The Plant Journal

In total internal reflection fluorescence (TIRF), the laser contacts the coverslip at a very shallow angle and gets completely reflected by the glass surface adjacent to the sample before it can hit the sample (right). This generates in the sample a very shallow evanescent field of the same wavelength as the laser, which excites the fluorophores in the sample.

One can also steepen the laser’s angle of incidence so it passes through the coverslip into the sample at a very shallow angle (centre). This essentially gives widefield fluorescence with much less out-of-focus illumination, yielding clearer images with less photodamage.

Mammalian cells with GFP localization in widefield (upper) and TIRF (lower). Sample preparation: Divya Dhasmana; principal investigator: Thorsten Wohland, Biophysical Fluorescence Laboratory; imaging: Tong Yan, Light Microscopy Core, Centre for Bioimaging Sciences.

TIRF has a large field of applications, such as single molecule imaging, imaging secretion processes, Fluorescence Recovery After Photobleaching (FRAP, FLIP), photoactivation, etc.

Comparison of LM techniques: