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Total Internal Reflection Fluorescence Microscopy

Optical microscopy is virtually the only means by which living cells and tissues can be studied with high spatial resolution. This has recently led to a return of light microscopy to the frontlines of biological research, with confocal, multiphoton, and total internal reflection fluorescence applications leading the way. Listed below are links to resources on the web for information about total internal reflection fluorescence microscopy (TIRFM) including microscope manufacturers, university laboratories, industrial imaging laboratories, technical white papers and tutorials.

Clive R. Bagshaw Laboratory - Housed in the Department of Biochemistry at the University of Leicester, Dr. Bagshaw's research interests include the molecular basis of muscular contraction studied with rapid reaction methodology. He also conducts an active research program in single molecule enzyme kinetics, fluorescence spectroscopy, including total internal reflection fluorescence microscopy.

BioElectroSpec - This corporation develops and manufactures spectroscopic and spectroelectrochemical biosensor systems for detection and control of biospecific protein-protein, receptor-ligand, protein-DNA, DNA-DNA, protein-membrane, and other biomolecular interactions. Their primary technologies are based on TIRF and ultraviolet-visible absorbance spectroscopy.

The Critical Angle - Part of a more extensive physics website entitled "The Physics Classroom", this discussion addresses the critical angle necessary for total internal refection. Specific examples and test questions are included.

Light Reflection and Refraction - Part of the Patterns in Nature section of the Arizona State University website, this discussion covers elementary aspects of total internal reflection, refraction, and reflection.

Molecular Motors Group - Based at the University of York, this group utilizes single molecule techniques to investigate the mechanisms of molecular motors. Among the techniques employed are total internal reflection fluorescence microscopy and laser tweezers.

Radiation pressure from evanescent wave measured - In an article written for OE Reports, British science writer Sunny Bains discusses a method of measuring light pressure to show the forces exerted by an evanescent wave. The system works measuring the 3D displacement of a particle to within a few nanometers and then factoring out other known forces to determine how much of the position change was due to radiation pressure.

Refraction and Reflection of Light - Sponsored by Bell College in the United Kingdom, this brief article is well illustrated and contains basic information about both phenomena.

Refraction, Snell's Law, and Total Internal Reflection - Sponsored by the Physics Department at Boston University, this site discusses specific details about the basic aspects of light refraction. The principles of Snell's law are covered as well as information about total internal reflection.

Single Molecule Imaging and Interaction Study Using Evanescent Wave Excitation - Available from International Scientific Communications in portable document format (PDF), this manuscript by X. Fang and W. Tan discusses how to configure a microscope for single molecule image using TIRF.

Synaptic Musings: Optical and biophysical methods allow a closer examination of the "nervous impulse" - Written by Susan Grammer, this article appears on the American Chemical Society website. The nicely written and illustrated text addresses examination of neurotransmitter release by synaptic vesicles using TIRFM.

Lukas K. Tamm Laboratory - Housed in the Molecular Physiology and Biological Physics Department at the University of Virginia, Dr. Tamm's laboratory investigates the structure and dynamics of membrane proteins and their interactions with lipid bilayers. The group employs total internal reflection fluorescence microscopy to study supported model membranes, membrane fusion, and membrane protein folding.

Total Internal Reflection - This site contains a nice discussion on the physics of total internal reflection, geared at the k-12 community. Topics covered include refraction, Snell's law, reflection, and total internal reflection with specific applications included as examples.

Total Internal Reflection - Sponsored by ThinkQuest, this discussion is part of Unit 8 in the Optics section. Topics covered include refraction, Snell's law, the critical angle, and total internal reflection. Examples include reflection in fiber optic pipes.

Total Internal Reflection - Complete with demonstrations and a thorough discussion of the phenomenon, this site is sponsored by the Physics Department at the University of Virginia.

Total Internal Reflection - A nice presentation with well-documented graphics, this short, but concise explanation of total internal reflection is sponsored by Georgia State University.

Total Internal Reflection Fluorescence Microscopy - This application note, written by Yoshihiro Kawano and Reinhard G. Enders of Olympus America Inc., reviews the basic principles of TIRFM with emphasis on instrumental configuration using high numerical aperture objectives.

Total Internal Reflection Microscopy - Written by Dr. Dennis C. Prieve, this tutorial is one of the most comprehensive treatments of TIRM available on the Internet. Also discussed are properties of the evanescent wave in the measurement of colloidal forces.

Tissue Culture Observation Chambers

Bellco Glass Culture Chambers - Bellco produces and distributes the Sykes-Moore culture chamber, designed to study of living cells by means of high magnification microscopy, phase contrast optics, TIRFM, or time lapse techniques. The chambers are used with stationary monolayer tissue cultures when the medium is changed intermittently.

Dvorak-Stotler Culture Chamber - The Dvorak-Stotler controlled environment culture chamber is an invaluable tool that offers the biomedical scientist the opportunity to fully utilize the light microscope for the in-depth study of biological materials. Specimen chambers of this type are ideal for imaging cells utilizing TIRFM techniques for cells grown in monolayer tissue culture.

Wilmad Glass - Specializing in high-quality optical glass and quartz cells for spectroscopy, Wilmad also produces rectangular cross-section microcapillary tubes and similar glass chambers that are ideal for observation of living cells with TIRFM.

Optical Components

Coherent Optics - Best known for its outstanding laser products, Coherent also distributes a large lineup of optical accessories including mirrors, beamsplitters, beam expanders, prisms, lenses, polarizers, and filters. The company also markets microscope components, pinholes, and apertures and has many downloadable PDF documents highlighting various products.

Newport Optics and Mechanics - Newport specializes in optical components, and is a distributor of prisms, beamsplitters, lenses, fiber optic couplers, vibration isolators, lasers, and detectors. The company's website also includes a series of tutorials on many aspects of optics, vibration control, image detection, and general photonics.

Rolyn Optics - In addition to coated and uncoated flint glass and standard index glass prisms, this optical company also distributes laser systems, beamsplitters, apertures, filters, laser beam expanders, mirrors, polarizers, and other components that are useful in configuring a microscope for TIRFM experiments.

Ross Optical Industries - Located in El Paso, Texas, Ross specializes in standard glass optical products such as lenses, mirrors, windows, prisms, and beamsplitters. The company also markets achromats, fused silica (quartz) lenses, and cylindrical lenses.

Turnkey TIRFM Systems

Olympus America - A leader in microscopes, optics, and digital imaging, Olympus offers a number of products that enable the investigator to construct a complete TIRFM instrument. Included are several high numerical aperture objectives, a laser illuminator, digital and classical photomicrography imaging, and both inverted and upright microscopes with infinity-corrected optical systems.

Till Photonics - Based in Germany, Till markets the Polychrome IV monochromatic xenon arc epi-fluorescence accessory, which is a multiwavelength illumination system that includes a monochromatizing device with an integrated light source. Coupled to a Imago camera system, proprietary imaging software, and an aftermarket laser system, Till products can be combined to produce a suitable instrument for TIRFM investigations.

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