TIRF/FRAP Microscope

Equipment Configuration

Illumation

• ROPER laser bench
  • 405nm, 30 mW
  • 491nm, 50 mW
  • 561nm, 50 mW
• Monochromator

Detectors

• Camera : Evolve 512 mounted on the DualView (Photometrics)

Objectives

Bloc Filters

• ET-GFP/mCherry

Associated Devices

• Temperature box and CO2 controler (Life Imaging Service)
• FRAP Module iLAS2 (Roper Scientific)

Techniques

• Fluorescence
• Live Imaging (2D, 3D)
• TIRF
• FRAP, Photo-activation

Applications

• Cell Biology

Principle

Total Internal Reflection Fluorescence (TIRF) Microscopy

Various mechanisms are often employed in fluorescence microscopy applications to restrict the excitation and detection of fluorophores to a thin region of the specimen. Elimination of background fluorescence from outside the focal plane can dramatically improve the signal-to-noise ratio, and consequently, the spatial resolution of the features or events of interest. Total internal reflection fluorescence microscopy (TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices. In practice, the most commonly utilized interface in the application of TIRFM is the contact area between a specimen and a glass coverslip or tissue culture container.

The concepts underlying TIRFM are not new, and much of the recent interest in, and enthusiasm for, the technique have come about due to technological advances that facilitate its use. The availability of complete ready-to-use instrumentation systems for employment of the method, as well as developments in fluorophore technology, such as genetically encoded fluorescent species, have made it possible to investigate a number of cell membrane and other surface processes in a direct manner that was not previously possible. More...