Efficient homogeneous illumination and optical sectioning for quantitative single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) relies on the switching of fluorescent molecules between a fluorescent and a dark state to achieve super resolution. This process is inherently dependent on the intensity distribution of the laser light used for both activation from the dark state and excitation of the bright state. Typically, laser light is coupled directly or via a single-mode fiber into the microscope, which leads to a Gaussian intensity profile in total internal reflection (TIR) or epi illumination. As a result, switching dynamics and brightness of the fluorescent molecules vary strongly across the field of view, impacting their localization precision and impeding quantitative analysis. Here we present a simple illumination scheme based on the use of a multimode fiber and a laser speckle-reducer, which results in a flat, homogeneous and speckle-free illumination across the entire field of view. In addition, we combined homogeneous multimode excitation of the sample with single-mode based TIR activation to simultaneously obtain the advantages of both approaches: uniform brightness of single fluorophores and TIR-like optical sectioning. (C) 2016 Optical Society of America