Quantitative Photo Activated Localization Microscopy: Unraveling the Effects of Photoblinking

Paolo Annibale,Aleksandra Radenovic,Stefano Vanni,Marco Scarselli,Ursula Rothlisberger,Rainer Heintzmann

doi:10.1371/journal.pone.0022678

Abstract

In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of β2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.

Highlights

Super-resolution techniques based on the sequential photoswitching/photo-activation [1,2,3,4,5,6,7,8,9] of single photoemitters have allowed detection of single molecules with spatial localization accuracy below 10 nanometers and Nyquist-Shannonlimited resolution [10] of approximately 20 nm
Starting from the complex photophysical features of one of the most recent and promising photoconvertible fluorescent proteins for Photo Activated Localization Microscopy (PALM) studies we have systematically investigated the effect of molecular photoblinking and fluorescence dark times on a typical PALM experiment
In this work we have proposed a method to obtain a reliable estimation of the number of photoblinking molecules present in a sample by comparing simulations tailored on single molecule photophysics and in vitro experiments

Summary

Introduction

Super-resolution techniques based on the sequential photoswitching/photo-activation [1,2,3,4,5,6,7,8,9] of single photoemitters have allowed detection of single molecules with spatial localization accuracy below 10 nanometers and Nyquist-Shannonlimited resolution [10] of approximately 20 nm. Depending on the fluorophore photophysics, excitation light power and acquisition frame rate, the choice of a certain dark time td during data analysis and fluorescent trace grouping will result in a number of molecular localizations that might be far from the actual number of labeled molecules.

Results

Conclusion