Cryogenic single-molecule active control microscopy with a photoactivatable fluorescent protein

Annina M. Sartor; Peter D. Dahlberg; Jiarui Wang; Saumya Saurabh; Lucy Shapiro; W. E. Moerner

doi:10.1117/12.2546333

13 February 2020 Cryogenic single-molecule active control microscopy with a photoactivatable fluorescent protein

Annina M. Sartor, Peter D. Dahlberg, Jiarui Wang, Saumya Saurabh, Lucy Shapiro, W. E. Moerner

Proceedings Volume 11246, Single Molecule Spectroscopy and Superresolution Imaging XIII; 112460G (2020) https://doi.org/10.1117/12.2546333
Event: SPIE BiOS, 2020, San Francisco, California, United States

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.

Conference Presentation

Citation Download Citation

Annina M. Sartor, Peter D. Dahlberg, Jiarui Wang, Saumya Saurabh, Lucy Shapiro, and W. E. Moerner "Cryogenic single-molecule active control microscopy with a photoactivatable fluorescent protein", Proc. SPIE 11246, Single Molecule Spectroscopy and Superresolution Imaging XIII, 112460G (13 February 2020); https://doi.org/10.1117/12.2546333

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