Measuring Nanometer Distances Between Fluorescent Labels Step-by-Step.

Super-resolution fluorescence microscopy methods are increasingly applied to study the structure of biological molecules within their natural context or at biomaterial interfaces. We here provide a protocol for Single-molecule High-Resolution Imaging with Photobleaching (SHRImP) that can be used to obtain information about the conformation of large proteins or other macromolecules at the single-molecule level. This procedure requires site-specific protein labeling with fluorescent dyes, immobilization and sample preparation, optimization of imaging buffer composition and microscope settings, and acquisition of short time-lapse movies that capture the stepwise bleaching behavior of individual molecules. We then describe a method for reliably determining the relative positions of labels from bleaching movies using the free image processing package Fiji (ImageJ) with the help of auxiliary macros that are provided as Supplementary Material. The presented approach allows for measuring intramolecular distance distributions in the range of a few to hundreds of nanometers and can be applied to a wide variety of biological systems.