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An integrated single- and two-photon non-diffracting light-sheet microscope.

Sze Cheung Lau1, Hoi Chun Chiu1, Luwei Zhao2

  • 1Department of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

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This study presents a novel fluorescence microscope for large-volume imaging, integrating single-photon and two-photon excitations. The microscope offers high-resolution imaging for both thin and thick biological samples, advancing live-cell and tissue research.

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Area of Science:

  • Biophotonics
  • Microscopy
  • Optical Imaging

Background:

  • Large-volume imaging requires advanced microscopy techniques.
  • Light scattering in thick samples limits resolution in traditional methods.
  • Simultaneous single-photon and two-photon excitation offers versatile imaging capabilities.

Purpose of the Study:

  • To develop an integrated fluorescence optical microscope for large volume imaging.
  • To combine single-photon and two-photon non-diffracting light-sheet excitations in one setup.
  • To achieve high-resolution imaging in both transparent and scattering biological samples.

Main Methods:

  • Designed a microscope accommodating visible (400-700 nm) and near-infrared (800-1200 nm) wavelengths.
  • Integrated line-Bessel sheet (LBS) for single-photon excitation and scanning Bessel beam (SBB) for two-photon excitation.
  • Utilized LBS for thin samples and SBB for deep-cell/tissue imaging with significant light scattering.

Main Results:

  • Achieved nearly identical lateral resolution of ~350 nm and axial resolution of ~270 nm for both excitation methods.
  • Demonstrated effective imaging for both transparent thin samples and scattering deep-cell/tissue samples.
  • Validated the combined LBS and SBB light-sheet approach in a single microscope.

Conclusions:

  • The integrated light-sheet microscope provides a versatile platform for large-volume, high-resolution imaging.
  • This technology enables high-speed 3D imaging of live cells and tissues.
  • The dual-excitation capability addresses challenges in imaging diverse biological sample types.