We have studied the dynamics of certain key processes near the plasma membrane inside two types of chemically-triggerable living cells using total internal reflection fluorescence microscopy (TIRFM). In TIRFM, a laser beam is incident upon the cell/glass-substrate interface from the glass side at an angle greater than the critical angle for total internal reflection. This creates an exponentially decaying evanescent field in the cell medium (with a characteristic depth of > 100 nm) capable of exciting fluorescence selectively from the membrane-proximal regions at cell/substrate contacts. Various ways of setting up the optics for such a system are discussed, involving the use of either prisms or very high aperture objectives.In one application of TIRFM, the motion of adrenalin-containing secretory granules in the immediate submembrane region of chromaffin cells is examined before and after chemical stimulation that causes the granules to release their contents to the cell exterior.
Digital holography of total internal reflection
