Zylbertal Asaph, Bianco Isaac H
University College London, Department of Neuroscience, Physiology & Pharmacology, London, United Kingdom.
Neurophotonics. 2024 Jul;11(3):035006. doi: 10.1117/1.NPh.11.3.035006. Epub 2024 Aug 7.
Light-sheet microscopy is a powerful imaging technique that achieves optical sectioning via selective illumination of individual sample planes. However, when the sample contains opaque or scattering tissues, the incident light sheet may not be able to uniformly excite the entire sample. For example, in the context of larval zebrafish whole-brain imaging, occlusion by the eyes prevents access to a significant portion of the brain during common implementations using unidirectional illumination.
We introduce mirror-assisted light-sheet microscopy (mLSM), a simple inexpensive method that can be implemented on existing digitally scanned light-sheet setups by adding a single optical element-a mirrored micro-prism. The prism is placed near the sample to generate a second excitation path for accessing previously obstructed regions.
Scanning the laser beam onto the mirror generates a second, reflected illumination path that circumvents the occlusion. Online tuning of the scanning and laser power waveforms enables near uniform sample excitation with dual illumination.
mLSM produces cellular-resolution images of zebrafish brain regions inaccessible to unidirectional illumination. The imaging quality in regions illuminated by the direct or reflected sheet is adjustable by translating the excitation objective. The prism does not interfere with visually guided behavior.
mLSM presents an easy-to-implement, cost-effective way to upgrade an existing light-sheet system to obtain more imaging data from a biological sample.
光片显微镜是一种强大的成像技术，它通过对单个样本平面进行选择性照明来实现光学切片。然而，当样本包含不透明或散射组织时，入射光片可能无法均匀地激发整个样本。例如，在斑马鱼幼体全脑成像的背景下，在使用单向照明的常见实现过程中，眼睛的遮挡会阻止对大脑很大一部分区域的观察。
我们引入了镜面辅助光片显微镜（mLSM），这是一种简单且廉价的方法，通过添加一个光学元件——一个微镜棱镜，就可以在现有的数字扫描光片设置上实现。该棱镜放置在样本附近，以生成第二条激发路径，从而观察到先前被遮挡的区域。
将激光束扫描到镜子上会产生第二条反射照明路径，从而避开遮挡。对扫描和激光功率波形进行在线调整，可实现双照明下样本的近均匀激发。
mLSM能够生成单向照明无法观察到的斑马鱼脑区的细胞分辨率图像。通过平移激发物镜，可以调整直接或反射光片照明区域的成像质量。该棱镜不会干扰视觉引导行为。
mLSM提供了一种易于实施、经济高效的方法，可对现有的光片系统进行升级，以便从生物样本中获取更多成像数据。
