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Wide-field bond-selective fluorescence imaging: from single-molecule to cellular imaging beyond video rate.

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  • 1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

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Summary

We developed Wide-Field Bond-selective Fluorescence-detected Infrared-Excited (WF-BonFIRE) spectro-microscopy for sensitive, bond-selective chemical imaging. This new technique achieves single-molecule detection and high-speed live-cell imaging, advancing chemical microscopy capabilities.

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Area of Science:

  • Chemical Imaging
  • Spectro-microscopy
  • Biophysics

Background:

  • Wide-field (WF) chemical microscopy lacks sensitivity for single-molecule detection.
  • WF fluorescence microscopy offers live-cell mapping but misses crucial chemical information.
  • Existing WF techniques struggle to combine sensitivity, chemical specificity, and speed.

Purpose of the Study:

  • Introduce Wide-Field Bond-selective Fluorescence-detected Infrared-Excited (WF-BonFIRE) spectro-microscopy.
  • Achieve single-molecule sensitivity with bond-selective contrast in WF imaging.
  • Enable high-speed, chemically specific live-cell imaging.

Main Methods:

  • Combined optimized imaging speed and field-of-view (FOV).
  • Implemented temporal-delay modulation for kilohertz imaging (up to 1500 Hz).
  • Demonstrated multicolor imaging and tracking using narrow-band signals.

Main Results:

  • Achieved single-molecule sensitivity with bond-selective contrast.
  • Successfully imaged single molecules, cells, astrocytes, and live neurons across 50 μm × 50 μm FOVs.
  • Monitored live *E. coli* motion with millisecond temporal resolution and performed multicolor tracking.

Conclusions:

  • WF-BonFIRE spectro-microscopy overcomes limitations of existing WF techniques.
  • The method enables high-speed, high-sensitivity chemical imaging.
  • WF-BonFIRE significantly expands possibilities for observing dynamic biological events at the molecular level.