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Microscopy Reference Library

Total Internal Reflection Fluorescence Microscopy (TIRFM)

Often referred to as evanescent wave microscopy, total internal reflection fluorescence microscopy (TIRFM) is proving to be a powerful technique for examining phenomena occurring at the plasma membrane in living cells and for imaging single molecules. TIRFM has grown in utility and popularity as manufacturers have provided increasingly sophisticated turnkey instrumentation coupled to advanced software interfaces. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of TIRFM and related methodology.

Schneckenburger, H.

Total internal reflection fluorescence microscopy: technical innovations and novel applications.  Current Opinion in Biotechnology 16: 13-18 (2005).  An excellent review that addresses current topics, including enhanced depth resolution, reduction of detection volumes, single molecules, as well as the biologically-relevant imaging techniques of endocytosis, focal adhesions, and ion channels. The article also discusses the application of synthetic fluorophores and fluorescent proteins to TIRFM.

Axelrod, D.

Total internal reflection fluorescence microscopy in cell biology.  Traffic 2: 764-774 (2001).  A review of TIRFM by the investigator who established the technique in the biological sciences. Dr. Axelrod discusses key physical concepts in TIRF and describes how the technique can be applied to the study of biochemical kinetics and single molecule dynamics at surfaces. Included is a description of several applications.

Toomre, D. and Manstein, D. J.

Lighting up the cell surface with evanescent wave microscopy.  Trends in Cell Biology 11: 298-303 (2001).  The authors discuss how TIRFM is being applied to important cellular processes taking place near the plasma membrane. Included is an overview of the technique, a discussion of advantages, key formulas, microscope configuration parameters, and several application examples relevant to cell biology.

Axelrod, D., Burghardt, T. P. and Thompson, N. L.

Total internal reflection fluorescence.  Annual review of Biophysics and Bioengineering 13: 247-268 (1984).  Written over a quarter century ago, this review was the first comprehensive treatise on TIRFM. The authors exhaustively describe TIR theory, detection strategies, numerous microscope optical designs, and numerous relevant applications.

Wazawa, T. and Ueda, M.

Total internal reflection fluorescence microscopy in single molecule nanobioscience.  Advances in Biochemical Engineering/Biotechnology 95: 77-106 (2005).  A comprehensive discussion of single molecule TIRF techniques that includes extensive sections on theory and instrumentation. Numerous applications in single molecule biology are also addressed, including motor proteins, enzymatic reactions, conformational dynamics, ion channels, and imaging in living cells.

Konopka, C. A. and Bednarek, S. Y.

Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex.  The Plant Journal 53: 186-196 (2008).  Presented by the authors as an alternative to TIRFM, this report describes a highly oblique illumination technique that utilizes subcritical incident angles to decrease background fluorescence. Included is a discussion of advantages over TIRFM and applications in living plant cells.

Tokunaga, M., Imamoto, N. and Sakata-Sogawa, K.

Highly inclined thin illumination enables clear single-molecule imaging in cells.  Nature Methods 5: 159-161 (2008).  An original research report describing illumination using a highly inclined, thin beam to increase image intensity while simultaneously decreasing background to provide high signal-to-noise ratios. The authors creatively apply this advanced TIRF technique to obtain high-resolution single molecules images in living cells.

Shaw, J. E., Oreopoulos, J., Wong, D., Hsu, J. C. Y. and Yip, C. M.

Coupling evanescent-wave fluorescence imaging and spectroscopy with scanning probe microscopy: challenges and insights from TIRF-AFM.  Surface and Interface Analysis 38: 1459-1471 (2006).  A thorough review on the integration of TIRFM with scanning probe microscopy for investigations of biological phenomena at interfaces. Included are discussions of resolution, TIRFM and scanning probe microscopy theory, instrumentation requirements, and techniques for image analysis.

Kobitski, A. Y., Heyes, C. D. and Nienhaus, G. U.

Total internal reflection fluorescence microscopy-a powerful tool to study single quantum dots.  Applied Surface Science 234: 86-92 (2004).  The authors describe TIRFM measurements using colloidal CdSe quantum dots at high resolution. Discussed are quantum dot blinking phenomena, extracting data from images, and an explanation of the observed behavior in terms of current physical models of quantum ionization and neutralization.

Axelrod, D.

Selective imaging of surface fluorescence with very high aperture microscope objectives.  Journal of Biomedical Optics 6: 6-13 (2001).  The original paper describing prismless "through-the-objective" TIRFM with high numerical aperture microscope objectives. Included are several schematic diagrams of microscope configurations, experimental demonstrations, and practical suggestions for implementing this technique.