Optimization of cell morphology measurement via single-molecule tracking PALM.

In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution...

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Main Authors: Nicholas A Frost, Hsiangmin E Lu, Thomas A Blanpied
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2012-01-01
Series:PLoS ONE
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22570741/?tool=EBI
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author Nicholas A Frost
Hsiangmin E Lu
Thomas A Blanpied
author_facet Nicholas A Frost
Hsiangmin E Lu
Thomas A Blanpied
author_sort Nicholas A Frost
collection DOAJ
description In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in interpretation of single-molecule data in living cells and provide a method to minimize artifacts in single-molecule experiments.
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spelling doaj.art-b54a937f4f654d01a4cd3e5c04d25d9f2022-12-21T22:43:37ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0175e3675110.1371/journal.pone.0036751Optimization of cell morphology measurement via single-molecule tracking PALM.Nicholas A FrostHsiangmin E LuThomas A BlanpiedIn neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in interpretation of single-molecule data in living cells and provide a method to minimize artifacts in single-molecule experiments.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22570741/?tool=EBI
spellingShingle Nicholas A Frost
Hsiangmin E Lu
Thomas A Blanpied
Optimization of cell morphology measurement via single-molecule tracking PALM.
PLoS ONE
title Optimization of cell morphology measurement via single-molecule tracking PALM.
title_full Optimization of cell morphology measurement via single-molecule tracking PALM.
title_fullStr Optimization of cell morphology measurement via single-molecule tracking PALM.
title_full_unstemmed Optimization of cell morphology measurement via single-molecule tracking PALM.
title_short Optimization of cell morphology measurement via single-molecule tracking PALM.
title_sort optimization of cell morphology measurement via single molecule tracking palm
url https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22570741/?tool=EBI
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