Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells

Simao Coelho, Jongho Baek, Matthew S. Graus, James M. Halstead, Philip R. Nicovich, Kristen Feher, Hetvi Gandhi, J. Justin Gooding, Katharina Gaus

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Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ∼1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

Original languageEnglish
Article numbereaay8271
Number of pages9
JournalScience Advances
Issue number16
Publication statusPublished - 15 Apr 2020
Externally publishedYes

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