Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy (TIRFM) is an elegant optical technique utilized to observe single molecule fluorescence at surfaces and interfaces. The technique is commonly employed to investigate the interaction of molecules with surfaces, an area which is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology.
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Total Internal Reflection Fluorescence Microscopy

Introduction and Theoretical Aspects

The basic concept of TIRFM is simple, requiring only an excitation light beam traveling at a high incident angle through the solid glass coverslip or plastic tissue culture container, where the cells adhere.

Basic Microscope Configuration

A wide spectrum of optical configurations was placed under scrutiny during the early stages of instrument development for total internal reflection fluorescence microscopy investigations (TIRFM).

TIRFM - Olympus Application Note

Olympus has designed a new high numerical aperture apochromatic objective specifically matched for total internal reflection fluorescence microscopy at high critical angles.

Alignment of Prism-Based TIRF Systems

The guidelines presented in this section are intended to serve a starting point offering an outline of the basic requirements for TIRFM microscope configuration using a prism, laser light source, and a focusing lens.

Alignment of Objective-Based TIRF Systems

Total internal reflection can be investigated utilizing high numerical aperture objectives (ranging between 1.4 and 1.65 in aperture), preferentially using an inverted tissue culture microscope.

Laser Fundamentals

Explained in this section are how basic laser systems operate throughstimulatedemission, and how they are designed to amplify this form of light to create intense and focused beams.

Laser Systems for Optical Microscopy

The lasers employed in optical microscopy are high-intensity monochromatic light sources, which are useful for a variety of techniques including lifetime imaging studies, photobleaching recovery, and TIRFM.

Olympus IX70 Microscope Cutaway Diagram

The Olympus IX70 inverted tissue culture microscope is a research-level instrument capable of imaging specimens in a variety of illumination modes including brightfield, darkfield, phase contrast, fluorescence, and DIC.

Contributing Authors

Daniel Axelrod - Department of Biophysics, University of Michigan, 930 North University Ave., Ann Arbor, Michigan 48109.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Mortimer Abramowitz, William K. Fester, Yoshihiro Kawano, and Reinhard G. Enders - Olympus America, Inc., Two Corporate Center Drive, Melville, New York, 11747.
John C. Long, Brian O. Flynn, Matthew Parry-Hill, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.