3D active stabilization for single-molecule imaging
- 2 December 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 16 (1), 497-515
- https://doi.org/10.1038/s41596-020-00426-9
Abstract
A key part of any super-resolution technique involves accurately correcting for mechanical motion of the sample and setup during acquisition. If left uncorrected, drift degrades the resolution of the final reconstructed image and can introduce unwanted artifacts. Here, we describe how to implement active stabilization, thereby reducing drift to ~1 nm across all three dimensions. In this protocol, we show how to implement our method on custom and standard microscopy hardware. We detail the construction of a separate illumination and detection path, dedicated exclusively to acquiring the diffraction pattern of fiducials deposited on the imaging slide. We also show how to focus lock and adjust the focus in arbitrary nanometer step size increments. Our real-time focus locking is based on kHz calculations performed using the graphics processing unit. The fast calculations allow for rapid repositioning of the sample, which reduces drift below the photon-limited localization precision. Our approach allows for a single-molecule and/or super-resolution image acquisition free from movement artifacts and eliminates the need for complex algorithms or hardware installations. The method is also useful for long acquisitions which span over hours or days, such as multicolor super resolution. Installation does not require specialist knowledge and can be implemented in standard biological laboratories. The full protocol can be implemented within ~2 weeks.Keywords
Funding Information
- Department of Education and Training | Australian Research Council (CE140100036, FL150100060, CE140100011)
- Department of Health | National Health and Medical Research Council (APP1059278)
This publication has 57 references indexed in Scilit:
- Non-Bias-Limited Tracking of Spherical Particles, Enabling Nanometer Resolution at Low MagnificationBiophysical Journal, 2012
- How the biotin–streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramerBiochemical Journal, 2011
- Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread functionProceedings of the National Academy of Sciences, 2009
- Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructureProceedings of the National Academy of Sciences, 2009
- Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolutionNature Methods, 2008
- Live-cell photoactivated localization microscopy of nanoscale adhesion dynamicsNature Methods, 2008
- New directions in single-molecule imaging and analysisProceedings of the National Academy of Sciences of the United States of America, 2007
- Imaging Intracellular Fluorescent Proteins at Nanometer ResolutionScience, 2006
- Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)Nature Methods, 2006
- Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolutionProceedings of the National Academy of Sciences of the United States of America, 2005