Albert-Smet Ignacio, Marcos-Vidal Asier, Vaquero Juan José, Desco Manuel, Muñoz-Barrutia Arrate, Ripoll Jorge
Department of Bioengineering and Aerospace Engineering, Universidad Carlos III de Madrid, Madrid, Spain.
Experimental Medicine and Surgery Unit, Instituto de Investigación Sanitaria del Hospital Gregorio Marañón, Madrid, Spain.
Front Neuroanat. 2019 Jan 25;13:1. doi: 10.3389/fnana.2019.00001. eCollection 2019.
Light-sheet fluorescence microscopy (LSFM) has been present in cell biology laboratories for quite some time, mainly as custom-made systems, with imaging applications ranging from single cells (in the micrometer scale) to small organisms (in the millimeter scale). Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution, properties that make them ideal for a large range of applications. These include the study of cellular dynamics, in particular cellular motion which is essential to processes such as tumor metastasis and tissue development. Experimental setups make extensive use of microdevices (bioMEMS) that provide better control over the substrate environment than traditional cell culture experiments. For example, to mimic conditions, experiment biochemical dynamics, and trap, move or count cells. Microdevices provide a higher degree of empirical complexity but, so far, most have been designed to be imaged through wide-field or confocal microscopes. Nonetheless, the properties of LSFM render it ideal for 3D characterization of active cells. When working with microdevices, confocal microscopy is more widespread than LSFM even though it suffers from higher phototoxicity and slower acquisition speeds. It is sometimes possible to illuminate with a light-sheet microdevices designed for confocal microscopes. However, these bioMEMS must be redesigned to exploit the full potential of LSFM and image more frequently on a wider scale phenomena such as motion, traction, differentiation, and diffusion of molecules. The use of microdevices for LSFM has extended beyond cell tracking studies into experiments regarding cytometry, spheroid cultures and lab-on-a-chip automation. Due to light-sheet microscopy being in its early stages, a setup of these characteristics demands some degree of optical expertise; and designing three-dimensional microdevices requires facilities, ingenuity, and experience in microfabrication. In this paper, we explore different approaches where light-sheet microscopy can achieve single-cell and subcellular resolution within microdevices, and provide a few pointers on how these experiments may be improved.
光片荧光显微镜（LSFM）已经在细胞生物学实验室中存在了相当长的时间，主要是作为定制系统，其成像应用范围从单细胞（微米尺度）到小型生物体（毫米尺度）。这类显微镜以其极低的光毒性水平以及高空间和时间分辨率而著称，这些特性使其成为众多应用的理想选择。这些应用包括细胞动力学研究，特别是细胞运动，而细胞运动对于肿瘤转移和组织发育等过程至关重要。实验装置广泛使用微器件（生物微机电系统），与传统细胞培养实验相比，这些微器件能更好地控制底物环境。例如，为模拟条件、实验生化动力学以及捕获、移动或计数细胞。微器件提供了更高程度的经验复杂性，但到目前为止，大多数微器件都设计用于通过宽视场或共聚焦显微镜进行成像。尽管如此，LSFM的特性使其成为活性细胞三维表征的理想选择。在使用微器件时，共聚焦显微镜比LSFM更广泛，尽管它存在光毒性更高和采集速度较慢的问题。有时可以用为共聚焦显微镜设计的光片照亮微器件。然而，这些生物微机电系统必须重新设计，以充分发挥LSFM的潜力，并更频繁地对更广泛的现象（如分子的运动、牵引、分化和扩散）进行成像。用于LSFM的微器件的应用已经从细胞追踪研究扩展到细胞计数、球体培养和芯片实验室自动化等实验。由于光片显微镜尚处于早期阶段，具备这些特性的装置需要一定程度的光学专业知识；而设计三维微器件则需要微加工方面的设备、独创性和经验。在本文中，我们探讨了光片显微镜在微器件内实现单细胞和亚细胞分辨率的不同方法，并提供了一些关于如何改进这些实验的建议。
