title: Refractive index measurements of single, spherical cells using digital holographic microscopy.
abstract: 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.
authors: Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany.
authors: Mirjam
authors: M
authors: Schürmann
authors: Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany.
authors: Jana
authors: J
authors: Scholze
authors: Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany.
authors: Paul
authors: P
authors: Müller
authors: Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK; Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany.
authors: Chii J
authors: CJ
authors: Chan
authors: Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany; Department of Physics, Creighton University, Omaha, NE, USA.
authors: Andrew E
authors: AE
authors: Ekpenyong
authors: Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK; Wellcome Trust/Medical Research Council Stem Cell Institute, Cambridge, UK.
authors: Kevin J
authors: KJ
authors: Chalut
authors: Biotechnology Center, Technische Universität Dresden, Tatzberg, Dresden, Germany; Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK.
authors: Jochen
authors: J
authors: Guck
mesh_terms: D048430:Cell Shape; D018922:HL-60 Cells; D006696:Holography / Q000379:methods*; D006801:Humans; D008853:Microscopy / Q000379:methods*; D012031:Refractometry / Q000379:methods*
keywords: Biomedical imaging; Cellular refractive index; Digital holographic microscopy; Marker-free; Quantitative phase microscopy