Multicamera 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 multicolour total internal reflection fluorescence (TIRF) microscope that enables simultaneous high-speed IRM and single molecule TIRF imaging. Our design utilises a camera for each channel (IRM and TIRF) allowing independent optimisation of camera parameters for the two different modalities. We illustrate its application by imaging unlabelled microtubules and GFP-labelled end-binding protein EB1, which forms comets on the tips of polymerising microtubules. Our design is easily implemented, and with minimal cost, making it accessible to any laboratory with an existing fluorescence microscope.