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PubMed2005

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins.

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.

Light↗
PubMed2003

Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium.

In recent years, fluorescence microscopy based on two-photon excitation has become a popular tool for biological and biomedical imaging. Among its advantages is the enhanced depth penetration permitted by fluorescence excitation with the near-infrared photons, which is particularly attractive for deep-tissue imaging. To fully utilize two-photon fluorescence microscopy as a three-dimensional research technique in biology and medicine, it is important to characterize the two-photon imaging parameters in a turbid medium. We investigated the two-photon point spread functions (PSFs) in a number of scattering samples. Gel samples containing 0.1-microm fluorescent microspheres and Liposyn III were used as phantoms mimicking the turbid environment often found in tissue. A full characterization of the two-photon PSFs of a water and oil immersion objective was completed in samples composed of 0, 0.25, 0.5, 1, and 2% Liposyn III. Our results show that up to depths of about 100 (oil) and 200 microm (water), the presence of scatterers (up to 2% Liposyn III) does not appreciably degrade the PSF widths of the objectives.

Computer Simulation↗
PubMed1994

Detection of Charcot-Leyden crystals by fluorescence microscopy of Papanicolaou-stained smears of sputum, bronchoalveolar lavage fluid, and bronchial secretions.

Fluorescence microscopy was used to examine Papanicolaou-stained smears of sputum and other secretions from the respiratory tract. Under these conditions Charcot-Leyden crystals (CLC) appear as bright yellow-green fluorescing needles. The study was performed to determine the value of this approach for the diagnosis of allergic lung diseases. The time taken to detect the crystals was recorded and the sensitivity of fluorescence microscopy for the detection of CLC was compared with light microscopy of the same samples. The data show that fluorescence microscopy is superior to light microscopy for the detection of CLC. The characteristic needle-shaped crystal can be recognized easily and fragments of crystals could be easily identified. In doubtful cases of allergic lung diseases, fluorescence microscopy may be used to supplement light microscopy for the detection of Charcot-Leyden crystals.

Adult↗
PubMed1984

Persistence of Nosema locustae Spores in Soil as Determined by Fluorescence Microscopy.

Nosema locustae, a protozoan parasite of grasshoppers, is used as a bioinsecticide. In the present study, the persistence of N. locustae spores in soil and the interaction of these spores with the indigenous soil microflora were examined with various forms of microscopy and staining. Fluorescence microscopy was found to be better than phase-contrast or bright-field microscopy for detecting and viewing spores in soil. Fluorescein isothiocyanate was a better fluorescent stain than acridine orange or fluorescein diacetate; water-soluble aniline blue did not stain spores. The eight bright-field microscopy stains tested (phenolic erythrosin, phenolic rose bengal, malachite green, crystal violet, safranin, Congo red, methyl red, and eosin B) were not satisfactory, as spore staining characteristics were either poor or masked by overstained soil debris. A procedure was developed which allowed spores to be extracted from soil with a peptone-phosphate buffer, recovered on a membrane filter, and stained with fluorescein isothiocyanate for microscopic counting. This procedure was used to assess the persistence of N. locustae spores in field and laboratory soils. The number of N. locustae spores in a laboratory model soil system persisted at a high level for over 8 weeks when the soil was incubated at 5 degrees C but exhibited a 1,000-fold decrease after 1 week of incubation at 27 degrees C. Persistence was related to the temperature-dependent activity of the indigenous soil microflora, which, on the basis of microscopic observations, appeared to prey on N. locustae spores. N. locustae spores were detected in an N. locustae-treated field soil at a low level consistent with the level for laboratory soil incubated at 27 degrees C, and they persisted at this level for over 2 months. No spores were detected on vegetation from this field or in the soil from an adjacent, nontreated control field. N. locustae-like spores were also detected in soil from nontreated fields supporting large grasshopper populations.

Journal Article↗
PubMed2004

Liquid domains in vesicles investigated by NMR and fluorescence microscopy.

We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Antigen receptor-mediated calcium signals in B cells as revealed by confocal fluorescence microscopy.

