Real-time 3D single molecule tracking.

Nature Communications
Shangguo HouKevin Welsher

Abstract

To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of locking on to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART is applied to actively track single Atto 647 N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385 bp) of single DNA molecules at rates up to 10 µm2/s. In addition, 3D-SMART is able to quantify the occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.

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Citations

Mar 16, 2021·The Journal of General Physiology·Kazuki ObashiShigeo Okabe
Mar 20, 2021·Analytica Chimica Acta·Daniel F Kienle, Daniel K Schwartz
Jun 3, 2021·International Journal of Molecular Sciences·Ai-Yu GuoWen-Qiang Wu
Aug 10, 2021·Current Opinion in Biomedical Engineering·Yuan-I ChenHsin-Chih Yeh
Oct 31, 2020·ACS Sensors·Pavel N MelentievVictor I Balykin
Sep 2, 2021·The Journal of Physical Chemistry Letters·Zhihong WeiYuxi Tian

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Methods Mentioned

BETA
light-scattering
PCR
in vitro transcription

Software Mentioned

DyPLoT
SMART
PicoQuant
3D
LabView

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