Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy

René Platzer,Florian Baumgart,Mario Brameshuber,Eva Sevcsik,Johannes B Huppa,Benedikt K Rossboth,Hannes Stockinger,Magdalena C Schneider,Gerhard J Schütz

doi:10.1038/s41467-020-18726-9

René Platzer, Florian Baumgart + Show 7 more

Open Access

https://doi.org/10.1038/s41467-020-18726-9

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Journal: Nature Communications	Publication Date: Oct 5, 2020
Citations: 25	License type: open-access

Affiliation: Medical University of Vienna, TU Wien

Abstract

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

Highlights

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions
We selected for this study PS-CFP2 and mEOS3.2 as photoswitchable model fluorophores in view of their wide use in PALM and their reportedly low to moderate blinking tendencies[24,27], and Abberior CAGE635 (CAGE635) and the recently developed PA
To be able to determine fluorophore-specific blinking properties with single-molecule resolution, we site- coupled PS-CFP2, mEOS3.2, PA-JF549, and CAGE635 via a biotin modification to monovalent and recombinant streptavidin, which was anchored by means of its three histidine tags to a gel-phase planar glass-supported lipid bilayer (SLB) consisting of 1,2-dioleoyl-sn-glycero-3-[(N-(5amino-1-carboxypentyl)iminodiacetic acid)succinyl]

Summary

Introduction

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. We reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps. Technique devised to resolve structures below the diffraction limit It relies on stochastic photoswitching of fluorophores, either irreversible or reversible, between a blue-shifted fluorescent or dark state and a red-shifted or bright fluorescent state[1,2]. Photoswitching only a minor fraction of fluorophores into the active state gives rise to well-separated single-molecule signals, which are localizable with a precision primarily determined by the signal-to-noise ratio[22]

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