Measuring Molecular Mass Densities at Subcellular Resolution Using Optical Diffraction Tomography

Biological systems intricately regulate their density and volume throughout their life cycles and in response to physiological changes. Mass density, as a fundamental physical quantity, plays significant roles in biological processes such as differentiation, cell growth, protein synthesis, and condensate formation. Loss of density homeostasis on the other hand can have severe consequences including cellular senescence and disease states. Recent developments in biophotonics now enable high-resolution density quantification, providing new insights into the biophysical properties of cells and subcellular structures. One such technique is optical diffraction tomography (ODT), which offers label-free, high-resolution measurements of mass density distribution based on refractive index (RI) measurements. In this chapter, we present a comprehensive guide to implementing ODT for quantitative characterization of mass density distribution in biological systems, including in vivo (adherent cell culture) and in vitro (Xenopus egg extract) samples. We begin by detailing the optical setups, emphasizing key considerations for optimizing tomography acquisition. Subsequently, we introduce preparation protocols tailored to biological samples in various types of sample carriers and offer guidance on standard image acquisition and data analysis procedures. Finally, we address the challenges posed by the linear relationship between RI and mass density in complex substances, offering strategies for overcoming these limitations.