A protocol for registration and correction of multicolour STED superresolution images

• Published in: Journal of microscopy (Oxford) 2017-08, Vol.267 (2), p.160-175
• Main Authors: HEBISCH, E., WAGNER, E., WESTPHAL, V., SIEBER, J.J., LEHNART, S.E.
• Format: Article
• Language: English
• Subjects: Aberration; Calcium; Calcium release channels; Cardiomyocytes; Color; Depletion; Diffraction; Emissions control; Fluorescence; Fluorescent dyes; Fluorescent indicators; Image acquisition; Image quality; Image registration; Imaging; Laser beams; Light diffraction; Localization; Microscopy; multicolour imaging; Optimization; Proteins; Quantitative analysis; Registers; Registration; Reproducibility; ryanodine receptor; Staining; STED microscopy; Stimulated emission; targeted superresolution microscopy
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1111/jmi.12556

Summary Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut‐shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co‐localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross‐correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co‐staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user‐friendly criteria, which – if fulfilled – support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co‐localisation analyses. Although the reference protocol is discussed exemplarily for two‐colour STED imaging, it can be readily expanded to three or more colours and STED channels. Lay description Multicolour imaging with fluorescent dyes was recently realized below the diffraction limit based on superresolution light microscopy. The first described method, STimulated Emission Depletion (STED), represents a targeted spatial approach for superresolution microscopy using doughnut‐shaped STED laser beams. For multicolour STED imaging, each colour channel requires precise image registration. This is particularly important when STED imaging is used for co‐