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

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.