A confocal fluorescence microscope was used to study the antigen receptor-mediated calcium signals in B cells. Anti-IgD binding to B lymphoma cells (BAL17) increased the intracellular calcium concentration with short lag times. Confocal fluorescence images of the fluo-3-loaded BAL17 cells showed that the intracellular calcium ion concentrations increased non-homogeneously, suggesting that the calcium signals transferred not only to the cytoplasm but also to the nucleus.

Antibodies↗
PubMed2004

Simultaneous two-photon spectral and lifetime fluorescence microscopy.

When a fluorescence photon is emitted from a molecule within a living cell it carries a signature that can potentially identify the molecule and provide information on the microenvironment in which it resides, thereby providing insights into the physiology of the cell. To unambiguously identify fluorescent probes and monitor their physiological environment within living specimens by their fluorescent signatures, one must exploit as much of this information as possible. We describe the development and implementation of a combined two-photon spectral and lifetime microscope. Fluorescence lifetime images from 16 individual wavelength components of the emission spectrum can be acquired with 10-nm resolution on a pixel-by-pixel basis. The instrument provides a unique visualization of cellular structures and processes through spectrally and temporally resolved information and may ultimately find applications in live cell and tissue imaging.

Animals↗
PubMed2006

[In vivo fluorescent microscopy of the liver microcirculation after embolization with a newly developed trans-proper hepatic arterial infusion animal model].

OBJECTIVE: To evaluate the value of the in vivo fluorescent microscopy in studying the changes of liver microcirculation after embolization with a newly developed animal model for tans-proper hepatic arterial infusion, and to summarize the method of making this animal model. METHODS: Ten Sprague-Dawley rats were used in this study. After a midline abdominal incision, microcatheter was placed into the gastroduodenal artery (GDA). The tip of the catheter was placed facing the orifice of proper hepatic artery. After infusions of 0.02%, 0.1%, 0.5%, 1% fluorescent sodium, fluorescent microscopy was used to evaluate the liver microcirculation. The image quality was then accessed. Embolization was obtained by injections of Lipiodol and Degradable Starch Microspheres (DSM) from the microcatheter. Corresponding changes of the liver microcirculation was evaluated by fluorescent microscopy. RESULTS: From the 10 rats, 8 animal models were successfully established. The microcirculation of the liver could be clearly visualized by the fluorescent microscopy. The optimal concentration of fluorescent sodium was 0.1%. The direct and indirect phenomena caused by embolic material could be evaluated by fluorescent microscopy. CONCLUSION: Fluorescent microscopy with the corresponding Trans-hepatic arterial infusion animal model is a valuable method to evaluate the microcirculation of the liver and can be used for the evaluation of changes of liver microcirculation caused by embolization material.

Animals↗
PubMed1996

Comparative analysis of CD4-mediated down-regulation of T cell adhesion to B cells by flow cytometry and fluorescence microscopy.

We have previously shown by means of fluorescence microscopy that antigen-independent adhesion of resting CD4 T cells to EBV-transformed B cells can be down-regulated by ligand interaction with CD4. In this study we used flow cytometry analysis of conjugate formation to confirm these findings. No conjugates between resting CD4 + T cells and B cells were initially detected in the latter method, because flow velocity in the flow chamber induces hydrodynamic elongation forces which disrupt low-affinity conjugates. After forcing cell conjugation by low-speed centrifugation of T and B cells, conjugates became detectable although in smaller numbers than in fluorescence microscopy. "Forced" cell conjugates had similar characteristics to their unforced counterparts, i.e., 37 degrees C temperature dependency, mediation by LFA-1/ICAM-1 and CD2/LFA-3 pathways, and transiency. The latter characteristic was at least partly mediated by CD4/HLA class II interaction, since adhesion of CD4 + T cells to HLA class II-B cells was more stable. In addition, adhesion was inhibited by anti-CD4 antibodies but not by an HLA DR-derived peptide known to inhibit unforced CD4 + T cell adhesion to B cells. This blocking effect was partially reproduced by reducing the centrifugation time prior to the adhesion assay. These results show that a) CD4-mediated down-regulation of T cell adhesion can be observed by means of two different techniques, and b) analysis of cell-cell adhesion after increasing centrifugation times (and possibly speeds) is a simple way of measuring adhesion forces semiquantitatively.

