[Fluorescence microscopy and fluorescence microphotography of neuroma, myoma and fibroma of the digestive tract].
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Digestive System Neoplasms↗
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Cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. A study using simultaneous scanning electron microscopy and fluorescence microscopy.
An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied immediately thereafter with the SEM part of the instrument. The cells were divided into six groups according to the number of microvilli and/or microridges present on their surface. A proportion of macrophages showed a DNA content more than occurs in diploid cells, which could indicate a future division. No special surface morphology could be detected in this cell type.
Animals↗
DNA fragmentation assessment by flow cytometry and Sperm-Bos-Halomax (bright-field microscopy and fluorescence microscopy) in bull sperm.
The aim of this study was to find the relationship between fertility (as 90-day non-return rates) and DNA fragmentation assessed by two techniques [sperm chromatin structure assay (SCSA) and Sperm-Bos-Halomax (SBH)]. Furthermore, other quality parameters were achieved (motility, morphological abnormalities, cytoplasmic droplets, viability, capacitation and acrosomal and mitochondrial status) and their correlations with fertility were analysed. Bulls were divided into three fertility groups: high [non-return rate (NRR) >or= 80], medium (80 < NRR >or= 70) and low (70 < NRR > 40). The results of this study indicate that there is a good correlation between fertility and different parameters of sperm quality (SBH and SCSA parameters, % of spermatozoa with head, neck and total abnormalities, and % of spermatozoa with proximal cytoplasmic droplets) and differences between fertility groups were observed in some of them (SBH and SCSA parameters and % of spermatozoa with head, neck and total abnormalities). In this sense, SBH parameters rendered good correlations with fertility (r = -0.42 using bright light microscope and r = -0.47 with fluorescence). Also, standard deviation of DNA fragmentation index (SD-DFI) and DFIh (cells with High DNA fragmentation index) showed good correlations with fertility (r = -0.41 and r = -0.29). No correlations were observed between SCSA and SBH parameters. A multiple regression shows that four parameters (% of proximal cytoplasmic droplets, % of intact acrosomes in total population, SD-DFI and percentage of fragmented DNA detected by bright light microscope) present a good predictive value of the fertility of sperm samples (r(2) = 0.34, p < 0.001).
Acrosome↗
Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments.
Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.
Adsorption↗
Determination of the chelatable iron pool of isolated rat hepatocytes by digital fluorescence microscopy using the fluorescent probe, phen green SK.
The intracellular pool of chelatable iron is considered to be a decisive pathogenetic factor for various kinds of cell injury. We therefore set about establishing a method of detecting chelatable iron in isolated hepatocytes based on digital fluorescence microscopy. The fluorescence of hepatocytes loaded with the fluorescent metal indicators, phen green SK (PG SK), phen green FL (PG FL), calcein, or fluorescein desferrioxamine (FL-DFO), was quenched when iron was added to the cells in a membrane-permeable form. It increased when cellular chelatable iron available to the probe was experimentally decreased by an excess of various membrane-permeable transition metal chelators. The quenching by means of the ferrous ammonium sulfate + citrate complex and also the "dequenching" using 2,2'-dipyridyl (2,2'-DPD) were largest for PG. We therefore optimized the conditions for its use in hepatocytes and tested the influence of possible confounding factors. An ex situ calibration method was set up to determine the chelatable iron pool of cultured hepatocytes from the increase of PG SK fluorescence after the addition of excess 2,2'-DPD. Using this method, we found 9.8 +/- 2.9 micromol/L (mean +/- SEM; n = 18) chelatable iron in rat hepatocytes, which constituted 1.0% +/- 0.3% of the total iron content of the cells as determined by atomic absorption spectroscopy. The concentration of chelatable iron in hepatocytes was higher than the one in K562 cells (4.0 +/- 1.3 micromol/L; mean +/- SEM; n = 8), which were used for comparison. This method allowed us to record time courses of iron uptake and of iron chelation by different chelators (e.g., deferoxamine, 1,10-phenanthroline) in single, intact cells.
Animals↗
Direct observation of frits and dynamic air bubble formation in capillary electrochromatography using confocal fluorescence microscopy.
