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Total Internal Reflection Fluorescence Microscopy

Section Overview:

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.

Review Articles

  • 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 through stimulated emission, 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.

Interactive Java Tutorials

  • Excitation by an Evanescent Wave

    TIRF investigates the interaction of molecules with surfaces, which is important to many disciplines in cell and molecular biology. Explore TIRFM excitation of fluorophores residing in the membranes of living cells in tissue culture.

  • Evanescent Field Penetration Depth

    Explore penetration depth of the evanescent field as a function of refractive index differences between the two phases surrounding the interface, the critical angle of incident illumination, and the laser excitation wavelength.

  • Polarized Light Evanescent Intensities

    The light intensity at a TIRFM interface is a function of the illumination angle of incidence and polarization of the incident light. See how field intensities vary as a function of critical angle and the refractive index of the medium.

  • Variable Prism Configurations

    Explore Total Internal Reflection Fluorescence Microscopy with a variable prism that morphs between a trapezoidal and cubic geometry with adjustable side angles and refractive index in this interactive tutorial.

  • Trapezoidal Prism Microscope Configuration

    Discover and learn about the effects of variations in refractive index and prism side angles on the critical angle and resulting incident laser angles in this featured interactive java tutorial.

  • Olympus TIRFM Fiber Illuminator Alignment

    Explore alignment of the input fiber connector with the microscope optical path in order to optimize the incident light angle through high numerical aperture objectives in this interactive tutorial.

  • High Numerical Aperture Objectives

    TIRFM instrument configurations lacking a prism have been developed to retrieve fluorescence information emitted by the specimen. See the effect of objective numerical aperture on incident angles in TIRFM.

  • Substage Prism Microscope Configuration

    A TIRFM instrument configuration that is compatible with simultaneous microinjection or patch clamp experiments utilizes a substage prism. Explore multiple TIR by the laser illumination source in designs of this type.

  • Olympus IX70 Microscope Light Pathways

    Discover illumination pathways in the Olympus IX70 research inverted tissue culture microscope. The microscope drawing shown in the applet illustrates a cut-away diagram of the Olympus TIRFM-IX laser illuminator.

  • Evanescent Field Polarization and Intensity Profiles

    See how changes in the incident angle effect wave intensity and the relationships between the electric field vectors of parallel/perpendicular components of the incident beam.

Literature References and Web Resources

  • Selected Literature References

    Listed in this section contains periodical location information about these articles, as well as providing a listing of selected original research reports from this cutting-edge field of research.

  • TIRFM Resources on the Web

    Although not as plentiful as other techniques, web resources on total internal reflection fluorescence microscopy will probably grow as the field becomes more popular.

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.

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