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      Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy

      , , , , , ,
      Optics Letters
      The Optical Society

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          Abstract

          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.

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          Most cited references13

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          Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.

          We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman, Brain Res 494: 65-74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement, B27, produced neuron survival above 60%, independent of plating density above 160 plated cells/mm2. For isolated cells (< 100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones.
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            Digital holography for quantitative phase-contrast imaging.

            We present a new application of digital holography for phase-contrast imaging and optical metrology. This holographic imaging technique uses a CCD camera for recording of a digital Fresnel off-axis hologram and a numerical method for hologram reconstruction. The method simultaneously provides an amplitude-contrast image and a quantitative phase-contrast image. An application to surface profilometry is presented and shows excellent agreement with contact-stylus probe measurements.
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              Phase contrast, a new method for the microscopic observation of transparent objects

              F ZERNIKE (1942)
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                Author and article information

                Journal
                OPLEDP
                Optics Letters
                Opt. Lett.
                The Optical Society
                0146-9592
                1539-4794
                2005
                2005
                March 01 2005
                March 01 2005
                : 30
                : 5
                : 468
                Article
                10.1364/OL.30.000468
                15789705
                6c7de65e-a903-45f4-ae6c-d5c84605c2d2
                © 2005
                History

                Molecular medicine,Neurosciences
                Molecular medicine, Neurosciences

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