Single molecule fluorescence imaging is a highly powerful technique to understand how proteins physically perform their function in a time course. To be able to observe single proteins, we specifically label them with organic fluorophores and shine a laser light to observe the emitted fluorescent light under a microscope. The emitted light is collected with a highly sensitive CCD camera for quantitative analysis.
Our fluorescence microscope is equipped with 488 nm, 532 nm and 633 nm laser lines for the excitation of wide variety of fluorescent probes. All three lasers are combined to follow the same optical path and focused to the back focal plane of the microscope objective (Nikon 100X, 1.49 NA). The beam is then shifted laterally along the focal plane that creates tilted and parallel laser beam emitted from the microscope objective. When the tilt is larger than the critical angle, transmitted light will be total internally reflected back from the glass/water boundary. Total Internal Reflection (TIR) of light creates an evanescent field which penetrates only 100 nm into the flow chamber.
TIRF is ideal to study surface immobilized cells and in vitro systems since it results in minimal fluorescent background and sensitively detects fluorescent photons. Compared to the scanning confocal setup, TIRF is a wide field illumination technique that readily allows simultaneous observation of many single molecules on the surface. Fluorescent photons are filtered and collected by an EMCCD camera (Ixon EM+, Andor Technology) which can detect 92% of incoming photons with a minimal electronic noise.
To specifically label the motors, we generated a cysteine-light dynein motor and conjugated it with malemide reactive probes. We also use genetic tags (e.g. HaloTag and SNAP Tag) to covalently link motors with TMR or biotin. To achieve high signal to noise ratio, we label biotinylated proteins with a streptavidin coated quantum dot that is ~20 times brighter and more stable than Cy3 and TMR dyes. Labeled motors were introduced into the flow chamber after immobilizing microtubules on the glass surface. In the presence of limited ATP, individual steps of motors can be discerned.