Article

Imaging with Total Internal Reflection Fluorescence Microscopy for the Cell Biologist

Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Journal of Cell Science (Impact Factor: 5.43). 11/2010; 123(Pt 21):3621-8. DOI: 10.1242/jcs.056218
Source: PubMed

ABSTRACT

Total internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biological applications, and is particularly well suited to analysis of the localization and dynamics of molecules and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within ~100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid exposure times. In this Commentary, we discuss the applications of TIRF to the study of cell biology, the physical basis of TIRF, experimental setup and troubleshooting.

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    • "Refer to the system's user manual for these procedures. For homebuilt systems, general TIRF setup procedures have been described previously [11, 12]. 2. Verify that laser intensities can be set to the predetermined values set for " observation " and " photobleaching " intensities. For homebuilt systems, this can be done by placing a light power meter in the light path and adjusting the laser power accordingly. "
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    • "To investigate reduced RhoJ activity, siRNA knockdown rather than a dominant-negative mutant was used to specifically reduce levels of this Rho GTPase; the promiscuous binding of GEF proteins to multiple related Rho GTPases would be likely to result in a dominant-negative mutant of RhoJ sequestering and inhibiting GEFs of Cdc42 or Rac (Debreceni et al., 2004; Schmidt and Hall, 2002). In order to track focal adhesions, human umbilical vein endothelial cells (HUVECs) were transduced with an RFP-tagged paxillin and subjected to total internal reflection fluorescence (TIRF) microscopy, which is suited for visualisation of structures close to the cell surface (Mattheyses et al., 2010). Paxillin is a well-characterised focal adhesion protein and this fusion has been previously used for studying focal adhesion dynamics (Berginski et al., 2011). "
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