SpeedyTrack: Direct microsecond wide-field single-molecule tracking and super-resolution mapping via CCD vertical...

Area of Science:

• Biophysics
• Microscopy techniques
• Single-molecule analysis

Background:

• Accurate tracking of single molecules is crucial for understanding biological processes.
• Existing methods often lack the temporal resolution or simplicity required for dynamic studies.

Purpose of the Study:

• To introduce SpeedyTrack, a novel strategy for high-temporal-resolution wide-field single-molecule tracking.
• To enable microsecond timescale imaging and trajectory analysis of freely diffusing molecules.

Main Methods:

• Utilizing the vertical charge shifting capability of electron-multiplying charge-coupled devices (EM-CCDs).
• Spatially encoding time information by staggering images across the CCD chip.
• Deconvolving spatial and temporal data for super-resolution mapping.

Main Results:

• Achieved 50 μs temporal resolution for single-molecule tracking in wide-field microscopy.
• Quantified diffusion coefficients up to 1,000 μm²/s.
• Enabled concurrent acquisition of diffusion trajectories and Förster resonance energy transfer (FRET) data.
• Resolved diffusion modes of fluorescent proteins in live cells with nanoscale resolution.

Conclusions:

• SpeedyTrack offers a simple, hardware-independent solution for microsecond single-molecule imaging and tracking.
• The method significantly enhances capabilities compared to existing techniques.
• Provides a facile approach for studying molecular dynamics and conformational changes.