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Proceedings Paper

Cryogenic single-molecule active control microscopy with a photoactivatable fluorescent protein
Author(s): Annina M. Sartor; Peter D. Dahlberg; Jiarui Wang; Saumya Saurabh; Lucy Shapiro; W. E. Moerner
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Paper Abstract

Single-molecule superresolution methods enable imaging of specifically-labeled biological samples with structures on length scales below the diffraction limit of visible light. Imaging samples at cryogenic temperatures (77 K) significantly reduces photobleaching, allowing more photons to be collected per emitter and thus improving the localization precision. Cryogenic single-molecule imaging also facilitates correlative imaging with cryogenic electron tomography (cryoET), which provides images of whole biological cells with high-resolution cellular contrast. Combining these two techniques by performing optical imaging under conditions that do not damage the sample for cryoET allows the combination of the high sensitivity and specificity from single-molecule fluorescence with the cellular context from cryoET. In this work, we use PAmKate, a red photoactivatable fluorescent protein, to perform cryogenic single-molecule imaging of proteins in the model organism Caulobacter crescentus at 77 K with sufficiently low illumination powers to prevent damage of the cryogenic sample. The enhanced number of photons detected allows localization precision to be improved to values below 10 nm.

Paper Details

Date Published: 13 February 2020
PDF: 7 pages
Proc. SPIE 11246, Single Molecule Spectroscopy and Superresolution Imaging XIII, 112460G (13 February 2020); doi: 10.1117/12.2546333
Show Author Affiliations
Annina M. Sartor, Stanford Univ. (United States)
Peter D. Dahlberg, Stanford Univ. (United States)
Jiarui Wang, Stanford Univ. (United States)
Stanford Univ. School of Medicine (United States)
Saumya Saurabh, Stanford Univ. School of Medicine (United States)
Lucy Shapiro, Stanford Univ. School of Medicine (United States)
W. E. Moerner, Stanford Univ. (United States)


Published in SPIE Proceedings Vol. 11246:
Single Molecule Spectroscopy and Superresolution Imaging XIII
Ingo Gregor; Felix Koberling; Rainer Erdmann, Editor(s)

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