Abstract
AbstractBackgroundNovel microscopic techniques which bypass the resolution limit in light microscopy are becoming routinely established today. The higher spatial resolution of super-resolution microscopy techniques demands for precise correction of drift, spectral and spatial offset of images recorded at different axial planes.MethodsWe employ a hydrophilic gel matrix for super-resolution microscopy of cellular structures. The matrix allows distributing fiducial markers in 3D, and using these for drift correction and multi-channel registration. We demonstrate single-molecule super-resolution microscopy with photoswitchable fluorophores at different axial planes. We calculate a correction matrix for each spectral channel, correct for drift, spectral and spatial offset in 3D.Results and discussionWe demonstrate single-molecule super-resolution microscopy with photoswitchable fluorophores in a hydrophilic gel matrix. We distribute multi-color fiducial markers in the gel matrix and correct for drift and register multiple imaging channels. We perform two-color super-resolution imaging of click-labeled DNA and histone H2B in different axial planes, and demonstrate the quality of drift correction and channel registration quantitatively. This approach delivers robust microscopic data which is a prerequisite for data interpretation.
Highlights
Novel microscopic techniques which bypass the resolution limit in light microscopy are becoming routinely established today
Distribution of fiducial markers in 3D Fiducial markers were frequently used for drift correction and image registration in single-molecule localization microscopy (Betzig et al 2006, Churchman et al 2005, Rust et al 2006, Shtengel et al 2009)
For drift correction and image registration, fiducial markers should be available in every imaging plane, and should not be attached to the target structure to avoid artifacts in single-molecule localization
Summary
Novel microscopic techniques which bypass the resolution limit in light microscopy are becoming routinely established today. Various techniques that bypass the resolution limit in light microscopy were established for cellular imaging (Galbraith and Galbraith 2011, Heilemann 2010, Hell 2009). These techniques, commonly summarized as super-resolution microscopy, resolve cellular structures up to the near-molecular level and still profit from the advantages of fluorescence microscopy, such as high contrast and live cell compatibility. At these small spatial scales, accurate correction for drift and spatial or spectral offsets is required. Drift correction through image correlation was demonstrated for 3D singlemolecule localization microscopy (McGorthy et al 2013)
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