Quantitative measurement of absolute cell volume and intracellular integral refractive index (RI) with Dual-wavelength Digital Holographic Microscopy (DHM)

Quantitative Phase Imaging techniques including DHM have been applied recently in the field of cell imaging to monitor and quantify non-invasively dynamic cellular processes modifying cell morphology and/or content. Concretely, the DHM phase signal is highly sensitive to cell thickness and intracellular integral RI variations associated with transmembrane water movements. As net water flow across the cell membrane leads at the same time to changes in cell thickness and intracellular RI, the interpretation of phase signal variations remains difficult. To overcome this drawback, we have developed a Dual-wavelength Digital Holographic Microscopy (DHM) setup allowing to separately measure, with a single CCD camera acquisition, thickness and integral RI of living cells. The method is based on the use of an absorbing dye that enhances the refractive index dispersion of the extracellular medium. Practically, two significantly different phase signals can be obtained when measuring at two appropriate wavelengths. From the two phase measurements, both cell RI and thickness can be univocally determined.