Department of Precision Machinery and Precision Instrumentation, University of Science and Technology of China, Anhui, Hefei, China.
Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Anhui, Hefei, China.
J Biophotonics. 2022 Jan;15(1):e202100185. doi: 10.1002/jbio.202100185. Epub 2021 Sep 14.
Dark-field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under-explored and so far, only meso-scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans- and epi-illumination configurations. Our findings suggest that the epi-illumination configuration is superior in both contrast and fidelity compared to trans-illumination, while having the added advantage of experimental simplicity and an "open top" for experimental intervention. Guided by the theoretical analysis, we constructed an epi-illumination dark-field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans.
暗场显微镜以提供高分辨率和对薄样品的直接可视化而著称。然而，其在厚样品上的性能和优化仍未得到充分探索，迄今为止，仅展示了整个生物体的中尺度信息。在这项工作中，我们仔细研究了透射和反射照明配置之间的差异。我们的发现表明，与透射照明相比，反射照明在对比度和保真度方面都具有优势，同时具有实验简单性和“开放式顶部”的附加优势，便于进行实验干预。在理论分析的指导下，我们构建了一个反射照明暗场显微镜，其测量的横向和轴向分辨率分别为 260nm 和 520nm。无需图像重建，即可直接观察整个生物体中的亚细胞结构，并通过同时进行荧光成像进一步确认。我们以 20 到 50fps 的成像速度可视化了快速的动态过程，例如秀丽隐杆线虫中的细胞分裂和咽泵。
