Fluorescence imaging total internal

Image dye in fluorescence or Total Internal Reflection Fluorescence microscopy using appropriate filter sets with 20 ms exposures and near-maximal multiplication on an EM-CCD camera see Note 17). An example of a cell migrating toward a micropipette filled with chemoattractant is shown in Fig. 4. 

Several other modes of fluorescence microscopy have been developed, including automated fluorescence image c3dometry (AFIC), fluorescence resonance energy transfer microscopy (FRETM), two-photon fluorescence microscopy, total internal reflection fluorescence microscopy, and standing-wave fluorescence microscopy. 

Tokunaga M, Kitamura K, Saito K, Iwane A H and Yanagida T 1997 Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy Biochem. Biophys. Res. Commun. 235 47-53... 

Total internal reflection fluorescence lifetime imaging microscopy... 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

Fig. 1 Real-time tracking of cell adhesion [42]. (a) Components of a total internal reflection fluorescent microscope (TIRFM). (b) The cell adhesion process (7) a cell approaches the surface, (2) the cell lands, (3) the cell attaches, and (4) the cell spreads out on the surface. The evanescent field was generated by total internal reflection of a laser beam at the glass-water interface. Cells with fluorescently labeled membranes (dashed lines) were plated on SAMs. Cell membranes within the evanescent field (solid line) were observed by TIRFM. Corresponding TIRFM images are shown below...

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

FIGURE 6.7. Epifluorescent images of (a) Texas Red labeled streptavidin confined in aqueous compartments above solid supported phospholipid bilayers containing 2 mol% biotinylated lipids. Each box contains a different aqueous phase small molecule as described in the text, (b) Same system as in (a), but fluorescence is excited by total internal reflection revealing that streptavidin is surface bound in boxes 1, 2, and 4, while the surface in box 3 remains protein free. 

FIGURE 6.11. (a) A total internal reflection fluorescence image of the same conditions as shown in Figure 6.10a. (b) Line scan of fluorescence intensity (dotted line) across the microchannels. 

FIGURE 2.17 Results of total internal reflection fluorescence (TIRF) measurements in which the discrete and processive stepping action of myosin can be seen clearly. SOURCE Reprinted with permission from Yildiz, A., J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin..2003. Myosin V walks hand-over-hand Single fluorophore imaging with 1.5-nm localization. Science 300 2061-2065. Copyright 2003 AAAS. 

Bowser, D. N., and Khakh, B. S. (2007). Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy. Proc. Natl. Acad. Sci. USA 104, 4212—4217. 

Fig. 15.1. Schematic representation of amyloid fibrils revealed by total internal reflection fluorescence microscopy, (a) The penetration depth of the evanescent field formed by the total internal reflection of laser light is 150nm for a laser light at 455 nm, so only amyloid fibrils lying parallel to the slide glass surface were observed. (b) Schematic diagram of a prism-type TIRFM system on an inverted microscope. ISIT image-intensifier-coupled silicone intensified target camera, CCD charge-coupled device camera...

SPR imaging was also employed to look at the same cholera toxin-GMl interaction on a supported bilayer by Philips et al. in array experiments [74]. Another investigative technique applied to the model ganglioside-cholera toxin system was total internal reflection fluorescence microscopy applied to spatially addressable supported bilayer membranes [75]. The recognition by HIV-1 surface glycoprotein gpl20 of glycosphingolipids partitioned within an SBM has also been looked at by total internal reflection fluorescence [76]. 

Thompson NL, Steele BL. Total internal reflection with fluorescence correlation spectroscopy. Nat. Protoc. 2007 2 878-890. Sund SE, Axelrod D. Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleach-ing. Biophys. J. 2000 79 1655-1669. 

The three-dimensional network of actin stress fiber, which is an association of actin filaments, provides mechanical support for the cell, determines the cell shape, and enables cell movement. Thus the shape change in the cell due to the laser tsunami can be examined by observing the laser-induced dynamics of fibers. The actin stress fiber was visualized by binding it with enhanced green fluorescence protein (EGFP), and monitored by total internal reflection fluorescence (TIRF) imaging [34]. 

Figure 28.7 Epi-fluorescence (a) and total internal reflection fluorescence (b—e) images of a single NIH3T3 living cell before (a, b) and after (c-e) inducing a laser tsunami.

On the other hand, optical microscopy, confocal microscopy, ellipsometry, scanning electron microscopy (SEM), scanning tunneling microscopy (STM), atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF) are the main microscopic methods for imaging the surface structure. There are many good books and reviews on spectroscopic and chemical surface analysis methods and microscopy of surfaces description of the principles and application details of these advanced instrumental methods is beyond the scope of this book. 

Total internal reflection fluorescence microscopy (TIRFM) is a promising alternative approach to low background fluorescence imaging [68], For excitation of molecules on a surface or within a thin slice of the sample, an evanescent optical field is used traveling along the interface between a medium with a high refractive index n, (typically a quartz glass prism) and... 

Key words Confocal fluorescence microscopy, Forster resonance energy transfer, Total internal reflection fluorescence microscopy, Single-molecule imaging, GPCR, Heterotrimeric G-proteins, Spatiotemporal dynamics... 

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