Totale interne reflectie fluorescentiemicroscopie

Total internal reflection fluorescence microscopy, or TIRF, selectively images fluorescent molecules in a sample close to a solid surface with a higher refractive index, such as a glass coverslip.

The excitation laser beam hits the coverslip at a critical angle, resulting in total internal reflection of the beam.

The reflected light does not enter the sample but creates a narrow electromagnetic field or an evanescent wave, which only illuminates fluorophores within 100 to 200 nanometers of the cell surface.

Because the evanescent wave rapidly decays as it moves further into the sample, most background fluorophores are not excited. So, the resulting images of the few fluorophore-tagged proteins near the solid surface are clear and sharp.

TIRF is routinely used for live-cell imaging, such as studying the movement of motor proteins like kinesin over the microtubules attached to the coverslip.