Two-photon fluorescence imaging of subsurface tissue structures with volume holographic microscopy

Area of Science:

• Biomedical Optics
• Microscopy Techniques
• Advanced Imaging

Background:

• Two-photon (2P) fluorescence imaging offers high-contrast, background-free imaging in scattering tissues.
• Acquiring multiplane images with conventional 2P imaging presents challenges.

Purpose of the Study:

• To design and implement a 2P-VHI system capable of high-contrast, optically sectioned multiplane imaging.
• To overcome limitations in multiplane imaging for scattering biological samples.

Main Methods:

• Developed a 2P-VHI system utilizing angularly multiplexed volume holographic gratings.
• Employed a femtosecond laser source for fluorescence excitation to achieve multifocal observation.
• Integrated nonlinear excitation for enhanced depth resolution and image contrast.

Main Results:

• Demonstrated imaging performance using standard fluorescent beads.
• Acquired two-depth resolved optical-sectioning images of fluorescently labeled thick mouse intestine samples.
• Quantified and compared the optical sectioning capability with conventional VHI systems.

Conclusions:

• 2P excitation in VHI systems significantly improves optical sectioning and reduces background noise.
• The integration of nonlinear fluorescence excitation offers unique advantages for VHI systems.
• The developed system has potential for creating multidepth optical sectioned spatial-spectral imaging systems.