Multi-camera Simultaneous Total Internal Reflection and Interference Reflection Microscopy

Interference Reflection Microscopy (IRM) is an optical technique that relies on the interference between the reflected light from an incident beam as it passes through materials of different refractive indices. This technique has been successfully used to image microtubules, biologically important biofilaments with a diameter of 25 nm. However, it is often desirable to image both the microtubule and microtubule interacting proteins simultaneously. Here we present a simple modification to a standard multi-color total internal reflection fluorescence (TIRF) microscope that enables simultaneous high-speed IRM and single molecule TIRF imaging. Our design utilizes a camera for each channel (IRM and TIRF) allowing independent optimization of camera parameters for the two different modalities. We illustrate its application by imaging unlabeled microtubules and GFP-labeled end-binding protein EB1 which forms comets on the tips of polymerizing microtubules. Our design is easily implemented, and with minimal cost, making it accessible to any laboratory with an existing fluorescence microscope.