Imaging TIRF

The major advantage of TIRF is that fluorophores outside the evanescent wave (typically more than 200 nm away from the surface) are not excited. Hence, TIRF has an intrinsic sectioning capability. Of interest is that the section capability (z-resolution) is far better than for confocal microscopy systems, which typically have a z-resolution of about 1 /mi. In addition and in contrast to confocal microscopy, TIRF does not cause out-of-focus bleaching because only the molecules at the surface will sense the evanescent wave. However, in comparison with confocal microscopy, a clear limitation of TIRF is that only one z-plane can be imaged the molecules immediately adjacent to the surface. As a consequence,... 

Since TIRF produces an evanescent wave of typically 80 nm depth and several tens of microns width, detection of TIRF-induced fluorescence requires a camera-based (imaging) detector. Hence, implementing TIRF on scanning FLIM systems or multiphoton FLIM systems is generally not possible. To combine it with FLIM, a nanosecond-gated or high-frequency-modulated imaging detector is required in addition to a pulsed or modulated laser source. In this chapter, the implementation with of TIRF into a frequency-domain wide-field FLIM system is described. 

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

Confocal microscopy (CM) is another microscope technique for apparent optical sectioning, achieved by exclusion of out-of-focus emitted light with a set of image plane pinholes. CM has the clear advantage in versatility its method of optical sectioning works at any plane of the sample, not just at an interface between substances having dissimilar refractive indices. However, other differences exist which, in some special applications, can favor the use of TIRF ... 

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. 

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. 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 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]. 

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. 

Breast Cancer Cell Movement Imaging Invadopodia by TIRF and IRM Microscopy 211... 

Imaging with Olympus TIRF Micr( cope... 

Fig. 1. TIRF microscope. (A) A view of the TIRF system. (B)View looking down on the back of the microscope (environmental chamber at bottom, back of the system at the top). Arrow b indicates the location of the Selection Prism containing the 80%/ 20% beamsplitter. Pulling the knob at the top to its full upwards position sets the beamsplitter to 100%/ 0% (all Epi-illumination input to the microscope). Pushing it downward sets the beamsplitter to 80% laser and 20% epifluo-rescence illumination. (B ) Location of the Field Diaphragm and the Field Stop on the epifluorescence arm of the split box. Two arrows AS and FS indicate Aperture Stop and Field Stop, respectively. Adjustment of these is vital for good IRM imaging. (B ) The laser input from which the screw white arroW) for TIRF angle adjustment projects. This pair of screws laterally translocates the laser path off center in the objective so that the angle of reflection is altered.

Here, we describe a detailed procedure to monitor cell adhesion by IRM imaging with standard epi-illumination, also see Note 11. And, we show a method to directly visualize the dynamic membrane localization of GPF fused c-Src(Y527F) by a combination of TIRF and IRM microscopy. 

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