We have developed a digital holographic microscope (DHM), in a transmission mode, especially dedicated to the quantitative visualization of phase objects such as living cells. The method is based on an original numerical algorithm presented in detail elsewhere [Cuche et al., Appl. Opt. 38, 6994 (1999)]. DHM images of living cells in culture are shown for what is to our knowledge the first time. They represent the distribution of the optical path length over the cell, which has been measured with subwavelength accuracy. These DHM images are compared with those obtained by use of the widely used phase contrast and Nomarski differential interference contrast techniques.

We have developed a digital holographic microscope (DHM), in a transmission mode, adapted to the quantitative study of cellular dynamics. Living cells in culture are optically probed by measuring the phase shift they produce on the transmitted wave front. The high temporal stability of the phase signal, equivalent to lambda/1800, and the low acquisition time (~20micros) enable to monitor cellular dynamics processes. An experimental procedure allowing to calculate both the integral refractive index and the cellular thickness (morphometry) from the measured phase shift is presented. Specifically, the method has been applied to study the dynamics of neurons in culture during a hypotonic stress. Such stress produces a paradoxical decrease of the phase which can be entirely resolved by applying the methodological approach described in this article; indeed the method allows to determine independently the thickness and the integral refractive index of cells.

Red blood cell (RBC) parameters such as morphology, volume, refractive index, and hemoglobin content are of great importance for diagnostic purposes. Existing approaches require complicated calibration procedures and robust cell perturbation. As a result, reference values for normal RBC differ depending on the method used. We present a way for measuring parameters of intact individual RBCs by using digital holographic microscopy (DHM), a new interferometric and label-free technique with nanometric axial sensitivity. The results are compared with values achieved by conventional techniques for RBC of the same donor and previously published figures. A DHM equipped with a laser diode (k 5 663 nm) was used to record holograms in an off-axis geometry. Measurements of both RBC refractive indices and volumes were achieved via monitoring the quantitative phase map of RBC by means of a sequential perfusion of two isotonic solutions with different refractive indices obtained by the use of Nycodenz (decoupling procedure). Volume of RBCs labeled by membrane dye Dil was analyzed by confocal microscopy. The mean cell volume (MCV), red blood cell distribution width (RDW), and mean cell hemoglobin concentration (MCHC) were also measured with an impedance volume analyzer. DHM yielded RBC refractive index n 5 1.418 AE 0.012, volume 83 AE 14 fl, MCH 5 29.9 pg, and MCHC 362 AE 40 g/l. Erythrocyte MCV, MCH, and MCHC achieved by an impedance volume analyzer were 82 fl, 28.6 pg, and 349 g/l, respectively. Confocal microscopy yielded 91 AE 17 fl for RBC volume. In conclusion, DHM in combination with a decoupling procedure allows measuring noninvasively volume, refractive index, and hemoglobin content of single-living RBCs with a high accuracy. ' 2008 International Society for Advancement of Cytometry Key terms digital holographic microscopy; laser scanning confocal microscopy; impedance volume analyzer; red blood cell volume; MCV; refractive index; mean corpuscular hemoglobin concentration; MCHC

A technique to perform two-wavelengths digital holographic microscopy (DHM) measurements with a single hologram acquisition is presented. The vertical measurement range without phase ambiguity is extended to the micron-range, thanks to the resulting synthetic wavelength defined by the beating of two wavelengths with a separation of about 80nm. Real-time dual-wavelength imaging is made possible by using two reference waves having different wavelengths and propagation directions for the hologram recording. The principle of the method is exposed and experimental results concerning a 1.2mum m high test sample as well as a moving micro-mirror are presented. To the extent of our knowledge, this is the first time that real-time synthetic beat-wavelength digital holography measurements are reported.

We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z 0 -factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC 50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.