Total Internal Reflection Fluorescence Microscopy Facility, Department of Physiology 
Mark S. Shapiro, Ph.D., DirectorTelephone: (210) 567-4328 Fax: (210) 567-4410 Email: shapirom@uthscsa.edu To make Total Internal Reflection Fluorescence (TIRF) microscopy and imaging available to the Department of Physiology, the Graduate School of Biomedical Sciences, and the UT Health Science Center community. TIRF Microscopy Total Internal Reflection Fluorescence (TIRF) microscopy was first used in the study of biological molecules in the 1980s by Daniel Axelrod and colleagues ( Axelrod, D., T.P. Burghardt, and N.L. Thompson, Total internal reflection fluorescence. Annu Rev Biophys Bioeng, 1984. 13: p. 247-68). The technique was further popularized by Wolfhard Almers and colleagues to study exocytosis of unitary neurotransmitter-containing vesicles (Steyer, J.A. and W. Almers, A real-time view of life within 100 nm of the plasma membrane. Nat Rev Mol Cell Biol, 2001. 2(4): p. 268-75.). TIRF illumination involves directing a laser beam at the interface between two transparent media of differing refractive indices at a glancing angle. 
By the laws of optics, at an angle greater than the critical angle determined by the ratio of the two refractive indices, the light beam is not primarily transmitted to the 2nd medium, but is instead reflected; however, not all the light energy is reflected; a component penetrates into the 2nd medium as an "evanescent wave" (EW) that decays exponentially in intensity over a distance of only several hundred nanometers. Thus, we can selectively excite only fluorophores located within ~300 nm of the plasma membrane by directing laser light at such a glancing angle through a special TIRF objective, otherwise known as "through-the-lens" TIRF illumination ( Axelrod, D., Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol, 2003. 361: p. 1-33). TIRF microscopy is therefore ideal for high-resolution study of events at the plasma membrane. Any molecules located deeper in the cytoplasm will not be illuminated.Our facility has two main systems mated together to constitute a superior integrated TIRF apparatus. The first is the Nikon evanescent wave imaging system. Evanescent waves created when laser light strikes the interface between the glass coverslip containing cells and aqueous solution are used to excite molecules in the sub-micron layer in contact with the coverglass. High numerical-aperture TIRF objectives make it possible to introduce laser illumination at incident angles greater than the critical angle (Θc) resulting in TIR, creating an evanescent wave immediately adjacent to the coverglass/specimen interface. The evanescent wave reaches less than 300 nm into the specimen and its energy drops off exponentially. Because the specimen is not excited beyond the evanescent wave, this imaging system can produce fluorescent images with an extremely high signal-to-noise (S/N) ratio. The second is a laser light delivery system assembled by Prairie Technologies. It consists of three lasers, a diode pumped solid-state (DPSS) emitting 442 nm, a 40-mW argon emitting 488 nm and 514 nm, and a green helium/neon laser emitting at 543 nm. These laser lines are ideal for the excitation of the popular fluorescent proteins CFP, GFP, YFP and dsRed, respectively. The lasers have a common output to an acoustic optical tunable filter (AOTF), which selects the laser line desired by tuning the AOTF crystal, controlled by MetaMorph software running on a desktop PC. A fiber-optic cable connects the output of the AOTF to the input of the Nikon TIRF microscope. 
TIRF illumination is selective for membrane molecules. CHO cells were co-transfected with membrane-targeted EGFP-F and cytoplasmic dsRed2. The top row are wide-field fluorescent images of a cell, using mercury lamp illumination. The bottom row are TIRF images of the same cell, excited by the 488 line of the Argon laser (left), or the 543 line of the HeNe laser (right). In TIRF, only the membrane-localized fluorophores are illuminated (courtesy of M.S. Shapiro and J.D. Stockand). 
ENaC Na+ channels are expressed in the membrane. TIRF images of COS-7 cells co-expressing eCFP-aENaC + eYFP-bENaC and untagged gENaC (top row), and eCFP-bENaC + eYFP-aENaC and untagged gENaC (bottom row). ECFP and EYFP emissions are pseudo-colored green or red, respectively (courtesy of J.D. Stockand). 
TIRF illumination shows membrane-clustering of K+ channels. CHO cells transfected with EYFP-tagged KCNQ5 K+ channels were illuminated under wide-field with a mercury lamp (left), or under TIRF with the 514 nm line of the Argon laser (right). The TIRF fluorescent micrographs reveal the clustering of the channels into puncta in the plasma membrane. Channels localized deeper in the cytoplasm are not illuminated (courtesy N. Gamper and M.S. Shapiro). Operating Guidelines The TIRF core facility is available to any Department of Physiology primary or cross-appointed faculty member free of charge, with the costs of the experiment (consumables, reagents) borne by the user. Any investigators outside of the Department of Physiology (either within or outside of the Health Science Center) can seek to use the facility by a collaborative arrangement with Physiology faculty. Priority will be given to investigators within the HSC. All users must be trained on the system to the satisfaction of the Director before any use. The equipment uses powerful lasers that can damage the eyes if used improperly, and all risks of use are assumed by the users. |