ABSTRACT
To situate the interest of digital holographic microscopy (DHM) in biology, let us recall that, in the past, phase contrast microscopy (Zernike 1937, 1942, 1949, Françon 1949) has played a dominant role in the observation of living cells and has permitted the ›rst precise determination of the refractive index of living cells (refractometry) (Barer and Joseph 1954, Barer 1957). At that time, the measuring procedure was quite complex and usually required the adjustment of the refractive index of an external medium in contact with the cell. ™ese determinations have allowed establishing a precise relationship between the index of refraction and the “dry mass” (Davies and Wilkins 1952) or density of the cell constituents: protein, nucleic acids and nucleoproteins, lipids and lipoproteins, but also with carbohydrates and inorganic constituents, salts, etc. ™e relationship between microscopic observations and basal physiologic parameters such as metabolism, cell growth, or decline and death could be ›rmly established. Moreover, a ›rst estimation of the optical dispersion laws could be established for proteins and opens the way to more precise characterization of cell constituents in the future. Technical adaptation of the design of the phase contrast microscope to a
re¦ection arrangement (re¦ection contrast microscopy) permitted a precise and quantitative determination of refractive index of cellular cytoplasm (Zernike 1942). Interpretations of tiny variations of index are possible in terms of the cellular structure.
