RT Journal Article
SR Electronic
T1 Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells
JF Science Advances
JO Sci Adv
FD American Association for the Advancement of Science
SP eaay8271
DO 10.1126/sciadv.aay8271
VO 6
IS 16
A1 Coelho, Simao
A1 Baek, Jongho
A1 Graus, Matthew S.
A1 Halstead, James M.
A1 Nicovich, Philip R.
A1 Feher, Kristen
A1 Gandhi, Hetvi
A1 Gooding, J. Justin
A1 Gaus, Katharina
YR 2020
UL http://advances.sciencemag.org/content/6/16/eaay8271.abstract
AB 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.