Back to Search Start Over

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

Authors :
Peter D. Dahlberg
Annina M. Sartor
W. E. Moerner
Saumya Saurabh
Jiarui Wang
Lucy Shapiro
Source :
Single Molecule Spectroscopy and Superresolution Imaging XIII.
Publication Year :
2020
Publisher :
SPIE, 2020.

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.

Details

Database :
OpenAIRE
Journal :
Single Molecule Spectroscopy and Superresolution Imaging XIII
Accession number :
edsair.doi...........d40b25cda5d0b4c743cf6fbb271d1492
Full Text :
https://doi.org/10.1117/12.2546333