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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Autores principales:	Nicholas A Frost, Hsiangmin E Lu, Thomas A Blanpied
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b54a937f4f654d01a4cd3e5c04d25d9f
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spelling	oai:doaj.org-article:b54a937f4f654d01a4cd3e5c04d25d9f2021-11-18T07:19:45ZOptimization of cell morphology measurement via single-molecule tracking PALM.1932-620310.1371/journal.pone.0036751https://doaj.org/article/b54a937f4f654d01a4cd3e5c04d25d9f2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22570741/?tool=EBIhttps://doaj.org/toc/1932-6203In 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.Nicholas A FrostHsiangmin E LuThomas A BlanpiedPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 5, p e36751 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Nicholas A Frost Hsiangmin E Lu Thomas A Blanpied Optimization of cell morphology measurement via single-molecule tracking PALM.
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.
format	article
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
title	Optimization of cell morphology measurement via single-molecule tracking PALM.
title_short	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_sort	optimization of cell morphology measurement via single-molecule tracking palm.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/b54a937f4f654d01a4cd3e5c04d25d9f
work_keys_str_mv	AT nicholasafrost optimizationofcellmorphologymeasurementviasinglemoleculetrackingpalm AT hsiangminelu optimizationofcellmorphologymeasurementviasinglemoleculetrackingpalm AT thomasablanpied optimizationofcellmorphologymeasurementviasinglemoleculetrackingpalm
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Optimization of cell morphology measurement via single-molecule tracking PALM.

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