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PubMed1993

Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation.

The use of fluorescence microscopy for investigating the three-dimensional structure of cells and tissue is of growing importance in cell biology, biophysics and biomedicine. Three-dimensional data are obtained by recording a series of images of the specimen as it is stepped through the focal plane of the microscope. Whether by direct imaging or by confocal scanning, diffraction effects and noise generally limit axial resolution to about 0.5 microns. Here we describe a fluorescence microscope in which axial resolution is increased to better than 0.05 microns by using the principle of standing-wave excitation of fluorescence. Standing waves formed by interference in laser illumination create an excitation field with closely spaced nodes and antinodes, allowing optical sectioning of the specimen at very high resolution. We use this technique to obtain images of actin fibres and filaments in fixed cells, actin single filaments in vitro and myosin II in a living cell.

3T3 Cells↗
PubMed1994

Miscibility of lipoteichoic acid in dipalmitoylphosphatidylcholine studied by monofilm investigations and fluorescence microscopy.

The miscibility of the bacterial amphiphile lipoteichoic acid, a constituent of the cytoplasmic membrane of Gram-positive bacteria, in dipalmitoylphosphatidylcholine has been investigated by classic monofilm measurements and fluorescence microscopy at the air-water interface of monofilms obtained by spreading mixtures of both amphiphiles on a water subphase. The isotherms indicated miscibility of both lipids at concentrations up to 30 mol% lipoteichoic acid, whereas at higher concentrations immiscibility was detected. Increasing the lateral pressure over a certain value, lipoteichoic acid is squeezed out of the monofilm. By fluorescence microscopy the influence of lipoteichoic acid on the domain shape of condensed dipalmitoylphosphatidylcholine phases has been studied. The balance between hydrophobic and hydrophilic forces in the mixtures of both amphiphiles is discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMedDate not supplied

Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy.

We have developed a procedure for labeling lipoproteins with the fluorescent probe 3,3'-dioctadecylindocarbocyanine (Dil) and have used Dil-labeled native and acetoacetylated lipoproteins to differentiate macrophages from fibroblasts in mixed cell culture. Lipoproteins labeled with this probe were suitable for the direct viewing of their binding and internalization by cells in vitro. The labeling technique has been applied to human low density lipoproteins (LDL) and to two canine cholesterol-induced lipoproteins: apo-E HDLc, which contain only the E apoprotein (apo-E), and beta-migrating, very low density lipoproteins (beta-VLDL), which contain apo-B and apo-E. The Dil-labeled lipoproteins showed specific high affinity binding to human fibroblasts via the LDL (apo-B, -E) receptors. The equilibrium dissociation constant for the binding of Dil-labeled apo-E HDLc and LDL were the same as for the respective native lipoproteins. The specific binding of Dil-labeled LDL and apo-E HDLc was further substantiated by fluorescence microscopy. When an excess of native (non-fluorescent) lipoproteins was added to the Dil-labeled lipoproteins, essentially no fluorescently labeled lipoproteins were seen associated with the cells. The Dil-labeled LDL, apo-E HDLc, and beta-VLDL, which were bound to the cells at 4 degrees C, were associated with the cell surface and were often observed in linear arrays. Cells that were either incubated with Dil-labeled lipoproteins at 4 degrees C and subsequently heated to 37 degrees C or incubated with the Dil-labeled lipoproteins at 37 degrees C showed internalized perinuclear fluorescence. When Dil-labeled LDL, apo-E HDLc, or beta-VLDL were treated with diketene to acetoacetylate their lysine residues, and then were incubated at 37 degrees C with mixtures of fibroblasts and mouse peritoneal macrophages in culture, the fibroblasts did not become fluorescently labeled. The macrophages became highly fluorescent, however. The acetoacetylation inhibited the interaction of the lipoproteins with the apo-B, -E receptors of fibroblasts and stimulated their uptake by macrophages. The use of fluorescently labeled native lipoproteins and chemically modified lipoproteins may allow the functional differentiation of macrophages from other cell types (e.g., fibroblasts and smooth muscle cells) in the arterial wall. This differentiation may be useful in determining the origin of the lipid-laden foam cells of atherosclerotic lesions.

Apolipoproteins↗
PubMed2006

Study of the cholesterol-GM3 ganglioside interaction by surface pressure measurements and fluorescence microscopy.

