  Several techniques have been employed for the direct visualization of
cytoskeletal filaments and their associated proteins.
Total-internal-reflection-fluorescence (TIRF) microscopy has a high
signal-to-background ratio, but it suffers from photobleaching and photodamage
of the fluorescent proteins. Label-free techniques such as interference
reflection microscopy (IRM) and interference scattering microscopy (iSCAT)
circumvent the problem of photobleaching but cannot readily visualize single
molecules. Here, we present a protocol for combining IRM with a commercial TIRF
microscope for the simultaneous imaging of microtubule-associated proteins
(MAPs) and dynamic microtubules in vitro. Our protocol allows for high-speed
observation of MAPs interacting with dynamic microtubules. This improves on
existing two-color TIRF setups by eliminating both the need for microtubule
labeling and the need for several additional optical components, such as a
second excitation laser. We image both channels on the same camera chip to
avoid image-registration and frame-synchronization problems. We demonstrate our
setup by visualizing single kinesin molecules walking on dynamic microtubules.
