DescriptionConventional microscopy allows robust imaging of cells used in many laboratories. Relatively large cell features such as the nucleus and some organelles are observable, but not clearly distinguishable. Best contrast is possible via fluorescence microscopy by labeling of individual proteins with fluorescent dyes. However, only a resolution of about 250 nm is possible due to the diffraction limit and details like the actin cytoskeleton cannot be resolved. Single Molecule Localization Microscopy (SMLM) based on direct Stochastic Optical Reconstruction Microscopy (dSTORM) overcomes this limitation and allows a resolutions down to 15 nm . By mounting a cylindrical lens in the optical detection pathway, dSTORM can be extended to the third dimension. Nevertheless, for imaging of the whole cell or even cell clusters the astigmatic point spread function (PSF) is insufficient in the axial direction and the sample has to be scanned axially at defined steps. Thereby, the sample is divided in multiples slices consisting of 10 000 - 20 000 frames of stochastically blinking single molecules at a defined axial step. However, due to the refractive index mismatch between sample medium and objective, the PSF is asymmetrically axially distorted due to spherical aberration. These aberrations cause large gaps between observed and real axial positions, especially for large image depths. We developed software tools for correction of these aberrations which allows the application of standard software for astigmatic 3D Super Resolution Microscopy analysis (e. g. RapidSTORM, ThunderSTORM, 3D STORM Tools). The calibration needed for z-position determination based on the elliptical distortion of the PSF is recalculated using an enhanced modeling of the PSF which accounts for the refractive index mismatch. The PSF model is determined by phase retrieval of the original calibration stack of single molecules using algorithms based on ZOLA-3D . The calibration PSF for each slice is simulated using the information from the phase retrieval and applied to create refractive-index-corrected calibrations at the specific axial height. Therefore, a reconstruction of the whole sample with less artifacts is yielded.
|Period||6 Jun 2019|
|Event title||YSESM 2019 - 17th Youth Symposium on Experimental Solid Mechanics: null|
|Location||Telč, Czech Republic|