Total Internal Fluorescence Microscopy (TIRF)

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nm, can be observed.

1. Objective

2. Emission beam (signal)

3. Immersion oil

4. Cover slip

5. Specimen

6. Evanescent wave range

7. Excitation beam

8. Quartz prism

A TIRFM uses evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface. The evanescent wave is generated only when the incident light is totally reflected at the glass-water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium.

In optics, evanescent waves are formed when waves travelling in a medium undergo total internal reflection at its boundary because they hit it at an angle greater than the critical angle.

Good for cell surface structures. Can only focus on space right next to coverslip.