TIRF Microscopy

Total Internal Reflections Fluorescence Microscopy is a special application of confocal (fluorescence) microscopy. TIRF microscopy typically uses a high numerical aperture (NA) objective.

by Dr. Jakob Bierwagen, AHF analysentechnik AG

Internal reflection of the excitation beam

Into this objective, the excitation light is coupled with an annular intensity distribution. This ensures that the light strikes the interface between cover glass and sample only at a very shallow angle.

Better Resolution

Since the refractive index of water (1.33) is lower than that of the cover glass (1.53), total internal reflection of the excitation beam occurs. Only the evanescent field of the excitation light reaches 100 – 200 nm into the sample. As a result, only the fluorescent markers that are within this evanescent field are excited. Consequently, a much better resolution of the images in the Z-dimension is achieved than would be achievable with normal confocal microscopy (500 – 600 nm).

Matching beamsplitters and filter sets for TIRF

However, a drawback is that one can only record directly on the cover glass. But this is e.g. interesting, if you want to observe processes directly on the cell membrane. In order to achieve the necessary beam quality for the annular intensity distribution, particularly flat beam splitters are required. As a rule, beam splitters are used which have a flatness of <λ/5. AHF offers you beam splitters of high quality and matching filter cubes for TIRF to make your experiment a success.