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

Research output: Chapter in Book/Report/Conference proceedingConference contribution


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.

Original languageEnglish (US)
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging XIII
EditorsIngo Gregor, Felix Koberling, Rainer Erdmann
ISBN (Electronic)9781510632554
StatePublished - 2020
EventSingle Molecule Spectroscopy and Superresolution Imaging XIII 2020 - San Francisco, United States
Duration: Feb 1 2020Feb 2 2020

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


ConferenceSingle Molecule Spectroscopy and Superresolution Imaging XIII 2020
Country/TerritoryUnited States
CitySan Francisco


  • Correlative imaging
  • Cryogenic
  • Cryopalm
  • Fluorescent protein
  • Photoactivation
  • Photophysics

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging
  • Biomaterials


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