Integrated multi-wavelength microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes
Daguan Nong, Zachary K. Haviland, Kate L. Vasquez Kuntz, Ming Tien, Charles T. Anderson, William O. Hancock
Abstract
We describe a multimodal microscope for visualizing processive enzymes moving on immobilized substrates. The instrument combines interference reflection microscopy (IRM) with multi-wavelength total internal reflectance fluorescence microscopy (TIRFM). The microscope can localize quantum dots with a precision of 2.8 nm at 100 frames/s, and was used to image the dynamics of the cellulase, Cel7a interacting with surface-immobilized cellulose. The instrument, which was built with off-the-shelf components and is controlled by custom software, is suitable for tracking other degradative enzymes such as collagenases, as well as motor proteins moving along immobilized tracks.