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Shiqi Wang

at Shenzhen Univ

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SPIE Journal Paper | 24 January 2024 Open Access

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

Yongqiang Chen, Chenggui Luo, Shiqi Wang, Yanping Li, Binglin Shen, Rui Hu, Junle Qu, Liwei Liu

JBO, Vol. 29, Issue 01, 016501, (January 2024) https://doi.org/10.1117/12.10.1117/1.JBO.29.1.016501

KEYWORDS: Bessel beams, Imaging systems, Biological imaging, In vivo imaging, Biomedical optics, Fluorescence imaging, Spatial light modulators, Image processing, Brain, 3D image processing

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SignificanceTwo-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.AimWe 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.ApproachComparative 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.ResultsIn 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.ConclusionsWe 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.

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