Microscope calibration protocol for single-molecule microscopy

Single-molecule microscopy allows for the investigation of the dynamics of individual molecules and the visualization of subcellular structures at high spatial resolution. For singlemolecule imaging experiments, and particularly those that entail the acquisition of multicolor data, calibration of the microscope and its optical components therefore needs to be carried out at a high level of accuracy. We propose here a method for calibrating a microscope at the nanometer scale, in the sense of determining optical aberrations as revealed by point source localization errors on the order of nanometers. The method is based on the imaging of a standard sample to detect and evaluate the amount of geometric aberration introduced in the optical light path. To provide support for multicolor imaging, it also includes procedures for evaluating the geometric aberration caused by a dichroic filter and the axial chromatic aberration introduced by an objective lens. Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI.

Automated summary

Single-molecule microscopy allows for high-resolution visualization of subcellular structures and investigation of individual molecular dynamics. Calibrating the microscope for multicolor imaging experiments at a nanometer scale is crucial for accurate results. This calibration method involves imaging a standard sample to detect and evaluate optical aberrations, including those caused by a dichroic filter and objective lens, to ensure high-quality imaging.