Abstract
Single molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample's ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm.
Highlights
In the last decade, super-resolution microscopy techniques have paved the way for resolving cellular structures in unprecedented detail [1]
The technique of single molecule localization microscopy (SMLM) appears well suited for structural biology, as it is based on localization coordinates of individual molecules rather than on pixelated images of recorded fluorescence intensities
In SMLM, dye molecules are linked to the biomolecule of interest and imaged under conditions, where only a small subset of dye molecules is visible at any time-point
Summary
Super-resolution microscopy techniques have paved the way for resolving cellular structures in unprecedented detail [1]. The assembly of biomolecules at the nanoscale plays a crucial role in their functionality and is key to our understanding of cellular processes. The technique of single molecule localization microscopy (SMLM) appears well suited for structural biology, as it is based on localization coordinates of individual molecules rather than on pixelated images of recorded fluorescence intensities. In SMLM, dye molecules are linked to the biomolecule of interest and imaged under conditions, where only a small subset of dye molecules is visible at any time-point.
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