Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging

Tao Huang, Carey Phelps, Jing Wang, Li Jung Lin, Amy Bittel, Zubenelgenubi Scott, Steven Jacques, Summer Gibbs, Joe Gray, Xiaolin Nan

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels. Images of the same molecule in the two channels are then combined to determine both the location and the identity of the molecule. The single-objective configuration of our approach allows for flexible sample geometry and the use of a live-cell incubation chamber required for live-cell SMT. Despite a lower photon yield, we achieve excellent spatial (20–40 nm) and spectral (10–15 nm) resolutions comparable to those obtained with dual-objective, spectrally resolved Stochastic Optical Reconstruction Microscopy. Furthermore, motions of the fluorescent molecules did not cause loss of spectral resolution owing to the dual-channel spectral calibration. We demonstrate SMT in three (and potentially more) colors using spectrally proximal fluorophores and single-laser excitation, and show that trajectories of each species can be reliably extracted with minimal cross talk.

Original languageEnglish (US)
Pages (from-to)301-310
Number of pages10
JournalBiophysical Journal
Volume114
Issue number2
DOIs
StatePublished - Jan 23 2018

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Lasers
Cell Tracking
Biological Phenomena
Photons
Calibration
Microscopy
Color
Fluorescence
Single Molecule Imaging

ASJC Scopus subject areas

  • Biophysics

Cite this

Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging. / Huang, Tao; Phelps, Carey; Wang, Jing; Lin, Li Jung; Bittel, Amy; Scott, Zubenelgenubi; Jacques, Steven; Gibbs, Summer; Gray, Joe; Nan, Xiaolin.

In: Biophysical Journal, Vol. 114, No. 2, 23.01.2018, p. 301-310.

Research output: Contribution to journalArticle

Huang, Tao ; Phelps, Carey ; Wang, Jing ; Lin, Li Jung ; Bittel, Amy ; Scott, Zubenelgenubi ; Jacques, Steven ; Gibbs, Summer ; Gray, Joe ; Nan, Xiaolin. / Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging. In: Biophysical Journal. 2018 ; Vol. 114, No. 2. pp. 301-310.
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