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At least 1,189 records · Page 66Linked to original sources
PubMed1994

Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion.

Influenza hemagglutinin (HA, strain A/PR/8/34) was purified and reconstituted into supported planar membranes in a two-step process: 1) HA was purified by C12E8 detergent solubilization followed by detergent removal with Biobeads; (2) the purified HA was then incorporated into "viroplanes," i.e. supported planar membranes which contained the viral membrane proteins. This step was accomplished by a spontaneous reaction of the HA-proteoliposomes with a phospholipid monolayer that was supported on a quartz microscope slide. The reconstitution of the HA into the planar membranes was followed by total internal reflection fluorescence microscopy (TIRFM) using fluorescein-labeled HA. By changing the solution concentration of HA, surface concentrations between 2.4 x 10(4) and 4.3 x 10(4) HA monomers/micron 2 were reached. Greater than 90% of all HA molecules were oriented with their ectodomain facing away from the substrate toward the large aqueous compartment of the measuring cell. Binding experiments with conformation-sensitive monoclonal antibodies against HA established that the reconstituted HA could undergo the low pH-induced conformational change in the supported bilayer. Binding of vesicles containing the fluorescent lipid analog N-(7-nitro-2,1,3-benzoxadiazol-4-yl)egg phosphatidylethanolamine was also measured by TIRFM. Vesicle binding was promoted when sialic acid-containing gangliosides or negatively charged lipids were included in these target membranes. Membrane fusion of the HA bound vesicles was monitored by measuring long range (over several micrometers) lateral diffusion coefficients of the lipids in the bound layer by fluorescence recovery after photobleaching. The vesicles did not fuse at pH 7.4, but efficient vesicle fusion occurred on the viroplanes after acidification of the environment with pH 5 buffer. This fusion reaction was only observed when the bound vesicles exceeded a critical threshold surface concentration. The successful reconstitution of membrane fusion sites in a planar supported membrane system opens new possibilities for studying fusion intermediates by localized spectroscopy and microscopy.

Animals↗
PubMed1991

Fusion of influenza virions with a planar lipid membrane detected by video fluorescence microscopy.

The fusion of individual influenza virions with a planar phospholipid membrane was detected by fluorescence video microscopy. Virion envelopes were loaded with the lipophilic fluorescent marker octadecylrhodamine B (R18) to a density at which the fluorescence of the probe was self-quenched. Labeled virions were ejected toward the planar membrane from a micropipette in a custom-built video fluorescence microscope. Once a virion fused with the planar membrane, the marker was free to diffuse, and its fluorescence became dequenched, producing a flash of light. This flash was detected as a transient spot of light which increased and then diminished in brightness. The diffusion constants calculated from the brightness profiles for the flashes are consistent with fusion of virus to the membrane with consequent free diffusion of probe within the planar membrane. Under conditions known to be fusigenic for influenza virus (low pH and 37 degrees C), flashes appeared at a high rate and the planar membrane quickly became fluorescent. To further establish that these flashes were due to fusion, we showed that red blood cells, which normally do not attach to planar membranes, were able to bind to membranes that had been exposed to virus under fusigenic conditions. The amount of binding correlated with the amount of flashing. This indicates that flashes signaled the reconstitution of the hemagglutinin glycoprotein (HA) of influenza virus, a well-known erythrocyte receptor, into the planar membrane, as would be expected in a fusion process. The flash rate on ganglioside-containing asolectin membranes increased as the pH was lowered. This is also consistent with the known fusion behavior of influenza virus with cell membranes and with phospholipid vesicles. We conclude that the flashes result from the fusion of individual virions to the planar membrane.

Hydrogen-Ion Concentration↗
PubMed2005

Fluorescence microscopy with super-resolved optical sections.

The fluorescence microscope, especially its confocal variant, has become a standard tool in cell biology research for delivering 3D-images of intact cells. However, the resolution of any standard optical microscope is at least 3 times poorer along the axis of the lens that in its focal plane. Here, we review principles and applications of an emerging family of fluorescence microscopes, such as 4Pi microscopes, which improve axial resolution by a factor of seven by employing two opposing lenses. Noninvasive axial sections of 80-160 nm thickness deliver more faithful 3D-images of subcellular features, providing a new opportunity to significantly enhance our understanding of cellular structure and function.

Cellular Structures↗
PubMed2003

Optical sectioning by two-pinhole confocal fluorescence microscopy.

A two-pinhole axially superresolving confocal fluorescence imaging system is presented. Based on the concept of subtractive incoherent imaging, the system described here is equipped with a zero-focus complex-transmittance pupil filter in one of the collector paths. The optical sectioning capacity of the system is 25% superior to that of a free-pupil one-pinhole instrument.

Image Enhancement↗
PubMed1975

The adaptation of fluorescence microscopy for Nocardia asteroides: a preliminary report.

The Fluorescence Microscope is extremely effective in revealing the presence and morphology of Nocardia asteroides. This has been effectively demonstrated when the Auramine Fluorochrome was decolourized with 1% H2SO4 and the interval of decolourization was limited to two minutes and thirty seconds. When Nocardia asteroids was seeded in sputum found to be negative for acid-fast organisms and in greatly diluted quantities after one hour intervals, its presence was still revealed with the Flourescence Microscope. The normally employed Kinyoun technique of acid-fastness was seen to be less effective under the same conditions.

Benzophenoneidum↗
PubMed2003

Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies.

Fluorescence imaging of two independently labelled proteins is commonly used to determine their co-localization in cells. Antibody-mediated crosslinking can mediate the patching of such proteins at the cell surface, and their co-localization can serve to determine complex formation among them. However, manual analysis of such studies is both tedious and subjective. Here we present a digital co-localization analysis that is independent of the fluorescence intensity, is highly consistent and reproducible between observers, and dramatically reduces the analysis time. The approach presented is based on a segmentation procedure that creates binary objects, and then determines whether objects belonging to two different groups (e.g. green- and red-labelled) are co-localized. Two methods are used to determine co-localization. The 'overlap' analysis defines two objects as co-localized if the centre of mass of one falls within the area of the other. The 'nearest-neighbour distance' analysis considers two objects as co-localized if their centres are within a threshold distance determined by the imaging modality. To test the significance of the results, the analysis of the actual images is tested against randomized images generated by a method that creates images with uncorrelated distributions of objects from the two groups. The applicability of the algorithms presented to study protein interactions in live cells is demonstrated by co-patching studies on influenza haemagglutinin mutants that do or do not associate into mutual oligomers at the cell surface via binding to AP-2 adaptor complexes. The approach presented is potentially applicable to studies of co-localization by other methods (e.g. electron microscopy), and the nearest-neighbour distance method can also be adapted to study phenomena of correlated placement.

Adaptor Protein Complex 2↗
PubMed1987

[Structural changes in contractile proteins of muscle fibers studied by polarization ultraviolet fluorescence microscopy. X. The effect of ATP, Ca2+, pH change and ionic strength of the washing solution on the structural state of thick filaments].

By the method of polarized ultraviolet fluorescence microscopy, effects of ATP, Ca2+, changes in pH and ionic strength of washing solution of the structural state of thick filaments in both actin-free muscle fibers of rabbit and anisotropic discs (A-discs) of glycerinated fibers of crab were studied. The dependence of tryptophan fluorescence anisotropy of thin filaments upon physico-chemical parameters (compounds) of washing solution has been found. The structural state of thick filaments was suggested to be influenced by ATP, Ca2+, changes in pH and ionic strength of washing solution.

Adenosine Triphosphate↗