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Results for “Fluorescence imaging”

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At least 163 records · Page 9Linked to original sources

PubMed2000

Microregional heterogeneity of non-protein thiols in cervical carcinomas assessed by combined use of HPLC and fluorescence image analysis.

Under low oxygen conditions, non-protein thiols (NPSHs, non-protein sulfhydryls) can effectively compete for DNA radicals sites and hence represent a potentially important cause of radiation resistance in the clinic. Intra- and intertumoral heterogeneity of glutathione (GSH) and cysteine were assessed in cryostat sections of multiple biopsies obtained from 10 cervical carcinomas by the combined use of a sensitive high-performance liquid chromatography (HPLC) method and a fluorescence image analysis technique to examine the spatial distribution of NPSHs in tumor tissue. Glutathione concentrations ranged from 1.98 to 4.42 mM; significant (> or =1 mM) concentrations of cysteine, a more effective radioprotector than GSH, were found in some tumors. By HPLC, the intratumoral heterogeneity of NPSHs was relatively small compared with the intertumoral heterogeneity. The histochemical stain 1-(4-chloromercuryphenoylazo)-2-napthol (mercury orange), which binds to GSH and cysteine, was used to determine the spatial distribution of NPSHs in tumor tissue. A comparison of NPSH levels in serial cryostat sections showed a close correlation between NPSH values determined by HPLC and mercury orange fluorescence quantification. Using fluorescence image analysis, an approximately 2-fold increase of NPSHs in tumor versus nonmalignant tissue was observed in the same section. Because some cervical carcinomas contain radiobiologically important levels of cysteine, agents that target the biochemical pathways maintaining tumor cysteine have therapeutic potential as adjuncts to radiotherapy in cervix cancer patients.

Adenocarcinoma↗

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↗

PubMed2006

Chlorophyll a fluorescence imaging of ozone-stressed Brassica napus L. plants differing in glucosinolate concentrations.

Brassicaceae are characterised by glucosinolates (GS), which appear to be involved not only in biotic but also in abiotic stress responses of plants. We investigated the effect of O (3) stress on leaf GS concentrations in two lines of BRASSICA NAPUS L., differing in GS content. Ozone fumigation decreased GS concentrations in leaves of B. NAPUS of one line. In control conditions, chlorophyll content, rates of saturating photosynthesis, and quantum yield of photosystem 2 differed between the two BRASSICA lines, but differences were smaller in O (3)-stress conditions, suggesting that the relationship between leaf GS concentration and sensitivity to abiotic stress merits further research. In agreement with other ecophysiological measurements, chlorophyll fluorescence imaging clearly distinguished both lines and in some cases also treatments. A method for analysis of fluorescence images accounting for the two-dimensional leaf heterogeneity is presented.

Brassica napus↗

PubMed1994

Fluorescence imaging of intracellular Ca2+.

The measurement of intracellular Ca2+ concentrations ([Ca2+]i) is of critical importance, because many cellular functions are tightly regulated by [Ca2+]i. The fluorescent indicator, fura-2, has been used frequently to measure [Ca2+]i because of its sensitivity and specificity, and because it can be loaded into living cells with little disruption of function. Most importantly, the peak excitation wavelength of fura-2 changes when it binds Ca2+. As a consequence, measurements of fluorescence at two excitation wavelengths can be used to obtain an estimate of [Ca2+]i that is independent of dye concentration and cell thickness. Fura-2 acetoxymethyl ester (AM) is a lipid-soluble derivative that is often used because of its ability to pass through cell membranes. There are, however, several problems with the use of fura-2 AM such as intracellular compartmentation and incomplete deesterification. The availability of low-light-level cameras and computer hardware for the digitization of fluorescent images has made quantitative fluorescence microscopy possible. This technique has shown a striking spatial heterogeneity of [Ca2+]i in a variety of cell types, and has revealed substantial new information on dynamic intracellular biochemistry and signal transduction. However, the current imaging technology is not fully developed because of dye and instrumentation limitations. Further development of techniques and new probes are required to improve temporal and spatial resolution.

Animals↗

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↗

PubMed1999

Live cell fluorescence imaging of T cell MEKK2: redistribution and activation in response to antigen stimulation of the T cell receptor.

T cell activation requires engagement of the T cell receptor (TCR) at the interface of conjugates formed with antigen-presenting cells. TCR engagement is accompanied by a redistribution of specific signaling molecules to the cytoplasmic region of the TCR complex. In this study, immunocytochemistry and live cell fluorescence imaging demonstrate that T cell MEK kinase 2 (MEKK2) is translocated to the T cell/antigen-presenting cell interface in response to antigen activation. MEKK2 translocation occurs more rapidly as the antigen concentration is increased. Biochemical activation of MEKK2 follows TCR stimulation, and expression of a dominant-negative MEKK2 inhibits TCR-mediated conjugate stabilization and ERK and p38 MAP kinase phosphorylation. Live cell fluorescence imaging thus enables characterization of signal transducers that are dynamically translocated following TCR engagement.

