Shenzhen University, College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Guangdong Province, Ministry of Education, Shenzhen, China.
J Biomed Opt. 2024 Jan;29(1):016501. doi: 10.1117/1.JBO.29.1.016501. Epub 2024 Jan 24.
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 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.
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.
Comparative 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.
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 experiments.
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.
双光子荧光显微镜(TPFM)用高斯光束激发,需要轴向层析扫描才能进行三维(3D)容积成像,这是一个耗时的过程,而缓慢的成像速度阻碍了其在大脑成像中的应用。贝塞尔焦点具有大的焦深和恒定的分辨率,便于将三维体积投影到二维图像上,从而大大提高了容积成像的速度。
我们旨在展示具有无旁瓣贝塞尔光束的 TPFM 的能力,为活体生物样本的研究提供有前途的工具。
在活的小鼠大脑和转基因斑马鱼中进行了比较成像,以评估 TPFM 和基于贝塞尔光束的 TPFM 的性能。此外,引入了一种利用零阶和三阶贝塞尔光束的图像差方法,以有效抑制旁瓣引入的背景干扰。
与传统的 TPFM 相比,基于贝塞尔光束的 TPFM 的成像通量和速度提高了 30 倍。此外,在活体生物样本中验证了图像差方法的有效性,导致图像对比度显著增强。重要的是,我们的无旁瓣贝塞尔光束的 TPFM 对轴向位移具有鲁棒性,这是实验中非常有价值的特性。
我们使用无旁瓣贝塞尔光束的 TPFM 实现了活体小鼠大脑和转基因斑马鱼血管的快速、高对比度和稳健的容积成像。