Imaging with Total Internal Reflection Fluorescence Microscopy for the Cell Biologist
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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- "To investigate the suitability of Lifeact-GFP in platelets, we isolated Pf4Cre Lifeact-GFP platelets from murine blood. We used total internal reflection (TIRF) microscopy, which images the first 100 nm of the bottom of the cells (Mattheyses et al., 2010) to examine realtime dynamics of F-actin. In this way we imaged spreading platelets forming filopodia (Fig. 1C, S3A and Supplementary Movie 2). "
ABSTRACT: We describe here the development and characterization of a conditionally inducible mouse model expressing Lifeact-GFP, a peptide that reports the dynamics of filamentous actin. We have used this model to study platelets, megakaryocytes and melanoblasts and we provide evidence that Lifeact-GFP is a useful reporter in these cell types ex vivo. In the case of platelets and megakaryocytes, these cells are not transfectable by traditional methods, so conditional activation of Lifeact allows the study of actin dynamics in these cells live. We studied melanoblasts in native skin explants from embryos, allowing the visualization of live actin dynamics during cytokinesis and migration. Our study revealed that melanoblasts lacking the small GTPase Rac1 show a delay in the formation of new pseudopodia following cytokinesis that accounts for the previously reported cytokinesis delay in these cells. Thus, through use of this mouse model, we were able to gain insights into the actin dynamics of cells that could only previously be studied using fixed specimens or following isolation from their native tissue environment.European journal of cell biology 05/2012; 91(11-12):923-9. DOI:10.1016/j.ejcb.2012.04.002 · 3.70 Impact Factor
Protein Interactions, 03/2012; , ISBN: 978-953-51-0244-1
- "A detailed protocol and trouble-shooting for FLIM is given by Periasamy (Sun, Day, and Periasamy 2011). Detailed reviews on the physical basis of TIRF and advanced applications are given by Axelrod and Rappoport (Mattheyses, Simon, and Rappoport 2010; Axelrod 2003; Axelrod 2008). "
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- "In TIRF the evanescent wave generated by a laser beam that is reflected at the interface between the coverglass and the aqueous medium above only excites fluorophores in very close proximity (100–500 nm) to the cover glass (Mattheyses et al., 2010). Thus, TIRF is particularly useful to observe events at the bottom of the cell with high contrast such as vesicle fusion with the plasma membrane or adhesion site dynamics (Hu et al., 2007;Toomre et al., 2000). "
ABSTRACT: The palette of fluorescent proteins (FPs) has grown exponentially over the past decade, and as a result, live imaging of cells expressing fluorescently tagged proteins is becoming more and more mainstream. Spinning disk confocal (SDC) microscopy is a high-speed optical sectioning technique and a method of choice to observe and analyze intracellular FP dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low-noise scientific grade-cooled charge-coupled device cameras, and can achieve frame rates of up to 1000 frames per second. In this chapter, we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy and provide a rationale for specific design choices. We also give guidelines of how other imaging techniques such as total internal reflection microscopy or spatially controlled photoactivation can be coupled with SDC imaging and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction.Methods in enzymology 01/2012; 504:293-313. DOI:10.1016/B978-0-12-391857-4.00015-X · 2.19 Impact Factor