Rapid, high-contrast, and steady volumetric imaging with Bessel-beam-based two-photon fluorescence microscopy

Abstract.

Significance

Two-photon fluorescence microscopy (TPFM) excited by Gaussian beams requires axial tomographic scanning for three-dimensional (3D) volumetric imaging, which is a time-consuming process, and the slow imaging speed hinders its application for in vivo brain imaging. The Bessel focus, characterized by an extended depth of focus and constant resolution, facilitates the projection of a 3D volume onto a two-dimensional image, which significantly enhances the speed of volumetric imaging.

Aim

We aimed to demonstrate the ability of a TPFM with a sidelobe-free Bessel beam to provide a promising tool for research in live biological specimens.

Approach

Comparative in vivo imaging was conducted in live mouse brains and transgenic zebrafish to evaluate the performance of TPFM and Bessel-beam-based TPFM. Additionally, an image-difference method utilizing zeroth-order and third-order Bessel beams was introduced to effectively suppress background interference introduced by sidelobes.

Results

In comparison with traditional TPFM, the Bessel-beams-based TPFM demonstrated a 30-fold increase in imaging throughput and speed. Furthermore, the effectiveness of the image-difference method was validated in live biological specimens, resulting in a substantial enhancement of image contrast. Importantly, our TPFM with a sidelobe-free Bessel beam exhibited robustness against axial displacements, a feature of considerable value for in vivo experiments.

Conclusions

We achieved rapid, high-contrast, and robust volumetric imaging of the vasculature in live mouse brains and transgenic zebrafish using our TPFM with a sidelobe-free Bessel beam.