Ultra-long anti-diffracting beam volume imaging using a single-photon excitation microscope

Area of Science:

• Microscopy and Imaging Technologies
• Biophotonics
• Optical Physics

Background:

• Conventional microscopy faces limitations in imaging thick, scattering biological samples due to limited axial range and resolution.
• Gaussian and Airy beams, commonly used in microscopy, have restricted focal lengths and scattering penetration capabilities.
• Developing advanced illumination strategies is crucial for high-resolution, deep-tissue volumetric imaging.

Purpose of the Study:

• To introduce and evaluate a novel volumetric single-photon excitation microscope utilizing an ultralong anti-diffracting (UAD) beam.
• To compare the imaging performance of UAD beams against conventional Gaussian and Airy beams in scattering media.
• To demonstrate the capability of the UAD microscope for dynamic volumetric imaging and micro-object localization.

Main Methods:

• Implementation of a single-photon excitation microscope with UAD beam illumination.
• Comparative imaging experiments using fluorescent microspheres in agarose gel under Gaussian, Airy, and UAD illumination modes.
• Layer-by-layer scanning of thick samples followed by 3D structural projection onto 2D images.
• Dynamic volumetric imaging of zebrafish cardiovascular systems and substance transport in blood vessels.

Main Results:

• The UAD beam demonstrated an axial imaging range approximately 14 times greater than Gaussian beams and 2 times greater than Airy beams, while maintaining narrow lateral width.
• Significantly increased volume imaging speed and deeper axial projection were achieved with the UAD beam due to its longer focal length.
• Successful dynamic volumetric imaging of zebrafish vasculature and substance transport was performed, showcasing the UAD microscope's capabilities.
• The UAD beam's trajectory was found suitable for micro-object localization.

Conclusions:

• The novel UAD microscope offers superior performance for volumetric imaging in scattering environments compared to conventional methods.
• The UAD beam's extended axial range and enhanced imaging speed are highly beneficial for studying dynamic biological processes in 3D.
• This technology holds promise for advanced applications in biological research, particularly in in vivo imaging and micro-object manipulation.