Digital holographic microscopy for study cellular dynamics

Digital holographic microscopy for study cellular dynamics Pierre Marquet, Benjamin Rappaz, Pierre MagistrettiDepartement of de PhysiologieUniversity of Lausanne, CH-1005 Lausanne, SwitzerlandPierre.marquet@unil.chEtienne Cuche, Yves EmeryLyncée Tec SA, Rue du bugnon 7, CH-1005 Lausanne, SwizerlandTristan Colomb, Frédéric Montfort, Florian Charrière, Anca Marian and Christian D. Depeursinge Ecole Polytechnique Fédérale de Lausanne, Institut d'imagerie et d’optique appliquée, CH-1015 Lausanne, Switzerland Abstract: Based on an original numerical reconstruction algorithm (E. Cuche et al. Appl. Opt. 38,6994 1999), we have developed a Digital Holographic Microscope (DHM), in a transmissionmode, allowing to investigate cellular structures and dynamics.2003 Optical Society of AmericaOCIS codes: (090.0090) Holography, (170.1530) Cell analysis1. IntroductionIn biology, the visualization of transparent specimens, including living cells, led to the development of optical contrast-enhancing imaging techniques. Among the numerous modalities of contrast-enhancing techniques which have been developed to visualize non invasively unstained transparent specimens, phase contrast (PhC), initially proposed by F. Zernicke as a means of image contrast1, as well as Normarski’s differential interference contrast2 (DIC) are available for high-resolution light microscopy3 and widely used in biology. Although, these technique reveal the structure of such transparent specimen, the phase information provided is qualitative.Unlike the PhC and DIC microscopy techniques, interferometric techniques present the great advantage of yielding quantitative measurements of parameters including the phase distribution produced by transparent specimens. However, whereas interferometric techniques are widely used in metrology, only few biological applications have been reported in biology. Recently, both a phase-shifting interferometry technique referred to as Fourier phase microscope4 and DHM5 have demonstrated their capacity to yield accurately absolute phase images of living cells. In this proceeding we present an interferometric technique, based on a digital holography principle, that allows one to measure the phase or the optical pathlength distribution (OPL) created by living cells and to monitor OPL variations induces by biological processes.Digital holography i.e., digital recording and numerical reconstruction of holograms, offers indeed new perspectives in imaging, because numerical processing of complex wave front allows to compute simultaneously the intensity and the phase distribution of the propagated wave6. In optical microscopy, digital holography7-9 has made possible to numerically focus on different object planes without using any opto-mechanical movements8,10. Moreover different lens aberrations8,11 can be corrected by a numerical procedure.2. MethodThe digital holographic microscope (DHM) that we have developed is basically a mach-Zehnder interferometer (Fig. 1). A laser beam is divided by a beam splitter (BS). The sample (S) is illuminated by one beam through a condenser (C). A microscope objective (MO) collects the transmitted light and forms the object wave (O), which interferes, in an off-axis geometry, with a reference wave R to produce an hologram intensity. The DHM method was realized with standard, commercial opto-mechanical components. A linearly polarized Helium-Neon laser (10 mW, λ = 633 nm) was used as coherent light source. Primary cultures of mouse cortical neurons were prepared according to Brewer12 and were observed through a 60×, 0.8 NA MO that enables a diffraction limited transverse resolution of around 0.6 µm (Ref 7) and a full field of view of 120×120 µm. Holograms (8 bits, 512 × 512 pixels) were recorded on a standard monochrome CCD video camera (PCO VX44-C). Acquisition,digitization and reconstruction of holograms were performed on a 2.66 MHz Pentium IV computer using a video frame grabber (IMAQ-PCI) within the LabVIEW environment (National Instruments). The measured irradiance at the neuronal plane was ~ 200 µW/cm 2, which is several orders of magnitude lower than the power used in classical confocal laser scanning microscope.Fig. 1: Basic configuration for digital holographic microscopy. Inset, schematic representation of cultured cells mounted ina closed perfusion chamber. M means mirror others abbreviations define in text.3. Results and discussionQuantitative phase images are obtained by DHM (Fig.2), according to an original procedure using a single recorded hologram 13. The value of each pixel i of the reconstructed phase image can be expressed as: ()(),,022()()id i c i m i c i m i m n z dz n D d n n d n D ππϕλλ=+−=−+∫ (1)where λ is the wavelength, z is the axial co-ordinate, i d is the cellular thickness corresponding to the pixel i, ,()c i n z is the function representing the value of the intracellular refractive index along the cellular