Spectral STORM: Multi-colour super-resolution imaging with a single excitation laser
Creators: name: Walde, Marie
name: Woehler, Andrew
Publisher: name: Zenodo
Year: 2018
Subjects: Fluorescence microscopy
Super-resolution microscopy
SMLM
dSTORM

description: Single-molecule localization microscopy allows us to observe cellular structures on the nanoscale. Due to the high frame numbers required to achieve good localization precision, multi-colour experiments can however be challenging. Here, we present an approach to record four standard organic fluorescent dyes simultaneously with a single excitation wavelength over a large field of view. This approach dissolves problems with image registration, chromatic aberrations and sample drift common to other multi-colour approaches on the nanoscale. <strong>Method: </strong>We use a 638 nm diode laser and a microlens-based Köhler integrator<em><sup>1</sup></em> for homogenous epi-illumination of the entire field of view. This ensures field-independent image resolution<em><sup>2</sup></em>. Stochastic emission events from samples labelled with ‎DyLight 633, Alexa Fluor 647, Biotium CF660 and CF680 are detected through an image splitter on a sCMOS camera. For 3D localization, a cylindrical lens introduces a depth-dependent astigmatism in one detection path. Microscope control and localization fitting are performed in open-source software µManger<em><sup>3</sup></em> and Fiji/ ThunderSTORM<em><sup>4</sup></em><em><sup>,</sup></em><em><sup>5</sup></em>. Subsequent custom spectral analysis assigns a false colour to each localized emission event based on its spectroscopic signature. In contrast to previous spectroscopic super-resolution microscopy efforts<em><sup>6,7</sup></em> we can achieve nanometer resolution in 3D with a single objective and camera over a field of view of 130 x 60 µm<sup>2</sup>. We use immunolabelling with standard organic dyes, which makes this method applicable to a wide range of molecular biology samples. The optical setup can be implemented on any epi-fluorescence microscope. We intend to publish the spectral analysis as an open source package in the near future. <strong>References</strong> Völkel, R. &amp; Weible, K. J. Laser Beam Homogenizing: Limitations and Constraints. <em>Proc. SPIE</em> <strong>7102</strong> (2008). Douglass, K. M.<em> et al.</em> Super-resolution imaging of multiple cells by optimized flat-field epi-illumination. <em>Nat. Photonics</em> <strong>10 </strong>(2016). Edelstein, A. D. <em>et al.</em> Advanced methods of microscope control using μManager software. <em>J. Biol. Methods</em> (2014). Ovesný, M. <em>et al.</em> ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. <em>Bioinformatics</em> <strong>30</strong> (2014). Schindelin, J. <em>et al.</em> Fiji: an open-source platform for biological-image analysis. <em>Nat. Methods</em> (2012). Dong, B. <em>et al.</em> Super-resolution spectroscopic microscopy via photon localization. <em>Nat. Commun.</em> <strong>7</strong> (2016). Zhang, Z.<em> et al.</em> Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. <em>Nat. Methods</em> <strong>12</strong> (2015).