Total internal reflection is an optical property that is harnessed for a number of practical applications. In molecular cell biology, total internal reflection fluorescence (TIRF) microscopy is an invaluable tool for imaging events close to the cell surface.
When light (or any propagating wave) reaches a medium boundary, it can be reflected or refracted. Total internal reflection occurs when the wave strikes the boundary above a particular critical angle. At this point, the wave is not refracted at all but is instead completely (totally) reflected. For total internal reflection to occur, the wave must be first travelling in a medium with a higher refractive index than the second medium it encounters – for example, a wave travelling in glass (such as a coverslip) will be totally internally reflected when it meets air (at the coverslip boundary).
Although the wave is reflected, it results in the creation of an evanescent field a very short distance on the other side of the boundary. This evanescent field decays exponentially with distance from the boundary.
TIRF microscopy exploits these principles to image cells mounted on a coverslip. A short wavelength light beam is reflected within the coverslip, at the coverslip-water interface, resulting in the generation of an evanescent field a very short distance (~100 nanometres) on the other side of the interface. This evanescent field will excite fluorophores very close to the surface of the cell. In living cells, this can be used to track the internalisation of macromolecules, signalling events, and other dynamic processes occurring at the cell surface.
The blog’s logo depicts a totally internally reflected light wave, with the letter “I” within the evanescent field.
Ironically, although I have extensive experience in light and fluorescence microscopy, I have not (at the time of writing) ever used TIRF microscopy myself. For why I thought it would be a good title for the blog, see the “Mandate” page.
More material (much of it paraphrased here) can be found on the following Wikipedia pages: