An integrated single- and two-photon non-diffracting light-sheet microscope

TL;DR

This paper presents a novel integrated microscope combining single-photon and two-photon non-diffracting light sheets, enabling high-resolution, large-volume 3D imaging of live cells and tissues with adaptable imaging depths.

Contribution

The development of a combined light-sheet microscope that seamlessly integrates single-photon and two-photon excitation for versatile, high-resolution biological imaging.

Findings

Achieved ~350/270 nm lateral/axial resolution for both imaging modes.

Demonstrated effective deep-tissue imaging with two-photon excitation.

Provided a versatile platform for live-cell and tissue imaging.

Abstract

We describe the apparatus of a fluorescence optical microscope with both single-photon and two-photon non-diffracting light sheets excitation for large volume imaging. With special design to accommodate two different wavelength ranges (visible: 400-700 nm, and near infrared: 800-1200 nm), we combine the line-Bessel sheet (LBS, for single-photon excitation) and the scanning Bessel beam (SBB, for two-photon excitation) light sheet together in a single microscope setup. For a transparent thin sample where the scattering can be ignored, the LBS single-photon excitation is the optimal imaging solution. When the light scattering becomes significant for a deep-cell or deep-tissue imaging, we use SBB light-sheet two-photon excitation with a longer wavelength. We achieved nearly identical lateral/axial resolution of about 350/270 nm for both imagings. This integrated light-sheet microscope may have a wide application for live-cell and live-tissue three-dimensional high-speed imaging.