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High speed functional imaging with source localized multifocal two-photon microscopy.

Peter Quicke1,2, Stephanie Reynolds3, Mark Neil2,4

  • 1Department of Bioengineering, Imperial College London, SW7 2AZ, UK.

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|October 20, 2018
PubMed
Summary
This summary is machine-generated.

This study introduces source-localized multifocal two-photon microscopy (MTPM) for faster, clearer brain imaging. The new method improves contrast and reduces crosstalk in scattering tissue, enabling high-speed functional imaging.

Keywords:
(100.0100) Image processing(110.0110) Imaging systems(180.0180) Microscopy

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Area of Science:

  • Neuroscience
  • Biophotonics
  • Microscopy

Background:

  • Multifocal two-photon microscopy (MTPM) enhances imaging speed by parallelizing fluorescence excitation.
  • Scattering in biological tissues, particularly mammalian brain, causes crosstalk, degrading image contrast and limiting imaging depth.
  • High-speed functional imaging is crucial for observing rapid neuronal activity.

Purpose of the Study:

  • To develop and validate a source-localized MTPM scheme for high-speed functional fluorescence imaging in scattering mammalian brain tissue.
  • To mitigate scattering-induced crosstalk and improve image contrast and signal-to-noise ratio (SNR) at depth.
  • To enable capture of faster and smaller functional signals than previously possible with MTPM.

Main Methods:

  • A rastered line array of beamlets was used for fluorescence excitation, with imaging performed by a complementary metal-oxide-semiconductor (CMOS) camera.
  • Crosstalk was reduced through temporal oversampling, structured illumination, and Richardson-Lucy deconvolution to reassign scattered photons.
  • Single images were reconstructed using maximum intensity projection of deconvolved image groups.

Main Results:

  • The source-localized MTPM achieved improved image contrast up to 112 μm in scattering brain tissue.
  • Functional crosstalk between pixels during neuronal calcium imaging was significantly reduced.
  • High signal-to-noise ratio (SNR) was maintained at frame rates above 50 Hz, even in sparsely labeled tissue.

Conclusions:

  • The developed non-descanned source-localized MTPM system enables high SNR, 100 Hz fluorescence transient capture in scattering brain tissue.
  • This advancement expands the capabilities of MTPM for imaging faster and smaller functional signals.
  • The method offers a promising tool for in vivo neuroscience research in complex biological environments.