Detection of acid-fast organisms in tissue sections by fluorescence microscopy.
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Fluorescence↗
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A COMPARATIVE STUDY OF LIGHT AND FLUORESCENT MICROSCOPY IN THE HISTOLOGIC EVALUATION OF MOTOR END PLATES.
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Cell Physiological Phenomena↗
Visualization of a specific sequence on a single large DNA molecule using fluorescence microscopy based on a new DNA-stretching method.
A method for analyzing large DNA which makes it possible to obtain spatial information on the positions of specific sequences along a DNA molecule has been developed. Making use of the fact that large DNA molecules are stably elongated under an alternating-current field in a concentrated linear polymer solution, the direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. The spatial positions of the fluorescent spots of the probe (fluorescence-labeled restriction endonuclease EcoRI) on DNA molecules were determined by image analysis. As expected, fluorescent spots of EcoRI were observed at certain positions on lambda DNA, where sequences to which EcoRI binds are located. Finally, the potential application of single large DNA molecule analysis using this DNA-stretching method is discussed.
Bacteriophage lambda↗
Detection of macrophage activity in atherosclerosis in vivo using multichannel, high-resolution laser scanning fluorescence microscopy.
Molecular and cellular mechanisms of atherogenesis and its treatment are largely being unraveled by in vitro techniques. We describe methodology to directly image macrophage cell activity in vivo in a murine model of atherosclerosis using laser scanning fluorescence microscopy (LSFM) and a macrophage-targeted, near-infrared fluorescent (NIRF) magnetofluorescent nanoparticle (MFNP). Atherosclerotic apolipoprotein E deficient (apoE -/-) mice (n=10) are injected with MFNP or 0.9% saline, and wild-type mice (n=4) are injected with MFNP as additional controls. After 24 h, common carotid arteries are surgically exposed and prepared for LSFM. Multichannel LSFM of MFNP-enhanced carotid atheroma (5x5-microm in-plane resolution) shows a strong focal NIRF signal, with a plaque target-to-background ratio of 3.9+/-1.8. Minimal NIRF signal is observed in control mice. Spectrally resolved indocyanine green (ICG) fluorescence angiograms confirm the intravascular location of atheroma. On ex vivo fluorescence reflectance imaging, greater NIRF plaque signal is seen in apoE -/- MFNP mice compared to controls (p<0.01). The NIRF signal correlates well with immunostained macrophages, both by stained surface area (r=0.77) and macrophage number (r=0.86). The validated experimental methodology thus establishes a platform for investigating macrophage activity in atherosclerosis in vivo, and has implications for the detection of clinical vulnerable plaques.
Animals↗
Site of docking and fusion of insulin secretory granules in live MIN6 beta cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy.
To determine the site of insulin exocytosis in the pancreatic beta cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 beta cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mm KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-beta-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 beta cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.
Animals↗
Recognition errors in the quantification of micro-organisms by fluorescence microscopy.
OBJECTIVES: An interlaboratory comparison of fluorescence microscopic counting of micro-organisms was carried out to assess counting errors in the measurement of micro-organisms in bioaerosols generated during handling of municipal waste. METHODS: Series of 27 replicate samples were collected in the work environment with a modified field exposure chamber. The preparation methods of three Scandinavian laboratories were compared. Four microscopists from these laboratories performed the counts which were also compared. Duplicate counting of identical microscopic fields allowed the assessment of recognition errors. RESULTS: The field exposure chamber collected replicate samples with a relative standard deviation of 5% when particles < or = 15 microm aerodynamic diameter were collected. Storage time of 40-200 days had no significant influence on the total micro-organism count. Differences between preparation methods were from 2 to 35% for bacteria, and from 15 to 35% for fungal spores when samples were analysed in Oslo; the results for fungal spore counts were significantly different (P < 0.01). These differences were not confirmed when samples were analysed in Umeå, Copenhagen and Oslo using those laboratories methods. These results can be explained by less efficient redispersion of aggregates when the Umeå and Copenhagen methods were recreated in Oslo yielding a greater number of innumerable aggregates. Differences between microscopists were minor for fungal spores (2-12%) but substantial for bacteria (4-53%). A major source of error was the recognition of bacteria which had a relative standard deviation (rsd) of 37% although a lower size limit of 0.75 microm was adopted for counting of bacteria. Fungal spores were recognised with much better precision (rsd 9%). CONCLUSIONS: Recognition errors of bacteria may be substantial and more specific fluorochromes are needed for fluorescence microscopic counting of micro-organisms.
Analysis of Variance↗
Effect of several fixatives on the density of concanavalin A binding sites of murine peritoneal macrophages determined by fluorescence microscopy and X-ray microanalysis.
The influence of various fixatives (glutaraldehyde, formaldehyde, OsO4) on the retention of antigenicity of peritoneal macrophages was studied by means of fluorescence microscopy, scanning electron microscopy, and X-ray microanalysis. After fixation with formaldehyde the Concanavalin A (Con A) binding sites were found to be highest, whereas the fixation with OsO4 results in loss of many binding sites. The fluorescence microscopic investigations of glutaraldehyde fixed cells is impaired by fixative induced fluorescence. Therefore X-ray microanalytical determinations show better results for these cells than fluorescence microscopic measurements. On contrary to direct labelling method the number of unspecific Con A binding sites on peritoneal macrophages labelled with the sandwich method is negligible.
