Abstract
Single-molecule microscopy allows for the investigation of the dynamics of individual molecules and the visualization of subcellular structures at high spatial resolution. For single-molecule 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.
Highlights
Single-molecule wide-field microscopy experiments have been widely used for a broad range of investigations in cell-biological studies [1]
Being able to achieve sub-nanometer uncertainty is critical for our current purposes, as we seek to assess the performance of microscopes and optical components by determining differences in the positional estimates of imaged point sources that will typically be on the order of nanometers
The advent of single-molecule microscopy has enabled researchers to estimate the position of single molecules at the nanoscale, allowing the tracking of the dynamics of single molecules and the visualization of fine details of subcellular structures
Summary
Single-molecule wide-field microscopy experiments have been widely used for a broad range of investigations in cell-biological studies [1]. Localization-based super-resolution microscopy can yield quantitative information on the spatial distribution of a molecule of interest and the spatial characteristics of cellular structures smaller than the diffraction limit [6,7,8,9]. Both types of experiments importantly depend on the estimation of the position of single molecules with a low level of uncertainty (i.e., a small variance or standard deviation) [10]. Being able to achieve sub-nanometer uncertainty is critical for our current purposes, as we seek to assess the performance of microscopes and optical components by determining differences in the positional estimates of imaged point sources that will typically be on the order of nanometers
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