Tool Manager: Carol Bayles
Location: Duffield 220
Total internal reflection fluorescence microscopy (TIRFM) exploits the unique properties of an induced evanescent wave in a very narrow region immediately adjacent to the interface between two media having different refractive indices. The most commonly utilized interface in the application of TIRFM is the contact area between an aqueous sample such as a cell and a glass coverslip.
When light strikes the interface of two media having different refractive indices at a sufficiently high angle, termed the critical angle, its refraction direction becomes parallel to the interface (90 degrees relative to the normal). At larger angles it is reflected entirely back into the first medium, creating the physical phenomenon of total internal reflection (TIR).
Although light no longer passes into the second medium, the reflected light generates a highly restricted electromagnetic field adjacent to the interface, in the lower-index medium. Fluorophores located in this vicinity can be excited by this evanescent field. The exponential falloff of the evanescent field intensity restricts the excitation of fluorophores to a region that is typically less than 100 nanometers in thickness. Since excitation of fluorophores in the bulk of the specimen is avoided, a very high signal-to-noise ratio is achieved, making it possible to detect single-molecule fluorescence by the TIRFM method.
- Nikon Ti-E/B automated inverted microscope with Perfect Focus and automated TIRF angle
- Nikon Elements software for image acquisition and controlling automated functions
- CFI APO 100x/1.45 oil immersion TIRF objective
- CFI Super Plan Fluor ELWD 40xc ADM long-working distance objective
- Andor iXON+ EMCCD Backthinned camera 512x512 (sufficient sensitivity for single molecule fluorescence measurements)
- Lasers at 405, 488, 561, 635 nm (up to 3 laser lines can be used simultaneously)
- X-Cite UV illuminator for conventional epifluorescence
- Transmitted light imaging with halogen lamp.
- Semrock Filter cubes for each laser line (405, 488, 561, 635), and a multicolor cube (405/488/561/635)
- Adaptable to FRET, FRAP, PALM experiments
- Can be combined with PicoPlus fluid atomic force microscope (AFM) measurements
Instrument protocols in the "downloads" menu to the right.