Animals↗
Automated recognition of patterns characteristic of subcellular structures in fluorescence microscopy images.
Methods for numerical description and subsequent classification of cellular protein localization patterns are described. Images representing the localization patterns of 4 proteins and DNA were obtained using fluorescence microscopy and divided into distinct training and test sets. The images were processed to remove out-of-focus and background fluorescence and 2 sets of numeric features were generated: Zernike moments and Haralick texture features. These feature sets were used as inputs to either a classification tree or a neural network. Classifier performance (the average percent of each type of image correctly classified) on previously unseen images ranged from 63% for a classification tree using Zernike moments to 88% for a backpropagation neural network using a combination of features from the 2 feature sets. These results demonstrate the feasibility of applying pattern recognition methods to subcellular localization patterns, enabling sets of previously unseen images from a single class to be classified with an expected accuracy greater than 99%. This will provide not only a new automated way to describe proteins, based on localization rather than sequence, but also has potential application in the automation of microscope functions and in the field of gene discovery.
Image Enhancement↗
Application of double staining technique and incident light fluorescence microscopy to immunofluorescence studies on renal diseases.
A method, by which the more precise localization of immunofluorescent deposits in the glomerulus is easily demonstrated with the fluorescence microscopy, was described. By means of this method, the deposits even in the Bowman's space could be identified, as well as the deposits in the subepithelial, subendothlial and mesangial regions. From this result, it was suggested that this method should be widely used for the immunofluorescence studies on human renal biopsied sections, in order to elucidate the relationship between the localization of deposits and renal diseases.
Animals↗
Organization of DNA in dipteran polytene chromosomes as indicated by polarized fluorescence microscopy.
Analysis of the polarization of fluorescence from Drosophila virilis polytene chromosomes stained with acridine orange suggests that the DNA in the interband regions of these chromosomes cannot be in a supercoiled configuration, but must lie parallel to the chromosome axis.
Acridines↗
A comparison of fluorescence microscopy with the Ziehl-Neelsen technique in the examination of sputum for acid-fast bacilli.
SETTING: National Tuberculosis Reference Laboratory in Dakar, Senegal. OBJECTIVES: Comparison of results with fluorescence and bright-field microscopy for acid-fast bacilli. METHODOLOGY: Two smears from 2,630 consecutive sputum specimens between January 1996 and June 1998 were prepared for blinded examination of one smear each by the Ziehl-Neelsen technique and fluorescence microscopy at 1,000x magnification. The time required to declare a slide as negative was determined for both techniques in a sample of 68 slides. RESULTS: Concordancewas 96.9% and 92.3% for diagnostic and follow-up examinations, respectively. The yield was similar with both techniques for specimens with at least 10 bacilli per 100 fields, but higher with fluorescence microscopy in those with fewer than 10 bacilli per 100 fields. The mean time required by fluorescence microscopy before declaring a slide as negative with the same magnification was 3 minutes 34 seconds, compared to 7 minutes 44 seconds with the Ziehl-Neelsen technique. CONCLUSIONS: The results obtained with one technique are highly reproducible by the other. Fluorescence microscopy appears to be more likely to detect bacilli in paucibacillary cases than bright-field microscopy, and it more than halves the required examination time.
Humans↗
Extended resolution wide-field optical imaging: objective-launched standing-wave total internal reflection fluorescence microscopy.
Standing-wave total-internal-reflection fluorescence (SW-TIRF) microscopy uses a super-diffraction-limited standing evanescent wave to extract the high-spatial-frequency content of an object through a diffraction-limited optical imaging system. The effective point-spread function is better than a quarter of the emission wavelength. With a 1.45 numerical aperture objective and 532 nm excitation wavelength, a Rayleigh resolution of approximately 100 nm can be achieved, which is better than twice the resolution of conventional TIRF microscopy. This first experimental realization of SW-TIRF in an objective-launched geometry demonstrates the potential for extended resolution imaging at high speed by using wide-field microscopy.
Equipment Design↗
Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations.
BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSC) are the routine techniques for fluorescent cell analysis. Recently, we developed a scanning fluorescent microscopy (SFM) technique. This study compares SFM to LSC (two slide-based cytometry, SBC, techniques) and FCM, in experimental and clinical settings. METHODS: For the relative cell-frequency determinations, HT29 colorectal cancer cells and Ficoll separated blood mononuclear cells (FSBMCs) were serially diluted (from 1:1 to 1:1,000) and measured by each of the three techniques. For the absolute cell number determinations (only for SBC) FSBMCs were smeared on slides, then HT29 cells were placed on the slide with a micromanipulator (5-50 cells). Tumor cells circulating in the peripheral blood were isolated by magnetic separation from clinical blood samples of colorectal cancer patients. All samples were double-stained by CD45 ECD and CAM5.2 FITC antibodies. For slides, TOTO-3 and Hoechst 33258 DNA dyes were applied as nuclear counter staining. RESULTS: In the relative cell frequency determinations, the correlations between the calculated value and measured values by SFM, LSC, and FCM were r(2) = 0.79, 0.62, and 0.84, respectively (for all P < 0.01). In the absolute cell frequency determinations, SFM and LSC correlated to a high degree (r(2) = 0.97; P < 0.01). CONCLUSIONS: SFM proved to be a reliable alternative method, providing results comparable to LSC and FCM. SBC proved to be more suitable for rare-cell detection than FCM. SFM with digital slides may prove an acceptable adaptation of conventional fluorescent microscopes in order to perform rare-cell detection.
Antiporters↗
[FLUORESCENCE MICROSCOPY IN THE STUDY OF ENDOCHONDRAL OSSIFICATION].
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Animals↗
A fluorescent microscopy study of biopsied muscles from infantile neuromuscular disorders.
The Acridine Orange (AO) stain for muscle biopsies is particularly useful to identify regenerating and ongoing hypertrophic muscle fibers under fluorescent microscopy. This method was applied to muscle biopsies from 65 patients who suffered from various childhood neuromuscular disorders. While normal fibers showed dull green cytoplasm with small green-yellow nuclei, striking fluorescent fibers were observed in eight cases of congenital muscular dystrophy (CMD) and 12 cases of Duchenne muscular dystrophy (DMD); these fibers were characterized as follows: (1) small fibers with big oval or spherical nuclei which fluoresced strongly with a bright orange color; (2) fibers of various sizes and different degrees of orange fluorescence; and (3) opaque fibers with bright yellow cytoplasm. The small diameter fibers in Werdnig-Hoffmann (WH) disease, nemaline myopathy, and congenital fiber type disproportion failed to show apparent AO-RNA fluorescence. Although all the atrophic fibers in Kugelberg-Welander (KW) disease showed a vague orange fluorescent color, this was obviously different from that of regenerating fibers seen in CMD and DMD. In addition to these findings, the hypertrophic fibers in a case of unclassified myopathy also showed moderate orange fluorescence around the entire periphery of the cytoplasm.
Adolescent↗
Expression-activity profiles of cells transfected with prostaglandin endoperoxide H synthase measured by quantitative fluorescence microscopy.
Transfection of cos-1 cells with either prostaglandin endoperoxide H synthase-1 (PGHS-1) or -2 (PGHS-2) results in a mixed population of cells containing a diverse range of expressed enzyme. The use of fluorescent substrates and antibodies, in conjunction with fluorescence microscopy, provides the means to quantitate expression and activity of the enzyme within individual cells. Data obtained from individual cells can be utilized to construct enzyme activity curves for a population of transfected cells. This method has been employed to prepare expression-activity profiles within a population of cos-1 cells expressing PGHS-1 or -2. A direct correlation was observed between enzyme expression and activity as measured in single cells. The data demonstrate that activity-expression analyses can now be performed within single adherent cells growing in tissue culture.
Amino Acid Sequence↗
Fluorescence microscopy of fresh tissue as a rapid technique for assessing early injury to mesophyll.
A technique was developed for sectioning fresh red spruce foliage (Picea rubens Sarg.) for use in fluorescence microscopy. This allowed rapid examination of mesophyll in 3-5 mm needle sections. Healthy, ozone treated and cold stressed needles were examined to assess the utility of this technique for early detection of damage. Healthy mesophyll cells fluoresced bright red, while injured cells fluoresced yellow-green in ozone treated needles, and yellow-orange green in ozone treated needles, and yellow-orange in frozen needles. Shifts in fluorescence wave-lengths may be useful for early detection of injury to mesophyll before it is evident by standard light or electron microscopy.
Cryopreservation↗
Localization of D1 dopamine receptors on live cultured striatal neurons by quantitative fluorescence microscopy.
Single neurons in culture express a heterogeneity of neurotransmitter receptor subtypes. The study of the effects of neurotransmitters on neuronal function is complicated by this heterogeneity. It would therefore be useful to be able to identify live neurons that express the receptors of interest and then use these neurons for functional studies. We have used quantitative fluorescence microscopy to identify single live striatal neurons that express D1 dopamine receptors. The binding of the fluorescent D1 dopamine receptor antagonist bodipy-SCH 23390 was measured in 2-3-week-old primary striatal cultures derived from fetal rats (embryonic day 18). Binding of bodipy-SCH 23390 to live neurons was displaced by (+)-butaclamol, dopamine or SCH 23390, indicating that it specifically labelled D1 dopamine receptors. However, the fraction of bodipy-SCH 23390 binding that was specific varied substantially among individual neurons indicating heterogeneity of D1 dopamine receptor expression. Interestingly, bodipy-SCH 23390 also specifically labelled discrete spots of receptors on the neuronal processes. This technique should prove useful in the study of the effects of dopaminergic drugs on neuronal function in primary culture.
Acids↗