Abstract
We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Teramura, Y. et al. EMBO J. 25, 4215–4222 (2006).
Yildiz, A. et al. Science 300, 2061–2065 (2003).
Douglass, A.D. & Vale, R.D. Cell 121, 937–950 (2005).
Kusumi, A., Sako, Y. & Yamamoto, M. Biophys. J. 65, 2021–2040 (1993).
Betzig, E. et al. Science 313, 1642–1645 (2006).
Hess, S.T. et al. Proc. Natl. Acad. Sci. USA 104, 17370–17375 (2007).
Wiedenmann, J. et al. Proc. Natl. Acad. Sci. USA 101, 15905–15910 (2004).
Kenworthy, A.K. et al. J. Cell Biol. 165, 735–746 (2004).
Jouvenet, N. et al. PLoS Biol. 4, 2296–2310 (2006).
Wieser, S., Moertelmaier, M., Fuertbauer, E., Stockinger, H. & Schutz, G.J. Biophys. J. 92, 3719–3728 (2007).
Vrljic, M., Nishimura, S.Y., Brasselet, S., Moerner, W.E. & McConnell, H.M. Biophys. J. 83, 2681–2692 (2002).
Ewers, H. et al. Proc. Natl. Acad. Sci. USA 102, 15110–15115 (2005).
Feder, T.J., Brust-Mascher, I., Slattery, J.P., Baird, B. & Webb, W.W. Biophys. J. 70, 2767–2773 (1996).
Nydegger, S., Khurana, S., Krementsov, D.N., Foti, M. & Thali, M. J. Cell Biol. 173, 795–807 (2006).
Acknowledgements
This project was supported by the Intramural Research Program of the US National Institute of Child Health and Human Development, National Institutes of Health, and performed while S.M. held a National Research Council Research Associateship Award at the National Institutes of Health. We thank D. Blair and A.D. Douglass for providing MATLAB code and helpful discussions.
Author information
Authors and Affiliations
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–4, Supplementary Methods (PDF 1939 kb)
Supplementary Video 1
Raw TIRF images of VSVG expressed in COS7 cells, near the center of the cell. Each image is a 50 msec exposure. Crosses mark peaks which meet the criteria used for sptPALM. On the left, crosses are overlaid on the raw data, on the right, only the crosses are shown. The size of a frame is 17 × 17 μm. (MOV 2583 kb)
Supplementary Video 2
Time-resolved PALM movie of VSVG-Eos expressed in COS7 cells. Each image is the sum of 100 frames (5 sec), with a shift of 25 frames (1.25 sec) between neighboring images. The entire movie corresponds to a time of 225 sec. This movie shows the overall distribution of VSVG-Eos in the cells under study and suggests that our imaging parameters are not detrimental to the cells since this time sequence is at the end of our image acquisition (10,000 frames, 50 msec exposures). It is possible that a live cell PALM super-resolution imaging approach involving a higher rate of molecular localization and higher density of molecules per frame can be achieved for describing the evolution of well-defined structures at considerably less than the diffraction limit. (MOV 2343 kb)
Rights and permissions
About this article
Cite this article
Manley, S., Gillette, J., Patterson, G. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5, 155–157 (2008). https://doi.org/10.1038/nmeth.1176
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.1176
This article is cited by
-
Temporal analysis of relative distances (TARDIS) is a robust, parameter-free alternative to single-particle tracking
Nature Methods (2024)
-
Cytosolic sorting platform complexes shuttle type III secretion system effectors to the injectisome in Yersinia enterocolitica
Nature Microbiology (2024)
-
Motion of VAPB molecules reveals ER–mitochondria contact site subdomains
Nature (2024)
-
Spectroscopic single-molecule localization microscopy: applications and prospective
Nano Convergence (2023)
-
Three-dimensional single particle tracking using 4π self-interference of temporally phase-shifted fluorescence
Light: Science & Applications (2023)