Adult↗
PubMed1990

Location of nuclear antigen(s) recognized by DSB389 MAb, a monoclonal antibody against desmin, observed by confocal laser scanning fluorescence microscopy.

The location of antigen(s) of DSB389 MAb, a monoclonal antibody which was raised against an intermediate filament protein, desmin, was investigated in HeLa S3 cells by indirect immuno-electron microscopy and by confocal laser scanning fluorescence microscopy. At interphase, the antigen(s) locates mainly in the intra-nuclear space adjacent to chromatin and sometimes near the nuclear periphery. At mitosis, they locate at the periphery of the chromosomes -first at each chromosome, then around the mass of chromosomes. The antigen(s) are thought to be a component of one type of nuclear matrix which is present outside both chromatin and chromosomes and which has some structural role(s) in organizing them in the nucleus or in the nuclear region.

Antibodies, Monoclonal↗
PubMed1989

Characterization of internalization and endosome formation of epidermal growth factor in transfected NIH-3T3 cells by computerized image-intensified three-dimensional fluorescence microscopy.

Computerized image-intensified fluorescence microscopy has been used to quantify routing and subcellular concentrations of rhodaminated EGF (Rh-EGF) during its receptor-mediated endocytosis in two transfected NIH-3T3 cell lines expressing 2 X 10(5) and 1.5 X 10(6) receptors per cell, respectively. A series of images were digitized by focusing at different depths through the volume of a single cell. The digitized pictures were corrected for fluorescence photobleaching, and removal of out-of-focus fluorescence contributions by deconvolution using the point spread function of the microscope optics (Agard, D. A., and J. W. Sedat. 1980. Proc. Soc. Photo-Opt. Instr. Eng. 264:110-117) allowed automatic computer analysis of the time dependence of endosomal vesicle size and fluorescence intensity in a live cell and also enabled the study of isolated vesicles. An increase in the amount of fluorescence bound to the cell surface, either by increasing the number of receptors expressed per cell or the concentration of Rh-EGF in the incubation drop, yielded an increase in the total fluorescence of internalized vesicles without an increase in their number and area. The linear relation between fluorescence intensity and area for vesicles at different times indicates that EGF concentration is conserved. This is compatible with fusion of small vesicles to form larger ones. However, as endocytosis proceeds, a twofold increase in the slope of the fluorescence vs. area plots is observed for larger vesicles, suggesting that active sorting causes the EGF to be concentrated. Alternatively, this factor could be produced by cumulative fluorescence contributions from stacked membranes. Since coated pits are internalized independent of their occupancy with EGF receptor, we propose that endocytosis does not involve a mechanism specifically recognizing occupied receptor but is rather triggered by a global intracellular event.

Animals↗
PubMed2006

Phototoxicity and fluorotoxicity combine to alter the behavior of neutrophils in fluorescence microscopy based flow adhesion assays.

The use of fluorescent probes that allow visualization of leukocyte-endothelial cell (EC) interactions has greatly informed our understanding of leukocyte recruitment. However, effects of these agents on the biological functions of leukocytes are poorly described, leading to concerns about the interpretation of such data. Here we used two flow-based neutrophil adhesion assays to compare the effects of phase contrast illumination (PCI) with high intensity illumination (HII) used for fluorescent microscopy, in the presence or absence of five commonly used fluorochromes. Isolated neutrophils were either (1) perfused across P-selectin to establish a population of rolling cells, which were subsequently activated with fMLP; or (2) perfused across EC activated with TNF-alpha. In the absence of fluorescent dyes, HII did not affect levels of leukocyte adhesion; however, subsequent neutrophil behavior was dramatically altered when compared with cells under PCI, for example, dramatically reducing their migration velocities. In the presence of fluorescent dyes, the effects of HII were exacerbated, although the precise nature of the biological effects of these probes was agent specific. Thus, for the first time, our experiments describe the effects of fluorescent microscopy on the separate stages of the neutrophil recruitment process and reveal a previously unsuspected effect of HII on neutrophil migration.

Cell Adhesion↗
PubMedDate not supplied

Fluorescence microscopy in the detection of early myocardial infarction.

