Imaging Dynamic Microtubules and Associated Proteins by Simultaneous Interference-Reflection and Total-Internal-Reflection-Fluorescence Microscopy

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.