Advancements in Single Molecule Localization Microscopy for Improved Multichannel, 3-Dimensional, and Structurally-Dense Imaging

Abstract

Super-resolution fluorescence microscopy now enables cellular detail to be probed at the nanometer scale using far-field optics with widely available instrumentation. However, single molecule localization microscopy (SMLM) techniques that rely on the stochastic on-off switching of individual fluorophores to achieve higher resolution face significant drawbacks when trying to achieve multicolor, 3-D, and structurally-dense imaging. This is due to limitations in the photoswitchable fluorophores currently available. Here, I provide a detailed discussion of photoswitchable fluorophores for SMLM and the challenges they create. I also present a new method, prSTORM, which is capable of using a single fluorophore to image all channels and achieve 3-D imaging at extended depths through the use of DNA barcodes. Additionally, I propose how this methodology can be extended to high structural density imaging without compromising resolution.