Optimized protocol for combined PALM-dSTORM imaging

Abstract

Multi-colour super-resolution localization microscopy is an efficient technique to study a variety of intracellular processes, including protein-protein interactions. This technique requires specific labels that display transition between fluorescent and non-fluorescent states under given conditions. For the most commonly used label types, photoactivatable fluorescent proteins and organic fluorophores, these conditions are different, making experiments that combine both labels difficult. Here, we demonstrate that changing the standard imaging buffer of thiols/oxygen scavenging system, used for organic fluorophores, to the commercial mounting medium Vectashield increased the number of photons emitted by the fluorescent protein mEos2 and enhanced the photoconversion rate between its green and red forms. In addition, the photophysical properties of organic fluorophores remained unaltered with respect to the standard imaging buffer. The use of Vectashield together with our optimized protocol for correction of sample drift and chromatic aberrations enabled us to perform two-colour 3D super-resolution imaging of the nucleolus and resolve its three compartments.

Characterizing Large-Scale Receptor Clustering on the Single Cell Level: A Comparative Plasmon Coupling and Fluorescence Superresolution Microscopy Study

“…In contrast, fluorescence superresolution microscopy techniques that utilize single-molecule photoactivation or photoswitching of fluorophores can map the receptor distribution below the diffraction limit. However, single-molecule localization microscopy techniques such as direct stochastic optical reconstruction microscopy (dSTORM) typically require acquisition of thousands of image frames − to reconstruct a superresolution image. The associated long acquisition times limit experimental throughput and make a statistical analysis of receptor clustering in a heterogeneous cell ensemble challenging.…”

Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique

“…Similar limitations exist for dSTORM, as many dyes with good blinking properties have different buffer requirements for inducing blinking, making it difficult, though not impossible, to combine these for multicolour imaging [49][50][51]. PALM and dSTORM can also be combined to increase multicolour options [52]. In all cases of multicolour SMLM it is important to consider balancing the brightness of different channels.…”

Super‐Resolution Fluorescence Microscopy Methods for Assessing Mouse Biology

“…In a recently published paper, Patel, Williamson, Owen, & Cohen (2021) discuss statistical methods to improve stochastic localization precision, mitigating for missed blinks and false positives, while Diekmann et al (2020) advise on improving speed and sensitivity of image acquisition. See Möckl & Moerner (2020) and Khater, Nabi, & Hamarneh (2020) for good reviews of SMLM techniques, Jimenez et al (2020) for practical advice regarding sample preparation and probes, and Glushonkov, Réal, Boutant, Mély, & Didier (2018) for a combined PALM-dSTORM stepwise protocol.…”

“…See Möckl & Moerner (2020) and Khater, Nabi, & Hamarneh (2020) for good reviews of SMLM techniques, Jimenez et al. (2020) for practical advice regarding sample preparation and probes, and Glushonkov, Réal, Boutant, Mély, & Didier (2018) for a combined PALM‐dSTORM stepwise protocol.…”