Fluorescent microscopy is amongst the many techniques devised for the post-mortem detection of early myocardial infarction. The method has the advantage of sensitivity, speed and simplicity, compared with more complex techniques such as enzyme histochemistry and electron microscopy. Disadvantages include the difficulty of permanent preservation of the sections and lack of sharp differentiation between normal and very early infarction--though the latter problem is common to most other methods, except enzyme histochemistry. There are two main techniques in fluorescent microscopy: The examination of sections specifically stained by fluorochrome dyes; The re-examination of haematoxylin-eosin sections for 'autofluorescence'.

Acridine Orange↗
PubMed1976

Study on development of Agaricus bisporus by fluorescent microscopy and scanning electron microscopy.

Two strains of Agaricus bisporus have been investigated by fluorescent microscopy and scanning electron microscopy. Somatic nuclei, stained with auramin O and examined by fluorescent microscopy, appear to be randomly distributed, divide asynchronously, and assume a nonclassical or "two-track" configuration during mitotic metaphase. A similar configuration has been observed for nuclei during meiosis, but early meiosis in A. bisporus appears to be classical, usually with nine pairs of chromosomes evident during prophase I. Scanning electron microscopy has been used to document developmental stages in the formation and germination of basidiospores. Two-spored basidia were predominant, but occasionally one- or three-spored forms were observed. Four-spored basidia were absent, and uninucleated basidiospores were exceedingly rare to absent.

Basidiomycota↗
PubMed1979

Fluorescence microscopy for detection of M. leprae in tissue sections.

The fluorescence method was compared with the Fite-Faraco method for detecting acid-fast microorganisms in paraffin sections of cases of leprosy. Biopsies were obtained from 50 cases of leprosy covering all varieties and at varying stages of treatment. The fluorescence method was better than the Fite-Faraco method; 22 biopsies showing acid-fast organisms in fluorescence microscopy and 20 in the Fite-Faraco method. Its superiority was evidenced in two cases in which the organisms were very scanty. Fluorescence microscopy can also be used to determine the Bacterial Index and the Morphologic Index of organisms. The Morphologic Index, however, was one and a half times higher than that obtained by the Fite-Faraco technic. The ease and speed of fluorescence microscopy appear to be a great advantage.

Diagnosis, Differential↗
PubMed1990

Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using single-wavelength fluorescence microscopy.

The purpose of this study was to use fluorescence microscopy to determine the viability and acrosome status of fresh and frozen-thawed human spermatozoa. Sperm cells were stained with the viability stains Hoechst 33258 (H33258) alone, or propidium iodide (PI) alone, and PI in combination with FITC-conjugated Pisum sativum agglutinin (PSA). The PSA stains the acrosome contents of permeabilized acrosome-intact sperm. Viability by fluorescence microscopy was compared to conventional eosin nigrosin staining. The overall viability using H33258 was not significantly different from that using PI. Therefore, PI was used in combination with PSA for simultaneous measurement of viability and acrosome status at the same excitation wavelength (488 nm). By combining PI and PSA, four subgroups of cells could be detected: group I, PI-neg/PSA-neg--viable, physiologic acrosome reacted (AR); group II, PI-neg/PSA-pos--viable, non-AR; group III, PI-pos/PSA-neg--nonviable, non-AR; group IV, PI-pos/PSA-neg--nonviable, degenerative AR. The postthaw sperm exhibited a significantly greater percent of sperm that were acrosome reacted (both viable and degenerative) (groups I and IV) than the fresh semen. We conclude that frozen-thawed sperm may undergo premature break-down of the acrosome prior to interaction with the oocyte, thus explaining the reduced fertility potential of cryopreserved semen.

Acrosome↗
PubMedDate not supplied

In situ observation of molecular diffusion in solid supports using two-photon fluorescence microscopy.

We demonstrate that two-photon fluorescence microscopy is a useful tool for monitoring the diffusion of molecules through polymeric solid supports. As a proof of principle, TentaGel beads were bound to a cover slip, and diffusion of Rhodamine 6G through single beads was observed in real time. Diffusion experiments performed in 40 different beads indicate that there is a considerable degree of heterogeneity in diffusion rates from bead to bead.

Color↗