Confocal fluorescence microscopy has been used to study the capillary electrochromatography (CEC) frits and dynamic air bubble formation under real chromatographic conditions. Confocal fluorescence microscopy provides a nondestructive way to view the three-dimensional structure of the frits with high spatial resolution. Frits prepared with four different procedures were studied: (1) sintering bare silica beads with sodium silicate; (2) sintering bare silica beads wetted with water; (3) sintering C18 beads wetted with water; and (4) sintering C18 beads wetted with water and then surfaced-recovered with C18. Frits prepared with sintering silicate-wetted beads have a high degree of heterogeneity, while the other three types of frits have similar, more homogeneous packing structures. Confocal fluorescence microscopy also provides sufficient temporal resolution for in situ observation of the dynamic processes in air bubble formation. In this study, air bubble formation is imaged during the reorganization process of the packing bed and is shown to occur close to the border between the packing bed and the outlet frit. Confocal fluorescence microscopy opens a new avenue in studying dynamic processes in situ in CEC separations.
Chromatography, Micellar Electrokinetic Capillary↗
Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy.
The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide-field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light-induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY-2 cell physiology. Our results show a non-linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY-2 cells loaded with oxidation-sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non-linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage.
Cells, Cultured↗
Chronology of cellular alterations during 7-ketocholesterol-induced cell death on A7R5 rat smooth muscle cells: analysis by time lapse-video microscopy and conventional fluorescence microscopy.
BACKGROUND: Time-lapse video microscopy was used to determine whether mitochondrial and nuclear changes (decrease in mitochondrial transmembrane potential, condensation, and/or fragmentation of the nuclei, morphologic features typical of apoptosis) occurring during 7-ketocholesterol-induced cell death on A7R5 rat smooth muscle cells took place before or after the loss of cell adhesion. In addition, changes in actin organization were followed by conventional fluorescence microscopy. METHODS: Morphologic, functional, and spatial changes at the mitochondrial level were investigated with 3,3'-dihexyloxacarbocyanine iodide and/or MitoTracker Red, and nuclear morphology was characterized by staining with Hoechst 33342. Actin fibers, which are major components of the filament network of the cytoskeleton, were visualized with phalloidin linked to fluorescein. The numbers of adherent and nonadherent cells were determined by cell counting. RESULTS: 7-Ketocholesterol-induced cell death was associated with a rapid alteration of actin fibers, a loss of intercellular junctions, and cell shape modifications. Analysis of mitochondrial transmembrane potential showed successively a hyperpolarization and a more or less pronounced progressive decrease followed by a dramatic drop associated with an increase in Hoechst 33342 staining, reflecting chromatin condensation and morphologic changes in the nuclei. CONCLUSIONS: During cell death induced by 7-ketocholesterol in A7R5 rat smooth muscle cells, the different methods of microscopy allowed us to establish that alterations of actin fibers and mitochondrial dysfunctions occurred before condensation and/or fragmentation of the nuclei, which preceded the loss of cell adhesion.
Actins↗
Fluorescence microscopy.
Although fluorescence microscopy permeates all of cell and molecular biology, most biologists have little experience with the underlying photophysical phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.
Animals↗
New modal wave-front sensor: application to adaptive confocal fluorescence microscopy and two-photon excitation fluorescence microscopy.
Confocal and multiphoton microscopes are particularly sensitive to specimen- or system-induced aberrations, which result in decreased resolution and signal-to-noise ratio. The inclusion of an adaptive optics correction system could help overcome this limitation and restore diffraction-limited performance, but such a system requires a suitable method of wave-front measurement. By extending the concept of a modal wave-front sensor previously described by Neil et al. [J. Opt. Soc. Am. A 17, 1098-1107 (2000)], we present a new sensor capable of measuring directly the Zernike aberration modes introduced by a specimen. This modal sensor is particularly suited to applications in three-dimensional microscopy because of its inherent axial selectivity; only those wave fronts originating in the focal region contribute to the measured signal. Four wave-front sensor configurations are presented and their input response is characterized. Sensitivity matrices and axial responses are presented.
Microscopy, Confocal↗
Time-resolved fluorescence microscopy.
In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.
Fluorescence Polarization↗
Intracellular pH and free calcium changes in single cells using quene 1 and quin 2 probes and fluorescence microscopy.
Photometric fluorescence microscopy has been used to measure intracellular pH (pHi) and free calcium concentrations [( Ca]i) in individual mouse thymocytes and 2H3 rat basophil leukaemic cells containing indicators for pH (quene 1) or calcium (quin 2). The pHi and [Ca]i measurements in individual 2H3 cells and mouse thymocytes and their responses to various stimuli were consistent with the corresponding data obtained from suspensions of these cells measured in a spectrofluorimeter. Photometric fluorescence microscopy of these indicators in individual cells provides a sensitive and fast method of following pHi and [Ca]i responses in individual cells.
