as the diagnostic standard in 272 samples from 67 symptomatic patients. The sensitivities for detecting low-grade transitional-cell carcinoma were 86% for SFM, 76% for QFIA, and 33% for Pap cytology. QFIA and SFM were significantly more sensitive at detecting bladder cancer than was Pap (0.01 greater than p greater than 0.001). Comparison of sensitivity obtained with bladder washings and urine samples showed that noninvasively obtained urines can be used. ANFI also detected recurrent and precancerous bladder lesions and kidney, ureter, and prostate lesions. This approach may prove generally useful in quantifying biochemical and immunological probes and should be broadly applicable as a research tool for studying the relationship of biochemical markers in the pathogenesis of disease and as a test for cancer control.

Carcinoma, Transitional Cell↗

PubMed1992

A digitized-fluorescence-imaging study of mitochondrial Ca2+ increase by doxorubicin in cardiac myocytes.

The objective of the present study was to investigate the role of mitochondrial Ca2+ in doxorubicin-induced cell injury. The effect of doxorubicin on cultured cells was investigated by digitized fluorescence imaging. The Ca2+ sensitive fluorescent dye fura-2 was used to estimate cytosolic, mitochondrial and total cellular Ca2+. Rhodamine 123 was used to estimate the mitochondrial membrane potential, and cellular ATP was determined by h.p.l.c. The data showed that doxorubicin induced greater-than-2-fold increases in mitochondrial Ca2+ before changes in cytosolic Ca2+ could be detected. An increase in mitochondrial Ca2+ paralleled the observed dissipation in mitochondrial membrane potential. Cellular ATP levels appeared to decrease as a result of mitochondrial dysfunction, which in turn produced greater-than-2-fold increases in cytosolic Ca2+. The data suggest that doxorubicin-induced alterations in mitochondrial Ca2+ homoeostasis are associated with a dissipation in energy conservation, which may result in cell injury.

Adenosine Triphosphate↗

PubMed1990

A quantitative assessment of F-actin content and distribution in untreated and butyric acid treated murine melanoma B16a tumour cells: a fluorescence image analysis study.

Image analysis of phallacidin, a fluorescent stoichiometric probe to F-actin, permitted the cytoskeletal-associated actin 'F-actin' to be visualized morphologically and to be divided into two groups, diffuse and filamentous. The filamentous actin group was categorized further into four subgroups according to the intensity of the phallacidin probe. F-actin groups and subgroups of untreated cells and cells treated with 1.5 mM butyrate acid were analysed independently. Butyric acid treatment significantly increased total actin, defined as diffuse actin, plus filamentous subgroup actins 1-4. Specifically, butyric acid-treatment increased filamentous subgroup actin 1.

Actins↗