Rai Mani Ratnam, Li Chen, Greenbaum Alon
Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill, Raleigh, NC 27695, USA.
Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27695, USA.
Biomed Opt Express. 2022 Apr 22;13(5):2960-2974. doi: 10.1364/BOE.454561. eCollection 2022 May 1.
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.
光片荧光显微镜（LSFM）是一种用于体内和体外标本三维成像的高速、高分辨率且光毒性极小的技术。LSFM具有光学切片功能，与组织透明化技术相结合时，有助于对厘米级标本进行微米级分辨率的成像。尽管LSFM应用广泛，但它仍面临两个影响图像质量的主要挑战，尤其是在对大体积标本进行高分辨率成像时。首先，光片照明平面和检测透镜焦平面需要共面，然而样品引起的像差会破坏这一要求并降低图像质量。其次，在检测路径中引入了样品引起的光学像差。当对整个生物体或结构复杂的标本（如耳蜗和骨骼，它们在软组织和硬组织之间有许多过渡）进行成像，或者对深度超过2毫米的标本进行成像时，这些挑战会加剧。为了解决这些挑战，人们开发了各种照明和像差校正方法，但尚未应用在照明和检测路径中都进行自适应校正的方法来改善LSFM成像。在这里，我们通过在定制的自适应LSFM上实施这两种校正技术来弥补这一差距。照明光束的角度特性由两个振镜扫描仪控制，同时在检测路径中放置一个可变形镜来校正像差。通过对整个猪耳蜗进行成像，我们比较并对比了这些校正方法及其对图像质量的影响。这些知识将极大地推动自适应LSFM领域以及对大量组织透明标本的成像研究。
