Optimization of cell morphology measurement via single-molecule tracking PALM
Open accessAbstract
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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Live super-resolution microscopy requires some thoughtful compromises between temporal and spatial resolution.
“In all cases, live super-resolution microscopy requires some thoughtful compromises between temporal and spatial resolution (Frost et al., 2012).”
From Barriers in the brain: resolving dendritic spine morphology and compartmentalization (2014) · cites this paper as Frost et al., 2012
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- Authors
- Frost, N. A., Lu, H. E., & Blanpied, T. A.
- Affiliations
- University of Maryland, Baltimore (US)
- Corresponding author
- Blanpied, Thomas A.
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2012-05-03
- Volume
- 7
- Issue
- 5
- Pages
- e36751
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3343014
- OpenAlex
- W2010021375
- Cited by
- 22
- References
- 43
- Citations by year
- 2025: 1 · 2022: 1 · 2020: 1 · 2019: 1 · 2018: 2 · 2016: 1 · 2015: 5 · 2014: 9 · 2013: 1
- FWCI
- 2.20
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Dana Foundation
- National Institutes of Health (MH080046)
- National Institute of Mental Health (F30MH086185, R01MH080046, MH080046, F32-MH086185, MH096376, R01MH096376, R37MH080046)
- National Center for Research Resources (S10RR024548)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Keywords
- dendritic spines, dendritic spine morphology, single-molecule tracking, photoactivated localization microscopy, Monte Carlo simulation, diffusion coefficient, localization bias, camera exposure time, pulsed excitation, neural processes, synaptic function
- MeSH terms
- Animals, Cells, Cultured, Computer Simulation, Image Processing, Computer-Assisted, Microscopy, Fluorescence, Monte Carlo Method, Neurons, Rats, Molecular Imaging, Time-Lapse Imaging
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