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High-density mapping of single-molecule trajectories with photoactivated localization microscopy

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.

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Figure 1: sptPALM imaging of Gag and VSVG expressed in live COS7 cells.
Figure 2: Analysis of single molecule trajectories in live COS7 cells.
Figure 3: Cluster analysis on the immobile fraction of Gag.

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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.

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Correspondence to Jennifer Lippincott-Schwartz.

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)

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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

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