Abstract
Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 μm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures - e.g. actin filaments, vesicles and mitochondria - in three dimensions.
Highlights
Over the past hundreds of years technical development in advanced microscopy designs and manufacturing improvement gave us the opportunity to visualize cells, cell organelles and other subcellular structures with high image quality and minimal aberration
The fluorescence intensities of single fluorescent molecules and nanoparticles registered by the two EMCCDs differ depending on the axial positions of the particles as a consequence of the anti-symmetric placement of the two spatial filters with respect to the focal plane
We demonstrated that the axial positions of fluorescent beads could be determined with a precision of 10–20 nm in x- and y- and 10–30 nm in z-direction by the usage of two spatial filters in the beam path of a two-camera super-resolution fluorescence microscope, which were placed before and behind the focal plane (figure 1(A)) the two otherwise symmetrical optical paths
Summary
Spatial filter and its application in threedimensional single molecule localization microscopy. View the article online for updates and enhancements. - Combination of structured illumination and single molecule localization microscopy in one setup Sabrina Rossberger, Gerrit Best, David Baddeley et al. - Understanding DNA organization, damage, and repair with super-resolution fluorescence microscopy Esther L Miriklis, Ashley M Rozario, Eli Rothenberg et al. - An introduction to optical super-resolution microscopy for the adventurous biologist J Vangindertael, R Camacho, W Sempels et al. This content was downloaded from IP address 163.1.196.105 on 29/01/2022 at 17:48
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