About the Total Internal Reflection Fluorescence (TIRF) Microscope

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

The special feature of TIRF microscopy is the employment of an evanescent field for fluorophore excitation. Unlike standard widefield fluorescence and laser scanning confocal microscopy, the evanescent field only excite fluorophores that are present at the bottom of the specimen adjacent to the glass water interface by about 100 nm -200nm starting from the coverslip/medium interface. This allows TIRF to gain a very high axial resolution. 

The following is a comparison of the few mentioned LM techniques mentioned above.

While TIRF microscopy requires the critical angle to establish an evanescence field that excites nearby fluorophores, users can conduct wide-field acquisition taking advantage of a tilted illumination to lower background blur that results from out of focus planes and to enhance the excitation illumination. This tilted/oblique illumination sectioning applies high incident angles but smaller than the critical angle, starting the TIRF domain, the angle of the excitation beam going through the sample is so high that the illuminated thickness is very thin (around 2μm), illustrated by the diagram below, with reference to the variable-angle epifluorescence (Courtesy of The Plant Journal).