Aminoquinolines↗
Image calibration in fluorescence microscopy.
A fluorescence image calibration method is presented based on the use of standardized uniformly fluorescing reference layers. It is demonstrated to be effective for the correction of non-uniform imaging characteristics across the image (shading correction) as well as for relating fluorescence intensities between images taken with different microscopes or imaging conditions. The variation of the illumination intensity over the image can be determined on the basis of the uniform bleaching characteristics of the layers. This permits correction for the latter and makes bleach-rate-related imaging practical. The significant potential of these layers for calibration in quantitative fluorescence microscopy is illustrated with a series of applications. As the illumination and imaging properties of a microscope can be evaluated separately, the methods presented are also valuable for general microscope testing and characterization.
Animals↗
Hormone binding by cells and cell fragments as visualized by fluorescence microscopy.
Fluorescent-labeled hormones offer an alternative approach to radio-labeling in studying the binding of hormones to intact cells or cell fragments. The binding of fluorescent-labeled hormones may be followed quantitatively by measurement of the polarization or the binding may be directly visualized in the fluorescence microscope. The binding of both fluorescein labeled prolactin and estradiol to a variety of whole cells or to microsomal fragments has been observed by fluorescence microscopy. No staining was observed with fresh cells whereas all cell types investigated, after freeze-thawing, stained at physiological levels (10-9M) of either hormone. Microsomal preparations from the mammary tissue of mid-pregnant rabbits likewise stained at low levels of prolactin. Inhibition of staining was not produced even by 10-6 M unlabeled hormone.
Cell Line↗
Probing individual molecules with confocal fluorescence microscopy.
Confocal fluorescence microscopy coupled with a diffraction-limited laser beam and a high-efficiency detection system has been used to study the diffusive movement and emission process of individual fluorescent molecules in the liquid phase at room temperature. The high detection sensitivity achieved at fast data acquisition speeds (greater than 1 kilohertz) allows real-time observation of single-molecule fluorescence without statistical analysis. The results show fluorescence-cycle saturation at the single-molecule level and multiple recrossings of a single molecule into and out of the probe volume as well as the triplet state.
Fluorescein↗
Fluorescence microscopy today.
Fluorescence microscopy has undergone a renaissance in the last decade. The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues. The impact of these and other new imaging methods in biophysics, neuroscience, and developmental and cell biology has been remarkable. Further advances in fluorophore design, molecular biological tools and nonlinear and hyper-resolution microscopies are poised to profoundly transform many fields of biological research.
Animals↗
Quantification of low density lipoprotein binding and cholesterol accumulation by single human fibroblasts using fluorescence microscopy.
Using fluorescence microscopy, we have quantified low density lipoprotein (LDL) binding by indirect immunofluorescence and cellular cholesterol with the fluorescent sterol-binding polyene, filipin, in individual cultured human fibroblasts from normal subjects and from patients with heterozygous and homozygous familial hypercholesterolemia. LDL binding by fibroblasts from heterozygous patients was about 40% of that of the normal cells, and cholesterol accumulation upon incubation with LDL was decreased to a similar degree. Most fibroblasts from homozygous patients bound no detectable LDL and only rare cells demonstrated any accumulation of cholesterol after incubation with LDL.
Cells, Cultured↗
Multiphoton fluorescence microscopy.
Multiphoton fluorescence microscopy has now become a relatively common tool among biophysicists and biologists. The intrinsic sectioning achievable by multiphoton excitation provides a simple means to excite a small volume inside cells and tissues. Multiphoton microscopes have a simplified optical path in the emission side due to the lack of an emission pinhole, which is necessary with normal confocal microscopes. This article illustrates examples in which this advantage in the simplified optics is exploited to achieve a new type of measurements. First, dual-emission wavelength measurements are used to identify regions of different phase domains in giant vesicles and to perform fluctuation experiments at specific locations in the membrane. Second, we show how dual-wavelength measurements are used in conjunction with scanning fluctuation analysis to measure the changes in the geometry of the domains and the incipient formation of gel domains when the temperature of the giant vesicles is gradually lowered.
Fluorescence Polarization↗