Local dimensionality determines imaging speed in localization microscopy

Patrick Fox-Roberts; Richard Marsh; Karin Pfisterer; Asier Jayo; Maddy Parsons; Susan Cox

doi:10.1038/ncomms13558

Local dimensionality determines imaging speed in localization microscopy

Patrick Fox-Roberts, Richard Marsh, Karin Pfisterer, Asier Jayo, Maddy Parsons, Susan Cox^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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Abstract

Localization microscopy allows biological samples to be imaged at a length scale of tens of nanometres. Live-cell super-resolution imaging is rare, as it is generally assumed to be too slow for dynamic samples. The speed of data acquisition can be optimized by tuning the density of activated fluorophores in each time frame. Here, we show that the maximum achievable imaging speed for a particular structure varies by orders of magnitude, depending on the sample dimensionality (that is, whether the sample is more like a point, a strand or an extended structure such as a focal adhesion). If too high an excitation density is used, we demonstrate that the analysis undergoes silent failure, resulting in reconstruction artefacts. We are releasing a tool to allow users to identify areas of the image in which the activation density was too high and correct for them, in both live-and fixed-cell experiments.

Original language	English
Article number	13558
Pages (from-to)	1-10
Journal	Nature Communications
Volume	8
Early online date	12 Jan 2017
DOIs	https://doi.org/10.1038/ncomms13558
Publication status	Published - Jan 2017

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Local dimensionality determines_FOX-ROBERTS_Published12January2017_GOLD VoR (CC-BY)Final published version, 1.33 MBLicence: CC BY

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AU - Pfisterer, Karin

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AU - Parsons, Maddy

AU - Cox, Susan

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AB - Localization microscopy allows biological samples to be imaged at a length scale of tens of nanometres. Live-cell super-resolution imaging is rare, as it is generally assumed to be too slow for dynamic samples. The speed of data acquisition can be optimized by tuning the density of activated fluorophores in each time frame. Here, we show that the maximum achievable imaging speed for a particular structure varies by orders of magnitude, depending on the sample dimensionality (that is, whether the sample is more like a point, a strand or an extended structure such as a focal adhesion). If too high an excitation density is used, we demonstrate that the analysis undergoes silent failure, resulting in reconstruction artefacts. We are releasing a tool to allow users to identify areas of the image in which the activation density was too high and correct for them, in both live-and fixed-cell experiments.

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Local dimensionality determines imaging speed in localization microscopy

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