The nature of the cholesterol/glycolipid interaction in rafts being poorly understood, the interaction of cholesterol with the GM(3) ganglioside has been studied by surface pressure measurements and fluorescence microscopy. Results have been compared to those obtained with sphingomyelin (SM)-cholesterol and palmitoyl-oleoyl-phosphatidylcholine (POPC)-cholesterol monolayers. The analysis of (pi-A) isotherms of mixed monolayers show a condensing effect of cholesterol on GM(3) molecules, in the same range than the effect observed with POPC and higher than the effect on SM. This is likely due to the similar state of GM(3) and POPC, since both molecules are in liquid expanded phases in our experimental conditions. The study of the cholesterol desorption induced by beta-cyclodextrin suggests also that the GM(3)-cholesterol interaction is rather weak as in the case of POPC-cholesterol interaction, and clearly lower than SM-cholesterol one. This lack of interaction is discussed in terms of nature of lipid chains and molecular shape, and suggests that no hydrogen bond is formed between GM(3) and cholesterol polar heads. Fluorescence microscopy performed on mixed GM(3)-cholesterol monolayers shows the presence, at surface pressure higher than 10 mN/m, of particular blurring patterns without defined boundary, which could be due to a partial solubilization in one phase of different phases observed at lower surface pressure, whereas SM-cholesterol and POPC-cholesterol monolayers are homogeneous at the lateral resolution of our microscopy set-up.

Cholesterol↗
PubMed2007

Hypothesis testing via integrated computer modeling and digital fluorescence microscopy.

Computational modeling has the potential to add an entirely new approach to hypothesis testing in yeast cell biology. Here, we present a method for seamless integration of computational modeling with quantitative digital fluorescence microscopy. This integration is accomplished by developing computational models based on hypotheses for underlying cellular processes that may give rise to experimentally observed fluorescent protein localization patterns. Simulated fluorescence images are generated from the computational models of underlying cellular processes via a "model-convolution" process. These simulated images can then be directly compared to experimental fluorescence images in order to test the model. This method provides a framework for rigorous hypothesis testing in yeast cell biology via integrated mathematical modeling and digital fluorescence microscopy.

Computational Biology↗
PubMed1981

Colloidal gold : a cytochemical marker for light and fluorescent microscopy and for transmission and scanning electron microscopy.

Gold sols are orange to violet, display electron dense properties and are capable of strong emission of secondary electrons. These properties enable gold particles to be used as specific markers in microscopy both at the low and high resolution level (light and fluorescent microscopy, scanning and transmission electron microscopy). Monodisperse colloidal gold can be produced by several methods in a size range of 5 nm to 150 nm. As a consequence, the gold method is well suited for multiple marking experiment at the high resolution level. Since gold markers bind non-specifically to a very low extent, the technique has found application in TEM for marking intracellular components on thin sections. Both the one step and the two step marking procedures have been utilized in the various modes of microscopy. Under appropriate conditions, gold particles can be labelled with a variety of macromolecules (polysaccharides, glycoproteins, proteins, lectins, antibodies), presumably through a noncovalent binding process. Generally the probes acquire the specific activity of the adsorbed macromolecule and their stability upon storage is good. A number of factors which influence the adsorption process are discussed in relation to the more general problems of adsorption of macromolecules onto metallic surfaces. The stability of gold markers is also best understood by the DLVO theory for disperse systems. The preparation, labelling, stabilization, stability and binding characteristics of gold markers are reviewed. Since the binding of gold probes to cell surfaces is primarily determined by the size of the particle, several problems related to steric hindrance and quantification of the method are also discussed. The advantages of the method over others are compared. The different modes of microscopy and the several gold methods available for marking cell surface and intracellular components are illustrated by micrographs.

Adsorption↗
PubMed2001

Detection of mitochondrial DNA in living animal cells with fluorescence microscopy.

The detection of mitochondrial DNA (mtDNA) in living human cells could be useful for understanding mitochondrial behaviour during cellular processes and pathological mtDNA depletions. However, until now, human mtDNA has not been visualized in living cells with fluorescence microscopy, although it has been easily detected in organisms with larger mtDNA. Previous reports have stated that mtDNA staining results in homogeneous fluorescence of mitochondria or that animal mitochondria are refractory to DAPI staining. This paper shows that mtDNA of cultured green monkey kidney CV-1 can be stained using a very low concentration of DAPI, then detected by a cooled Photometrics CCD camera with 14-bit resolution detection. Indeed, under these conditions CV-1 cells have small fluorescent spots in the cytoplasm that colocalize with mitochondria, even after mitochondrial movements, uncoupling by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and swelling. These observations have been reproduced for the human fibroblast foreskin cell line HS68. These results and known properties of DAPI as a specific DNA stain strongly suggest that mtDNA can be detected and visualized by fluorescence microscopy in human living cells, with potential developments in the study of mtDNA in normal and pathological situations.

