Dataset Information

Quantitative photo activated localization microscopy: unraveling the effects of photoblinking.

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 ?2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.

SUBMITTER: Annibale P

PROVIDER: S-EPMC3144238 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Quantitative photo activated localization microscopy: unraveling the effects of photoblinking.

Annibale Paolo P Vanni Stefano S Scarselli Marco M Rothlisberger Ursula U Radenovic Aleksandra A

PloS one 20110726 7

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 plas ...[more]

PMID: 21818365

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Project description:Localization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (t(Thresh) and d(Thresh)) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems.

Dataset Information

Quantitative photo activated localization microscopy: unraveling the effects of photoblinking.

Publications

Quantitative photo activated localization microscopy: unraveling the effects of photoblinking.

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