Point by Point: An Introductory Guide to Sample Preparation for Single‐Molecule, Super‐Resolution Fluorescence Microscopy

Abstract

Single‐molecule, localization‐based, super resolution microscopy is able to reveal detailed subcellular structures and protein distributions below the classical ∼250‐nm diffraction limit of light, but utilizing this technique effectively requires a combination of careful sample preparation, data acquisition, and data analysis, which can be daunting to novice researchers. In this protocol, detailed instructions on preparation of robust reference samples for super‐resolution microscopy, including the cytoskeleton (microtubules), membrane‐bound organelles (peroxisomes), and scaffold proteins (clathrin), are provided. These samples also constitute a representative subset of imaging targets of interest to biological researchers and highlight the differences and similarities in sample preparation. © 2015 by John Wiley & Sons, Inc.

Advances in Imaging Plant Cell Dynamics

“…approximately 20 nm), but these techniques suffer from extreme limitations related to sample stability and fluorophore performance during acquisition. Since they investigate fluorophore localization at tens of nanometer precision, it is essential that the sample should be very firmly immobilized, and the examined structures must exhibit negligible dynamics during the thousands of time frames needed for image acquisition (Halpern et al, 2015). The two most important fluorophore properties affecting the performance of SMLM are the number of photons detected for every switching event (transition between on/off states) and the duty cycle of the fluorophore, which represents the fraction of time that the fluorophore spends in the on state.…”

About samples, giving examples: Optimized Single Molecule Localization Microscopy

“…We will summarize the specific procedures and tweaks we use during imaging and briefly explain our processing workflow ( Fig.2 C-D). This will complement the existing reviews and methodological articles on how to perform STORM and DNA-PAINT (Allen et al, 2013;Halpern et al, 2015;Hoess et al, 2018;Pereira et al, 2015;Schnitzbauer et al, 2017;J. Xu et al, 2017).…”

“…In STochastic Optical Reconstruction Microscopy (STORM), high intensity illumination and an oxygen-deprived, reducing buffer is generally used to induce sparse blinking of organic fluorophores commonly used for immunolabeling (Heilemann et al, 2008;Rust et al, 2006). Single-color STORM is readily compatible with classical immunolabeled samples, by using Alexa Fluor 647-coupled secondary antibodies (Halpern et al, 2015). Multi-color STORM is not as straightforward, as it is a challenge to find two distinct fluorophores that have good blinking characteristics in the same environment, and inducing photoswitching of fluorophores outside of the far-red channel usually require high laser power illumination (Dempsey et al, 2011;Lehmann et al, 2016).…”

Multicolour localization microscopy by point-spread-function engineering

“…Samples were placed in a custom coverglass slide holder (Tokai Hit Co. Ltd., Japan) and warmed to room temperature and then placed in blinking media 32 (700µg/mL Glucose Oxidase, 50µg/mL Catalase, 0.55M Glucose, 140mM 2-Mercaptoethanol, all Sigma in 0.1M pH 8.0 Tris·HCl buffer, Invitrogen). The sample was scanned at low-intensity laser illumination for suitable regions and then imaged with high intensity (10kW/cm 2 ) 641 nm light and (5kW/cm 2 ) 514 nm light, with a rising intensity of the 405 nm activation laser (0-500W/cm 2 ) 32 .…”