Androstadienes↗

PubMed2005

High-resolution Hadamard transform microscope fluorescence imaging: quantifying the DNA content in single cells.

This study presents a novel miniaturized Hadamard transform fluorescence imaging microscope, by combining a conventional fluorescence microscope with Hadamard transform multiplexing encoding using a one-dimensional movable mask to realize spatial resolution and a linear CCD for multichannel detection. The microscope can provide high-resolution automatically-generated 0-255 gray level HT images for morphological analysis and visualization of a single cell, and normalized HT images for cellular quantitative measurements. The microscope's imaging capability was applied to measure the DNA content in human lymphocyte, chicken erythrocyte and eel erythrocyte, and a comparative study was performed. The results show that the calibrated DNA content in a chicken erythrocyte is 2.32 pg when human lymphocyte is used as the standard, and eel erythrocyte may be a potentially reliable and novel standard for determining DNA contents in other species because it has a stable DNA value of 2.06 pg, with a CV of 4.3% when 20 eel erythrocytes are measured. The results also demonstrate that the HT imaging microscope should be valuable in the fields of medicine and cell biology.

DNA↗

PubMed1999

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability.

A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.

Animals↗

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↗

PubMed2006

Fluorescence imaging of cardiac propagation: spectral properties and filtering of optical action potentials.

Fluorescence imaging using voltage-sensitive dyes is an important tool for studying electrical propagation in the heart. Yet, the low amplitude of the voltage-sensitive component in the fluorescence signal and high acquisition rates dictated by the rapid propagation of the excitation wave front make it difficult to achieve recordings with high signal-to-noise ratios. Although spatially and temporally filtering the acquired signals has become de facto one of the key elements of optical mapping, there is no consensus regarding their use. Here we characterize the spatiotemporal spectra of optically recorded action potentials and determine the distortion produced by conical filters of different sizes. On the basis of these findings, we formulate the criteria for rational selection of filter characteristics. We studied the evolution of the spatial spectra of the propagating wave front after epicardial point stimulation of the isolated, perfused right ventricular free wall of the pig heart stained with di-4-ANEPPS. We found that short-wavelength (<3 mm) spectral components represent primarily noise and surface features of the preparation (coronary vessels, fat, and connective tissue). The time domain of the optical action potential spectrum also lacks high-frequency components (>100 Hz). Both findings are consistent with the reported effect of intrinsic blurring caused by light scattering inside the myocardial wall. The absence of high-frequency spectral components allows the use of aggressive low-pass spatial and temporal filters without affecting the optical action potential morphology. We show examples where the signal-to-noise ratio increased up to 150 with <3% distortion. A generalization of our approach to the rational filter selection in various applications is discussed.

Action Potentials↗

PubMed2006

Wavelength division scanning for two-photon excitation fluorescence imaging.

We investigate wavelength division scanning for two-photon excitation fluorescence imaging. Two-photon imaging using lateral wavelength division scanning is demonstrated. In addition, we theoretically analyse the spatial and temporal properties of a femtosecond laser beam focused by a Fresnel lens and investigate the feasibility of axial scanning using wavelength division.

Microscopy, Confocal↗

PubMed2004

Time-wavelength two-dimensional femtosecond fluorescence imaging.

We report a new femtosecond time-resolved fluorescence spectrometer that enables us to observe fluorescence intensity as a time-wavelength two-dimensional image in a single measurement. This method utilizes a time-to-space conversion technique and fluorescence sum-frequency mixing with a femtosecond gate pulse. It provides a fluorescence image covering temporal and spectral spans of approximately 2 ps and approximately 60 nm, respectively. Calibration of the time and intensity axes of the image is made by use of a long-lived dye fluorescence. The two-dimensional fluorescence image of beta-carotene obtained demonstrates the high potential of this method for quantitative studies of ultrafast excited-state dynamics.

Journal Article↗

PubMed1997

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies.

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK1 cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144. 15-37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K+, Na+ and Ca2+). Structurally damaged cells revealed lower K+ and higher Na+ and Ca2+ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.

Animals↗

PubMed1998

Clinical multi-colour fluorescence imaging of malignant tumours--initial experience.

