Key Words: time-lapse imaging, widefield, GFP, fluorophore, N.A., oil immersion, SRIC, resolution, signal-to-noise-ratio, fluorescence, TIRF, environmental control
Definition:White-light TIRF is a form of TIRF microscopy that uses a mercury light source for excitation
TECHNOLOGY:
As in TIRF imaging, white-light TIRF exploits total internal reflection to confine excitation of fluorophores to a very thin plane at the contact between the specimen and the cover glass or tissue culture vessel. By confining excitation energy to this thin section, a high signal-to-noise-ratio is obtained, making it possible to observe single molecule fluorescence.
The use of a mercury light source for TIRF enables easy switching between white-light TIRF, epi-fluorescence, SRIC and thin-layer fluorescence (where increasing the angle of incidence to slightly more than that used in TIRF allows a deeper range of observation in the area close to the coverslip). As mercury illumination has a broad wavelength range, switching filters enables TIRF observations at a variety of wavelengths.
APPLICATIONS:
As with other TIRF modes, white-light TIRF enables nanometre-scale observations of molecular dynamics in living cells.
MICROSCOPE CONFIGURATION:
White-light TIRF can be configured on Nikon's Ti series inverted microscopes.
RECOMMENDED SYSTEM:
Nikon Ti series inverted microscope with environmental control, Nikon's dedicated TIRF Apo 60x NA 1.49 oil and TIRF Apo 100x 1.49 oil immersion objectives, and high performance epi-filter cassette holder. The TIRF Apo objectives correct for spherical aberration induced by temperature changes. Appropriate light sources include 100W mercury lamp and Nikon's Intensilight mercury-fibre illumination system
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