thickness i d ,,,01()id c i c i i n n z dz d =∫is the intracellular mean refractive index (,c i n ) along the cellular thickness i d , n m the constant refractive index of the surrounding medium and D is the thickness of the flow chamber (inset of Fig 1). The product n m D is a reference value that can be measured anywhere in the vicinity of the cell. Monitoring this reference value is important because it enables the compensation of mechanical or thermal instabilities of the set-up during the experiment.For each pixel i, the component of the signal, which accounts for the cell specific contribution to the optical pathlength (),c i m i n n d − (Eq. 1), depends on the cell thickness ()i d , the intracellular mean refractive index (,c i n ) and the refractive index of the perfusion solution ()m n whose value n m = 1.3336 ± 0.0002 has been measured with an Abbe refractometer at λ= 633 nm. By assuming, in first approximation, a constant and homogeneous cellular refractive index n c = 1.37514 , one can estimate that a phase shift of 10° corresponds to a cellular thickness of ~0.45 µm. This translates into a thickness of 1-3 µm for the neuronal processes and of ~8-10 µm for the cell body and allows a 3D representation of living neurons (Fig.2). With the assumption of a constant intracellular refractive index (which can be discussed given a certain degree of heterogeneity of constituents), these estimations of cell morphology give realistic values of typical neuron dimensions. Using the same assumption for refractive index, and based on the phase measurement accuracy of 2-4 degrees, we obtain a cellular axial (or vertical) accuracy of ~160-320 nm.Fig. 2 Perspective image in false colors of living neurons in culture. Neuronal bodies and processes are clearly visible.Color bar represent optical path length in degreesThe phase measurements accuracy results mainly from the presence of coherent noise originating from defects on and in optical elements, particularly in glass coverslips on which neurons were plated. On the other hand, the standard deviation of the phase temporal variations over 60-min periods has a value of σ = 0.3°. This standard deviation corresponds to the measurement of time-varying morphological cellular processes with an axial accuracy of ~30 nm.In summary DHM provides a non-invasive means of measuring quantitative phase distribution created by living cells in their natural environment. This quantitative distribution contains information concerning both morphology and refractive index of the observed specimen. In addition, the fast acquisition time and the high temporal stability permit to monitor rapid time-varying cellular processes with a sub-wave length axial accuracy offering attractive possibilities for the visualization of cellular dynamics.4. AcknowledgementsThis work was supported by grants n° #31-51882.97, #21-67068.01 and #205320-103885/1 from the Swiss National Science Foundation’.5. References1. F. Zernike, “Phase-contrast, a new method for microscopic observation of transparent objects, Part I”, Physica, 11, 686 (1942).2.G. Nomarski, “Differential microinterferometer with polarized waves”, J.Phys.Radium, 16, 9 (1955).3.M. Pluta, advanced light microcoscopy, vol 2, (Elsevier Science publishing CO., INC 1988)4.G. Popescu et al., ‘Fourier phase microscopy for investigation of biological structure and dynamics’, Optics Letters, 29 (21), 2503-2505,(2004)5.P marquet et al., ‘Digital holographic microscopy : a non invasive contrast imaging technique allowing quantitative visualization of livingcells with subwavelength axial accuracy’, in press Optics Letters, (2005)6.U. Schnars, and W.P.O. Jüptner, “Digital recording and numerical reconstruction of holograms”,Meas. Sci. & Tech., 13, R85-R101 (2002)7.T. Zhang, I. Yamaguchi, ”Three-dimensional microscopy with phase-shifting digital holography”, Otha, Opt. Lett. 23, 1121-1223 (1998).8. E. Cuche, P. Marquet, and C. Depeursinge, “Simultaneous amplitude and quantitative phase contrast microscopy by numericalreconstruction of Fresnel off-axis holograms”, Appl. Opt. 38, 6994-7001 (1999)9.G. Indebetouw, and P. Klysubun, “Spatiotemporal digital microholography”, J. Opt. Soc. Am. A, 18, 319-325 (2001)10. F. Dubois, L. Joannes, J.-C. Legros, “Improved three dimensional imaging with a digital holography microscope with a source of partialcoherence”,Appl. Opt. 38, 7085 (1999)11. A. Stadelmaier, and J.H. Massig, “Compensation of lens aberrations in digital holography“, Opt. Lett., 25, 1630-1632 (2000)12.G.J. Brewer, J.R Torricelli, E.K. Evege, P.J., Price, ”Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a newserum-free medium combination”, J. Neurosci. Res. 35, 567-576 (1993)13. E. Cuche, F. Bevilaqua and C. Depeursinge, “Digital holography for quantitative phase contrast imaging“, Opt. Lett. 24, 291-293 (1999).14. A. Dunn, R. Richards-Kortum, “Three-dimensional of light scattering by cells”, IEEE J. Select. Top. Quant. Elec., 2, 898, (1996).。