Waveguide-based total internal reflection fluorescence microscope enabling cellular imaging under cryogenic conditions.

Abstract

Total internal reflection fluorescence (TIRF) microscopy is an important imaging tool for the investigation of biological structures, especially the study on cellular events near the plasma membrane. Imaging at cryogenic temperatures not only enables observing structures in a near-native and fixed state but also suppresses irreversible photo-bleaching rates, resulting in increased photo-stability of fluorophores. Traditional TIRF microscopes produce an evanescent field based on high numerical aperture immersion objective lenses with high magnification, which results in a limited field of view and is incompatible with cryogenic conditions. Here, we present a waveguide-based TIRF microscope, which is able to generate a uniform evanescent field using high refractive index waveguides on photonic chips and to obtain cellular observation at cryogenic temperatures. Our method provides an inexpensive way to achieve total-internal-reflection fluorescence imaging under cryogenic conditions.