Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy

Area of Science:

• Biomedical Optics
• Microscopy
• In Vivo Imaging

Background:

• In vivo fluorescent cellular imaging of deep internal organs is limited by light scattering and signal degradation in biological tissues.
• Existing microscopy techniques struggle to achieve cellular resolution deep within scattering tissues.

Purpose of the Study:

• To develop an advanced imaging technique for high-resolution in vivo visualization of deep internal organs.
• To overcome the challenges of light penetration and signal diffusion in deep tissue imaging.

Main Methods:

• Combined two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM).
• Utilized Bessel beams for enhanced penetration and SIM for improved resolution and background reduction.
• Imaged live samples up to 600 microns wide with a penetration depth of 500 microns.

Main Results:

• Achieved clear cellular resolution imaging at depths beyond 200 microns in scattering tissues.
• Significantly reduced diffused background noise in deep tissue light-sheet imaging.
• Demonstrated successful in vivo two-color imaging of zebrafish kidney glomeruli and vasculature.

Conclusions:

• The combined two-photon Bessel light-sheet SIM technique significantly enhances deep tissue imaging capabilities.
• This method provides unprecedented clarity for visualizing cellular structures in deep internal organs of live organisms.
• Enables advanced research in developmental biology and disease mechanisms within intact biological systems.