Total internal reflection fluorescence microscopy (TIRFM) was used to image single molecules with evanescent waves.
The molecules were excited by an evanescent wave with different intensities. Single molecules were imaged on
low-noise high-quantum-yield charge-coupled device (CCD) cameras. Two-step photobleaching behavior was observed.
Duration from fluorescent spots appearing to disappearing was counted. The duration was decided by the speed of
photobleaching. Peak intensity was counted as exciting intensity change. The proportion relationship between the
reciprocal of duration and exciting intensity was obtained. The emitted intensity and duration of fluorescein were
compared with GFP. A single molecules emit the same number photons was proved.
Single-molecule/particle tracking technology is becoming a powerful tool to the noninvasive study of membrane
property and membrane molecular processes. Here, we used total internal reflection fluorescence microscopy combined
with oblique illumination fluorescence microscopy to investigate green fluorescence protein within living cell. Total
internal reflection illumination allowed the observation of molecules in the cell membrane of living cell since the
penetrate depth is adjusted to about 100nm, and oblique illumination is allowed the observation of molecules/particles
both in the cytoplasm and apical membrane, which suggested this combination would be promising to investigate protein
dynamics through the whole cell. Not only individual protein molecule/particle tracks have been analyzed quantitatively
to reveal the motion of protein, but also statistical analysis has been done to substantiate the observation of protein
dynamics. Our data that involved up to 700 trajectories in several hundreds cells are indicated that at least four modes of
motion are presented, that is directed motion, normal diffusion (random walk), binding motion and corralled motion.
Detailed analysis as well as statistical analysis is presented in the paper.
The two-photon absorption properties of a new dye have been experimentally studied using the comparable two-photon induced fluorescence method with a pulsed titanium-sapphire laser. This new dye has a two-photon absorption cross-section of 1.7 x 10-48 cm4s/photon at 790nm.