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Erythrocytes volume and refractive index measurement with a Digital Holographic Microscope
B. Rappaza, A. Barbulb, F. Charrièrec, J. Kühnc, P. Marquetd, R. Korensteinb, C. Depeursingec and P. Magistrettia,d
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EPFL, Brain Mind Institute, Lausanne, 1015, Switzerland Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Ramat Aviv 69 978, Tel-Aviv, Israel c Ecole PolytechniqueFédérale de Lausanne (EPFL), Imaging and Applied Optics Institute, Lausanne, 1015, Switzerland d Centre de Neurosciences Psychiatriques, Département de psychiatrie DP-CHUV, Site de Cery, Prilly-Lausanne, 1008, Switzerland email: Benjamin.Rappaz@epfl.ch
ABSTRACT

a

Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram, quantitative phaseimage 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. We have recently proposed a new and efficient decoupling procedure allowing to directly obtain separate measurements of thethickness and the integral refractive index of a given living cell. Consequently, it has been possible to accurately measure (with a precision of 0.0003) the mean refractive index and the volume of living erythrocytes. Here, application of this decoupling procedure on erythrocyte allows to measure a refractive index of 1.40 and a mean volume of about 106 μm3. Keywords: digital holography, erythrocyte,cell imaging, refractive index

INTRODUCTION
Digital holographic microscopy (DHM) we have developed is a technique allowing to obtain, from a single recorded hologram, quantitative phase images of living cell dynamics with interferometric accuracy (Cuche et al., 1999). The optical phase shift induced by a given sample on the transmitted wavefront can be regarded as a powerful endogenouscontrast agent, as it contains information about both the thickness and the refractive index of the sample. We have proposed (Rappaz et al., 2005) a decoupling procedure, based on a concept initially proposed by (Barer, 1957; Evans and Fung, 1972), allowing to directly calculate from the quantitative phase signal the corresponding cell morphology and integral refractive index related to the intracellularcontent, notably proteins.. This procedure is particularly useful for measuring, in the same experimental conditions, both cell morphology changes and their associated integral refractive index modifications occurring during biological processes (Rappaz et al., 2005). Erythrocytes are composed (Mazeron et al., 1997) of hemoglobin (32%, refractive index: n = 1.615) water (65%, n = 1.333) andmembrane components (3%, n = 1.6) and do not contain any nucleus. They can be characterized by their volume, shape and refractive index. Those parameters, altered in various pathological processes, can be used as good indicators to (Mohandas and Evans, 1994).

CELL PREPARATION
100-150 μl of blood was drawn from healthy laboratory personnel by fingerpick, collected and diluted at a ratio of 1:10(v/v) in cold HEP buffer (15 mM HEPES pH 7.4, NaCl 130 mM, KCl 5.4 mM and 10 mM glucose). Blood cells were sedimented at 200 g, 4 oC for 10 min and buffy coat with upper 20% of erythrocytes were gently removed. Red blood cells were washed twice in HEP buffer (1000 g X 2 min at 4 oC). Finally, erythrocytes were suspended in HEPA buffer (15 mM HEPES pH 7.4, 130 mM NaCl, 5.4 mM KCl, 10 mM glucose, 1 mMCaCl2, 0.5 mM MgCl2 and 1 mg/ml bovine serum albumin) at 0.2 % hematocrit. The erythrocyte suspension was introduced into the experimental perfusion chamber consisting of two cover glasses separated by spacers 1.2 mm thick, and incubated for 20 min at 37 oC. This allows the erythrocytes to adhere to the glass coverslip. Unattached cells were removed by gently perfusing the chamber with HEPA...