TIRF-microscopy

Total internal reflection fluorescence (TIRF) microscopy, fluorescence in situ hybridization (FISH), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM). 

Figure 7.10. TIRF microscopy with prisms fixed to the substrate plate. (Adapted from Weis el alS 2y)...

W. M. Reichert and G. A. Truskey, Total internal reflection fluorescence (TIRF) microscopy. I. Modeling cell contact region fluorescence, J. Cell Sci. 96, 219-230 (1990). 

Fig. 11. Scanning electron micrographs (a-d) shown sequential stages in the early part of the adhesion process for mouse fibroblasts from initial contact with a surface to the assumption of a more or less final morphology. The cytoskeleton has the ability to change cell shape quickly and an individual cell may pass from the initial spherical form to the final flattened one in a few minutes. The initial adhesion process at the points of contact between cell and surface is also very rapid but there are subsequent changes at the adhesion sites affecting the nature and strength of the bonds which may continue for many hours. These can be studied by TIRF microscopy...

For biotechnological purposes, it is necessary not only that cells remain viable but also that adhesion is maintained so that a cell can be found in the expected position. The adhesion pattern that a cell has adopted before freezing is crucial to successful cryo-preservation. Adhesion patterns can be visualised by (TIRF microscopy, [63]) mentioned above and also by confocal laser scanning fluorescence (CLSM, [64]) microscopy. We have investigated the adhesion patterns of fibroblasts as a function of adhesion time and various surface treatments [65]. Some examples are shown in Fig. 19. 

Cali, C., Marchaland,J., Regazzi, R., and Bezzi, R (2008). SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale Imaging analysis at the single-vesicle level with TIRF microscopy.. . Neuroimmunol. 198, 82—91. 

Fig. 12.6. Direct determination of mechano-chemical coupling of myosin, (a) An experimental arrangement for simultaneous measurement. Single-headed myosin in the co-filament with an excess myosin rod is immobilized on the pedestal of the slide glass. An actin filament is manipulated by a trapped laser through two beads attached at both ends to interact with a myosin head in the correct arrangement. The ATP turnover is measured by monitoring the fluorescence from Cy3-ATP (Cy3-ADP) associated to and dissociated from the myosin head using TIRF microscopy, (b) Time trajectory of the displacement of the myosin head and the ATP turnover. The upper, middle, and bottom trace show the time course of displacements, changes in stiffness, which was calculated from the variance of the thermal motion of the beads, and changes in the fluorescence intensity of Cy3-nucleotide at the position of the myosin head...

Fig. 15.7. Self-assembly of nanoparticles to patterns of binding sites, (a) The transfer DNA is modified with biotin, so that patterns of specific binding sites are created with SMCP. Fluorescent nanoparticles carrying streptavidin self-assemble to these patterns and form superstructures, (b) The formation of superstructures is observed online with TIRF microscopy, (c) The patterns of binding sites were created with different size and incubated with nanoparticles fluorescing at different wavelengths. Also, the scale bar is formed in this way. The pictures are standard deviations of TIRF microscopy image series recorded at 20 Hz...

The use of an evanescent wave to excite fluorophores selectively near a solid-fluid interface is the basis of the technique total internal reflection fluorescence (TIRF). It can be used to study theadsorption kinetics of fluorophores onto a solid surface, and for the determination of orientational order and dynamics in adsorption layers and Langmuir-Blodgett films. TIRF microscopy (TIRFM) may be combined with FRAP ind FCS measurements to yield information about surface diffusion rates and the formation of surface aggregates. 

Fig. 4. An example of an HL-60 cell crawling toward a mIcropIpette visualized with DIG and TIRF microscopy. Asterisk Indicates micropipette tip.

Key words Breast cancer cell migration, invasion, invadopodia. Total Internal Reflection Fluorescence (TIRF) microscopy. Interference Reflection Microscopy (IRM)... 

Incubate the cells on the MatTek dish in a humidifled, 37°C, 5% COj incubator or on the microscope stage in an environmental chamber with similar conditions for about 3 h and then start to observe with Olympus TIRF microscopy. 

Imaging the Dynamics of GPCR and G-Protein Subunits at the Single-Molecule Level by TIRF Microscopy... 

The evanescent field used for fluorescence excitation in TIRF microscopes declines exponentially with increasing distance from the reflecting surface. In practice, fluorescence will be recorded from a layer extending about 100 nm from the plasma membrane of an attached cell into its interior, this means with minimal background contributed by the bulk of the cytoplasm. The cortical actin network of Dictyostelium cells is located within the layer illuminated by the evanescent wave. Therefore, TIRF microscopy visualizes the actin network structures with a brilliance that is unmatched by any other technique. 

Key words Actin purification and assembly, Actin-binding proteins, Dictyostdium, NEM-myosin II, TIRF microscopy... 

Analysis of Actin Assembly by In Vitro TIRF Microscopy 403... 

Using In Vitro TIRF Microscopy to Wsualize Actin Polymerization... 

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