Animals↗
PubMed2001

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast.

Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle of incidence, to obtain quantitative information on the distance of the fluorescent object from the surface. We report the fabrication of hybrid surfaces consisting of nm layers of SiO(2) on lithium niobate (LiNbO(3), n = 2.3). Supported lipid bilayer membranes can be assembled and patterned on these hybrid surfaces as on conventional glass. By varying the angle of incidence of the excitation light, we are able to obtain fluorescent contrast between 40-nm fluorescent beads tethered to a supported bilayer and fluorescently labeled protein printed on the surface, which differ in vertical position by only tens of nm. Preliminary experiments that test theoretical models for the fluorescence-collection factor near a high refractive index surface are presented, and this factor is incorporated into a semiquantitative model used to predict the contrast of the 40-nm bead/protein system. These results demonstrate that it should be possible to profile the vertical location of fluorophores on the nm distance scale in real time, opening the possibility of many experiments at the interface between supported membranes and living cells. Improvements in materials and optical techniques are outlined.

Contrast Sensitivity↗
PubMed1998

Fluorescence microscopy of rat embryo sections stained with haematoxylin-eosin and Masson's trichrome method.

The fluorescence pattern induced by haematoxylin-eosin (HE) and Masson's trichrome (MT) staining methods on paraffin sections of rat embryos (from 13 to 18 days old) has been studied. Using optimal excitation (green light, 545 nm), HE- or MT-stained sections showed a selective red emission of the acidophilic tissue components, which was due to eosin Y in the case of HE and to acid fuchsin and/or xylidine ponceau in the case of MT. The fluorescence intensity induced by these anionic dyes was variable and related to the substrate nature and the embryo age. Whereas in young embryos only the immature red blood cells showed a noticeable fluorescence, in the oldest embryos there were also other tissue components that selectively fluoresced with these dyes, in particular fibre lens cells, elastic fibres, zymogen granules and muscle cells. Spectrofluorometric studies on free dyes and densitometric analysis of protein blots confirmed microscopical observations. Our results indicate that the standard HE and MT staining methods can be used in recognizing the appearance of specific protein structures in embryonic tissues by means of fluorescence microscopy.

Animals↗
PubMed2005

Micrometer-sized supported lipid bilayer arrays for bacterial toxin binding studies through total internal reflection fluorescence microscopy.

In this article, we present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy. The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated distearoylphosphatidylcholine:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside G(T1b) or G(M1). The ganglioside-populated SLB arrays were then exposed to either cholera toxin B subunit or tetanus toxin C fragment. Binding was assayed on planar substrates by total internal reflection fluorescence microscopy down to 100 pM concentration for cholera toxin subunit B and 10 nM for tetanus toxin fragment C. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is influenced by the microenvironment of the SLB and the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions.

Bacterial Toxins↗
PubMed1988

Analysis by fluorescence microscopy and flow cytometry of monoclonal antibodies produced against cell surface antigens.

The reactivity of seven monoclonal antibodies against the surface antigens of the murine myeloma cell line Sp2/O-Ag 14 was simultaneously analyzed by fluorescence microscopy and flow cytometry. To 1 X 10(6) Sp2/O-Ag 14 cells were added 200 microliters of the monoclonal antibody and the mixture was incubated at 4 degrees C for 30 min. After washing twice with PBS, the Sp2/O-Ag 14 cells were incubated at 4 degrees C for 30 min with a 1:400 dilution of fluoresceinated goat anti-mouse antibodies. Sp2/O-Ag 14 cells were ready for analysis after washing the cells 3 X with PBS. By fluorescence microscopic analysis different patterns of reactivity with monoclonal antibodies were detected. These patterns were identified as: smooth annular, dot-like annular, dot-like patches, diffuse and homogeneous. The observed patterns may represent different cell surface epitopes being recognized by the monoclonal antibodies. Flow cytometry analysis with the EPICS V system showed reactivity of seven monoclonal antibodies with Sp2/O-Ag 14 cell surface epitopes, which ranged from 79 to 90%. Compared to fluorescence microscopy, flow cytometry provides a faster, more sensitive and more accurate quantitative measurement of the reactivity of different monoclonal antibodies against Sp2/O-Ag 14 cell surface antigens.

Animals↗
PubMed2003

Identification of gap junction blockers using automated fluorescence microscopy imaging.

