Schürmann Mirjam, Scholze Jana, Müller Paul, Chan Chii J, Ekpenyong Andrew E, Chalut Kevin J, Guck Jochen
Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany.
Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK; Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany.
Methods Cell Biol. 2015;125:143-59. doi: 10.1016/bs.mcb.2014.10.016. Epub 2015 Jan 8.
In this chapter, we introduce digital holographic microscopy (DHM) as a marker-free method to determine the refractive index of single, spherical cells in suspension. The refractive index is a conclusive measure in a biological context. Cell conditions, such as differentiation or infection, are known to yield significant changes in the refractive index. Furthermore, the refractive index of biological tissue determines the way it interacts with light. Besides the biological relevance of this interaction in the retina, a lot of methods used in biology, including microscopy, rely on light-tissue or light-cell interactions. Hence, determining the refractive index of cells using DHM is valuable in many biological applications. This chapter covers the main topics that are important for the implementation of DHM: setup, sample preparation, and analysis. First, the optical setup is described in detail including notes and suggestions for the implementation. Following that, a protocol for the sample and measurement preparation is explained. In the analysis section, an algorithm for the determination of quantitative phase maps is described. Subsequently, all intermediate steps for the calculation of the refractive index of suspended cells are presented, exploiting their spherical shape. In the last section, a discussion of possible extensions to the setup, further measurement configurations, and additional analysis methods are given. Throughout this chapter, we describe a simple, robust, and thus easily reproducible implementation of DHM. The different possibilities for extensions show the diverse fields of application for this technique.
在本章中，我们介绍数字全息显微镜（DHM），这是一种用于确定悬浮液中单个球形细胞折射率的无标记方法。折射率在生物学背景下是一个决定性的度量。已知细胞状态，如分化或感染，会导致折射率发生显著变化。此外，生物组织的折射率决定了其与光相互作用的方式。除了这种相互作用在视网膜中的生物学相关性外，生物学中使用的许多方法，包括显微镜技术，都依赖于光与组织或光与细胞的相互作用。因此，使用DHM确定细胞的折射率在许多生物学应用中具有重要价值。本章涵盖了实施DHM的重要主题：设置、样品制备和分析。首先，详细描述了光学设置，包括实施过程中的注意事项和建议。接下来，解释了样品和测量准备的方案。在分析部分，描述了一种用于确定定量相位图的算法。随后，利用悬浮细胞的球形形状，给出了计算其折射率的所有中间步骤。在最后一部分，讨论了设置的可能扩展、进一步的测量配置和其他分析方法。在本章中，我们描述了一种简单、稳健且易于重现的DHM实施方法。不同的扩展可能性展示了该技术的多样应用领域。
