Quantitative analysis of illumination and detection corrections in adaptive light sheet fluorescence microscopy
Quantitative analysis of illumination and detection corrections in adaptive light sheet fluorescence microscopy
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DOI：
10.1364/boe.454561
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发表时间：
2022-05-01
影响因子：
3.4
通讯作者：
Greenbaum, Alon
中科院分区：
文献类型：
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作者：
Rai, Mani Ratnam;Li, Chen;Greenbaum, Alon
光片荧光显微镜（LSFM）是一种用于体内和体外标本3D成像的高速，高分辨率和最小光毒性技术。LSFM具有光学切片，当与组织清除技术相结合时，它有助于以微米分辨率对厘米级标本进行成像。尽管LSFM无处不在，但它仍然面临着影响图像质量的两个主要挑战，特别是在以高分辨率成像大体积时。首先，光片照明平面和检测透镜焦平面需要共面，然而样本引起的像差可能违反该要求并降低图像质量。其次，在检测路径中引入样本诱导的光学像差。当对整个生物体或结构复杂的样本（例如表现出从软组织到硬组织的许多转变的耳蜗和骨骼）进行成像时，或者当对深度（> 2 mm）进行成像时，这些挑战会加剧。为了解决这些挑战，已经开发了各种照明和像差校正方法，但是还没有应用照明和检测路径两者中的自适应校正来改善LSFM成像。在这里，我们通过在定制的自适应LSFM上实现两种校正技术来弥合这一差距。照明光束的角度特性由两个检流计扫描器控制，而可变形反射镜位于检测路径中以校正像差。通过对猪全耳蜗的成像，比较和对比了这些校正方法及其对图像质量的影响。这些知识将极大地有助于自适应LSFM领域，以及大体积组织清除标本的成像。(C)2022 Optica出版集团根据Optica开放获取出版协议的条款
Light-sheet fluorescence microscopy (LSFM) is a high-speed, high-resolution and minimally phototoxic technique for 3D imaging of in vivo and in vitro specimens. LSFM exhibits optical sectioning and when combined with tissue clearing techniques, it facilitates imaging of centimeter scale specimens with micrometer resolution. Although LSFM is ubiquitous, it still faces two main challenges that effect image quality especially when imaging large volumes with high-resolution. First, the light-sheet illumination plane and detection lens focal plane need to be coplanar, however sample-induced aberrations can violate this requirement and degrade image quality. Second, introduction of sample-induced optical aberrations in the detection path. These challenges intensify when imaging whole organisms or structurally complex specimens like cochleae and bones that exhibit many transitions from soft to hard tissue or when imaging deep (> 2 mm). To resolve these challenges, various illumination and aberration correction methods have been developed, yet no adaptive correction in both the illumination and the detection path have been applied to improve LSFM imaging. Here, we bridge this gap, by implementing the two correction techniques on a custom built adaptive LSFM. The illumination beam angular properties are controlled by two galvanometer scanners, while a deformable mirror is positioned in the detection path to correct for aberrations. By imaging whole porcine cochlea, we compare and contrast these correction methods and their influence on the image quality. This knowledge will greatly contribute to the field of adaptive LSFM, and imaging of large volumes of tissue cleared specimens. (C) 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement