TIRFM-Description and Images

TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY (TIRFM)

TIRFM selectively illuminates subcellular features close (within ~300 nm) to the plasma membrane at cell-glass coverslip contact regions without interference from optical signals from deeper within the cell. When light traveling through glass passes through an aqueous interface it is bent (refracted). As the angle of incidence of the light increases, the light propagated into the aqueous layer is increasingly bent until a 'critical angle' is achieved when light is no longer propagated. Beyond this critical angle two phenomena occur:

· Propagated light is totally reflected back into the glass at the interface.

· An 'evanescent' field (light) penetrates into the aqueous phase that decays exponentially with a characteristic distance 'd', which depends on the indices of refraction of the glass and aqueous phase, the incident angle and the wavelength of the light. The characteristic distance in our experiments is ~80 nm.

The fluorophores within the evanescent field are selectively excited. Because the intensity of the excitation light decays rapidly with distance from the glass interface, movements of labeled organelles perpendicular to glass interface of as little as 4 nm (the chromaffin granule diameter is 300 nm) can be accurately measured by determining changes in organelle fluorescent intensity with time. TIRFM also has the potential to detect protein-protein interactions in the vicinity of the plasma membrane by using fluorescence resonance energy transfer (FRET).

See following figures.