Gap junctions coordinate electrical signals and facilitate metabolic synchronization between cells. In this study, the authors have developed a novel assay for the identification of gap junction blockers using fluorescence microscopy imaging-based high-content screening technology. In the assay, the communication between neighboring cells through gap junctions was measured by following the redistribution of a fluorescent marker. The movement of calcein dye from dye-loaded donor cells to dye-free acceptor cells through gap junctions overexpressed on cell surface membranes was monitored using automated fluorescence microscopy imaging in a high-throughput compatible format. The fluorescence imaging technology consisted of automated focusing, image acquisition, image processing, and data mining. The authors have successfully performed a high-throughput screening of a 486,000- compound program with this assay, and they were able to identify false positives without additional experiments. Selective and pharmacologically interesting compounds were identified for further optimization.

Animals↗
PubMed1991

Drosophila gastrulation: analysis of cell shape changes in living embryos by three-dimensional fluorescence microscopy.

The first event of Drosophila gastrulation is the formation of the ventral furrow. This process, which leads to the invagination of the mesoderm, is a classical example of epithelial folding. To understand better the cellular changes and dynamics of furrow formation, we examined living Drosophila embryos using three-dimensional time-lapse microscopy. By injecting fluorescent markers that visualize cell outlines and nuclei, we monitored changes in cell shapes and nuclear positions. We find that the ventral furrow invaginates in two phases. During the first 'preparatory' phase, many prospective furrow cells in apparently random positions gradually begin to change shape, but the curvature of the epithelium hardly changes. In the second phase, when a critical number of cells have begun to change shape, the furrow suddenly invaginates. Our results suggest that furrow formation does not result from an ordered wave of cell shape changes, contrary to a model for epithelial invagination in which a wave of apical contractions causes invagination. Instead, it appears that cells change their shape independently, in a stochastic manner, and the sum of these individual changes alters the curvature of the whole epithelium.

Animals↗
PubMed1989

[Use of very low density light fluorescence microscopy: study of real-time of the development of chromatin of unicellular mouse embryos].

Video enhanced fluorescence microscopy coupled to digitized image processing was used to follow the dynamics of chromatin changes in live one cell mouse embryos. The experimentation on live material was made possible through the use of very low concentrations of the DNA specific fluorophore, Hoechst 33342, and very low irradiation intensities, which maintain a good viability of embryos.

Animals↗
PubMed2002

Interactions of the 18.5-kDa isoform of myelin basic protein with Ca(2+)-calmodulin: in vitro studies using fluorescence microscopy and spectroscopy.

The interactions of the 18.5-kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using fluorescence microscopy and spectroscopy. Two forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a hexahistidine-tagged recombinant murine product (rmMBP), with only minor differences in behaviour being observed. Fragments of each protein generated by digestion with cathepsin D (EC 3.4.23.5) were also evaluated. Using fluorescence microscopy, it was shown that MBP and CaM interacted in the presence of Ca2+ under a variety of conditions, including high urea and salt concentrations, indicating that the interaction was specific and not merely electrostatic in nature. Using cathepsin D digestion fragments of MBP, it was further shown that the carboxyl-terminal domain of MBP interacted with Ca(2+)-CaM, consistent with our theoretical prediction. Spectroscopy of the intrinsic fluorescence of the sole Trp residue of MBP showed that binding was cooperative in nature. The dissociation constants for formation of a 1:1 MBP-Ca(2+)-CaM complex were determined to be 2.1 +/- 0.1 and 2.0 +/- 0.2 microM for bMBP/C1 and rmMBP, respectively. Fluorescence spectroscopy using cathepsin D digestion fragments indicated also that the carboxyl-terminal region of each protein interacted with Ca(2+)-CaM, with dissociation constants of 1.8 +/- 0.2 and 2.8 +/- 0.9 microM for the bMBP/C1 and rmMBP fragments, respectively. These values show a roughly 1000-fold lower affinity of MBP for CaM than other CaM-binding peptides, such as myristoylated alanine-rich C-kinase substrate, that are involved in signal transduction.

Amino Acid Sequence↗
PubMed2006

High-throughput fluorescence microscopy for systems biology.

In this post-genomic era, we need to define gene function on a genome-wide scale for model organisms and humans. The fundamental unit of biological processes is the cell. Among the most powerful tools to assay such processes in the physiological context of intact living cells are fluorescence microscopy and related imaging techniques. To enable these techniques to be applied to functional genomics experiments, fluorescence microscopy is making the transition to a quantitative and high-throughput technology.

Animals↗