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
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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- "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). "
ABSTRACT: RhoJ is a RhoGTPase expressed in endothelial cells and tumour cells which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; siRNA-mediated knockdown of RhoJ increased focal adhesion disassembly time, while expression of an active mutant (daRhoJ) decreased it. Further, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast-2-hybrid, which depended on the Spa homology domain of GIT1. GIT1, GIT2, β-PIX and RhoJ all co-localised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of GIT1/2 or β-PIX phenocopying RhoJ knockdown. RhoJ knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give novel insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.Journal of Cell Science 06/2014; 127(14). DOI:10.1242/jcs.140434 · 5.43 Impact Factor
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- "To further address this presumption, we performed TIRF microscopy capable of visualizing at high lateral resolution the cell-substrate adhesion interface (up to 0.1 µm above the cell substrate ). In these experiments, VSVG-YFP, which was transiently expressed in MDCK cells, served as a fluorescent marker of the host cell basal plasma membrane . "
ABSTRACT: Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.PLoS ONE 10/2013; 8(10):e78431. DOI:10.1371/journal.pone.0078431 · 3.23 Impact Factor
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- "In addition to its advantages for smSRM microscopy, the methodology presented will be useful for a range of advanced fluorescence microscopy methods, such as 3D structured illumination microscopy , , , two-photon scanning fluorescence correlation spectroscopy methods (e.g. Number and Brightness analysis, or N&B) , , total internal reflection microscopy (TIRM) , or single-particle counting or counting experiments (e.g. slim field microscopy, or SFM) , . "
ABSTRACT: Bacteria have evolved complex, highly-coordinated, multi-component cellular engines to achieve high degrees of efficiency, accuracy, adaptability, and redundancy. Super-resolution fluorescence microscopy methods are ideally suited to investigate the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. These techniques require the long-term stability of samples, high signal-to-noise-ratios, low chromatic aberrations and surface flatness, conditions difficult to meet with traditional immobilization methods. We present a method in which cells are functionalized to a microfluidics device and fluorophores are injected and imaged sequentially. This method has several advantages, as it permits the long-term immobilization of cells and proper correction of drift, avoids chromatic aberrations caused by the use of different filter sets, and allows for the flat immobilization of cells on the surface. In addition, we show that different surface chemistries can be used to image bacteria at different time-scales, and we introduce an automated cell detection and image analysis procedure that can be used to obtain cell-to-cell, single-molecule localization and dynamic heterogeneity as well as average properties at the super-resolution level.PLoS ONE 10/2013; 8(10):e76268. DOI:10.1371/journal.pone.0076268 · 3.23 Impact Factor
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