Abstract
While fluorescence microscopes and atomic force microscopes are widely used to visualize, track, and manipulate single biomolecules, the resolution of these methods is limited by sample drift. To minimize drift, active feedback methods have recently been used to stabilize single molecule microscopes on the sub-nanometer scale. However, these methods require high intensity lasers which limits their application in single molecule fluorescence measurements. Furthermore, these feedback methods do not track user-defined regions of the sample, but rather monitor the relative displacement of an unknown point on a fiducial marker, which limits their use in biological force measurements. To overcome these limitations, we have developed a novel method to image, track and stabilize a sample using low laser intensities. We demonstrate the capabilities of our approach by tracking a user-chosen point on a fiducial marker at 8.6 kHz and stabilizing it with sub-nanometer resolution. We further showcase the application of our method in single molecule fluorescence microscopy by imaging and stabilizing individual fluorescently-tagged streptavidin proteins under biologically relevant conditions. We anticipate that our method can be easily used to improve the resolution of a wide range of single molecule fluorescence microscopy and integrated force-fluorescence applications.
Highlights
662-Pos Molecular Counting by Photon Statistics in Confocal Fluorescence Imaging Marcelle Koenig, Caroline Berlage, Paja Reisch, Christian Oelsner, Felix Koberling, Haisen Ta, Rainer Erdmann
We find that elastic fibers can be visualized by Third harmonic generation (THG) microscopy using Verhoeff Van Gieson (VVG) staining while collagen is directly highlighted by THG imaging of picrosirius red staining
We show that the coupling of cellular volumes and cell cycle times in combination with a mechanically guided arrangement process is key for a fail-safe embryogenesis of C. elegans [Biophys
Summary
662-Pos Molecular Counting by Photon Statistics in Confocal Fluorescence Imaging Marcelle Koenig, Caroline Berlage, Paja Reisch, Christian Oelsner, Felix Koberling, Haisen Ta, Rainer Erdmann. As tissue inherently exists as a three dimensional structure, it is important to develop histological methods to investigate it in such form while maintaining the high spatial resolution of light microscopy. Using single plane illumination microscopy (SPIM) and simulations, we have explored how physical cues determine the cell arrangement in the early embryogenesis of C. elegans.
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