Total internal reflection fluorescence microscopy for high-resolution imaging of cell-surface events.

The wavelength of light imposes a physical limit of approximately 400 nm on the maximum resolution that can be achieved using light microscopy. This unit will describe the use of total internal reflection fluorescence microscopy (TIR-FM), or evanescent wave microscopy, an approach that partially overcomes this physical limit and permits one to selectively image just those fluorophores in the optical plane (along the z axis) within 50 nm of the cell surface. TIR-FM works by means of limiting the depth of penetration of the excitation light within this narrow region. This narrow excitatory plane not only provides a high signal-to-noise ratio but also minimizes the photodamage to the cell.