PURPOSE: The detection of malignant tumours relies on a variety of diagnostic procedures including X-ray images and, for hollow organs, endoscopy. The purpose of this study was to present a new technique for non-invasive tumour detection based on tissue fluorescence imaging. MATERIAL AND METHODS: A clinically adapted multi-colour fluorescence system was employed in the real-time imaging of malignant tumours of the skin, breast, head and neck region, and urinary bladder. Tumour detection was based on the contrast displayed in fluorescence between normal and malignant tissue, related to the selective uptake of tumour-marking agents, such as haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), and natural chromophore differences between various tissues. In order to demarcate basal cell carcinomas of the skin, ALA was applied topically 4-6 h before the fluorescence investigation. For urinary bladder tumour visualisation (transitional cell carcinoma of different stages including carcinoma in situ), ALA was instilled into the bladder 1-2 h prior to the study. Malignant and premalignant lesions in the head and neck region were imaged after i.v. injection of HPD (Photofrin). Finally, the extent of in situ and invasive carcinomas of the breast was investigated in surgically excised specimens from patients that received a low-dose injection of HPD 24 h prior to the study. The tumour imaging system was coupled to an endoscope. Fluorescence light emission from the tissue surface was induced with 100-ns-long optical pulses at 390 nm, generated from a frequency-doubled alexandrite laser. With the use of special image-splitting optics, the tumour fluorescence, intensified in a micro-channel plate, was imaged in 3 selected wavelength bands. These 3 images were processed together to form a new optimised-contrast image of the tumour. This image, updated at a rate of about 3 frames/s, was mixed with a normal colour video image of the tissue. RESULTS: A clear demarcation from normal surrounding tissue was found during in vivo measurements of superficial bladder carcinoma, basal cell carcinoma of the skin, and leukoplakia with dysplasia of the lip, and in in vitro investigations of resected breast cancer. CONCLUSIONS: The initial clinical experience of using multi-colour fluorescence imaging has shown that the technique has the potential to reveal malignant tumour tissue, including non-invasive early carcinoma and also precancerous tissue. Further investigations are needed to fully develop the method.

Adult↗

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↗

PubMed1994

Measurement of absolute tracer concentrations in tissue sections by using digital imaging fluorescence microscopy. Application to the study of plasma protein uptake by the arterial wall.

Digital imaging fluorescence microscopy (DIFM) of tissue sections was used to quantify uptake of labelled plasma proteins by the arterial wall. Several aspects of the measuring system were investigated so that absolute tracer concentrations and their local variation could be derived from digitized images. These investigations may be relevant to other studies employing DIFM. Nonlinearities were found to arise from offsets in the video digitizers, from background fluorescence and stray light within the microscope and from the transfer characteristics of the intensified CCD camera. Camera gain controls showed complex behaviour. Camera output fell substantially for several hours after switching on and was affected by room temperature. Large spatial variations in response were caused by the geometry of the microscope optics and by the image intensifier. However, the ratios between areas were not affected by light intensity or camera gain settings. Measured intensities were independent of the depth-wise location of fluorophores within tissue sections but they were affected by the emission from objects outside the measuring area. Photobleaching of tracer varied significantly over the range of excitation intensities and durations used but was not concentration dependent. Methods used to correct these effects and obtain a uniform, linear and constant relationship between concentration and grey level are described. Using the system and appropriate corrections, in vivo uptake of sulphorhodamine-B-labelled serum albumin by the rabbit aortic wall was investigated. Results obtained for the mean uptake of tracer and its local variation were quantitatively similar to those previously obtained with nonmicroscopic methods.

Animals↗

PubMed2006

Multicolor fluorescence imaging for early detection of the hypersensitive reaction to tobacco mosaic virus.

The physiological status of plants can nowadays be promptly monitored with non-invasive methods. This opens the possibility to continuously follow-up plant performance and permits to detect stress-induced deviations presymptomatically. Upon stress, plants may synthesize specific compounds, depending on the causal agent. Such compounds may alter the absorption of the light impinging on plant leaves, hence the spectrum of reflected, re-emitted, and transmitted light changes. UV-excited fluorescence imaging specifically allows visualization of the accumulation of phenolic compounds, e.g. those associated with the hypersensitive response to pathogens. By using imaging at regular intervals (time-lapse series) of tobacco mosaic virus (TMV) infection in resistant tobacco we aimed at the description and quantification of the kinetics of blue-green fluorescence compared to the visual development of the disease. Presymptomatic responses to TMV infection were observed with a multicolor fluorescence and reflectance imaging setup. The onset of increases in blue-green and chlorophyll fluorescence were comparable in timing, although further symptom development was strikingly different. Compounds known to accumulate during the hypersensitive response and displaying blue-green fluorescence revealed different dynamics of fluorescence evolution in time. The multichannel imaging system permitted to discern the key components salicylic acid and scopoletin. In contrast, for the compatible interaction between TMV and non-resistant tobacco, no presymptomatic responses were detected on inoculated leaves. This work proves the potential of multispectral imaging to unveil stress-associated signatures, and the power of blue-green fluorescence imaging to monitor accumulation of secondary compounds.

Fluorescence↗

PubMed2005

Fluorescence imaging with one nanometer accuracy: application to molecular motors.

We introduce the technique of FIONA, fluorescence imaging with one nanometer accuracy. This is a fluorescence technique that is able to localize the position of a single dye within approximately 1 nm in the x-y plane. It is done simply by taking the point spread function of a single fluorophore excited with wide field illumination and locating the center of the fluorescent spot by a two-dimensional Gaussian fit. We motivate the development of FIONA by unraveling the walking mechanism of the molecular motors myosin V, myosin VI, and kinesin. We find that they all walk in a hand-over-hand fashion.

Biophysical Phenomena↗