Cellular and Integrative Physiology
Total Internal Reflection Fluorescence Microscopy Facility, Department of Cell and Integrative Physiology Location: South Texas Research Facility
Mark S. Shapiro, Ph.D., Director
Telephone: (210) 562-4029
Goal: To make Total Internal Reflection Fluorescence (TIRF) microscopy and imaging available to the Department of Cell and Integrative Physiology, the Graduate School of Biomedical Sciences, and the UT Health Science Center community.
Total Internal Reflection Fluorescence Microscopy Facility (TIRF)
Mark S. Shapiro, Ph.D
Tel: (210) 562-4045
What is 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: 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" 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 (such as the nucleus or Golgi) will not be illuminated.
Our facility, located in the Neuroscience Wing of the STRF on the Greehey campus, 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 (c), 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 with distance from the glass/saline interface. 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.
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).
Example of a Fluorescence Resonance Energy Transfer (FRET) experiment performed on CHO cells co-transfected with CFP-tagged KCNQ1-5 potassium ion channels and YFP-tagged A-kinase Anchoring Protein (AKAP) 79. Under TIRF illumination, the excitation light penetrates only 300 nm into the cells. The left two, and right two, columns are images of CFP emission, or YFP emission, respectively, from the cells before and after selective photobleaching of the YFP fluorophores. The CFP emission images are shown in “rainbow” pseudocolor for clarity in assaying intensity. The data show that AKAP79 is intimately associated (within 50 Ǻ) with KCNQ2-5, but not KCNQ1. From Bal et al., J. Neuroscience, 2010. 10;30(6):2311-23
The TIRF core facility is available to any Department of Cellular and Integrative 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 (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, training required by the UT Health Safety Office, and all risks of use are assumed by the users.
The second is a state-of-the-art laser light delivery system assembled by Coherent Technologies. It consists of four solid-state diode lasers of 30-50 mW each, emitting laser lines at 442 nm, 488 nm, 514 nm and 647 nm. These laser lines are ideal for the excitation of the popular fluorescent proteins CFP, GFP, YFP and mStrawberry, respectively, as well as the popular Alexa Fluor dyes at these wavelengths, and popular dyes used to conjugate to secondary antibodies, such as FITC, TRITC, Texas Red, Cy3, Cy2, Cy5, etc. Excitation of DAPI is also possible on this system, but is not ideal.
The lasers have a common output, 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. Recently, the ANDOR iXon Ultra EMCCD Camera, with pixels 512 x 512, 16 µm pixels, BI + Vis AR coating, 10 MHz, cooled to -100º C, capable of imaging individual molecules conjugated with an appropriate fluorescent protein or dye.