Progress and perspectives in digital holographic microscopy applied to life sciences

Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram, quantitative phase image of living cell with interferometric accuracy. Specifically the optical phase shift induced by the specimen on the transmitted wave front can be regarded as a powerful endogenous contrast agent, depending on both the thickness and the refractive index of the sample. The quantitative phase images allow the derivation of highly relevant cell parameters, including dry mass density and spatial distribution. Thanks to a decoupling procedure, cell thickness and intracellular refractive index can be measured separately. Consequently, cell morphology, shape as well as cell membrane fluctuations can be accurately monitor. As far as red blood cell are considered, Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), two highly relevant clinical parameters, have been measured non-invasively at a single cell level. The DHM nanometric axial and microsecond temporal sensitivities have permitted to measure the red blood cell membrane fluctuations (CMF) over the whole cell surface. In addition, the development of live-cell multimodality microscope combining fluorescence with digital holographic microscopy is presented. The biophysical cell parameters derived from the quantitative phase information in combination with the numerous different specific fluorescent cellular probes allow to this multimodality microscope to address various important issues in cell biology.