Zhao Ming, Zhang Han, Li Yu, Ashok Amit, Liang Rongguang, Zhou Weibin, Peng Leilei
College of Optical Sciences, the University of Arizona, 1630 East University Blvd., Tucson, AZ 85721, USA.
Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, MI 48109, USA.
Biomed Opt Express. 2014 Mar 31;5(5):1296-308. doi: 10.1364/BOE.5.001296. eCollection 2014 May 1.
In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.
对深部内部器官进行体内荧光细胞成像极具挑战性,因为激发光需要穿透强散射组织,且发射信号会因组织散射引起的光子扩散而显著衰减。我们报告称,通过将双光子贝塞尔光片显微镜与非线性结构照明显微镜(SIM)相结合,光片显微镜可以对宽度达600微米的活体样本进行成像,穿透深度为500微米,并且可以减少深部组织光片成像中的漫射背景,从而在超过200微米的深度获得细胞分辨率的清晰图像。我们展示了斑马鱼肾脏原肾肾小球和脉管系统的体内双色成像,通过双色双光子贝塞尔光片SIM能够高度清晰地揭示位于鱼体中心的细胞结构。