Evaluating holotomography as a new way to measure the refractive index of phytoplankton

The refractive index of phytoplankton directly affects their optical properties, particularly backscattering, and by extension, their observation by remote sensing. It has notably been linked to intracellular carbon concentration, making it an important factor for ocean carbon cycling monitoring. Measuring refractive indices of inhomogeneous particles has historically been challenging, with the most common approach being the immersion of particles in liquids of different known refractive indices until they become difficult to discern under a microscope or until the turbidity attains a minimum. Other ways include inversion of scattering from a flow cytometer using the Lorentz-Mie theory. However, these methods do not allow the observation of sub-cellular refractive index structures, which are believed, from modelling studies, to strongly influence backscattering. A new technology, holotomography, consisting of a 3-dimensional microscope measuring the scattering of a 520 nm laser beam at 45° to calculate refractive indices at a resolution of 200 nm, allows measuring the refractive index of particles in more details than it was ever possible before. We share our observations of phytoplankton with holotomography to evaluate the limits and possibilities of the new technology. We found that holotomography allows detailed measurements of phytoplankton cellular structure and of the repartition of the refractive indexes within cells and also cell assemblages. Limits of the technique include spatial resolution and optimization is necessary to observe thin outer cellular structures. Furthermore, the method allows the measurement of the volumes of intracellular structures as well as the observation of the three-dimensional shapes of the cells and of their internal structures. These parameters, obtained with holotomography, will be included in optical models to evaluate their impact on model closure.