Etienne Cuche

Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms.

We present a digital method for holographic microscopy involving a CCD camera as a recording device. Off-axis holograms recorded with a magnified image of microscopic objects are numerically reconstructed in amplitude and phase by calculation of scalar diffraction in the Fresnel approximation. For phase-contrast imaging the reconstruction method involves the computation of a digital replica of the reference wave. A digital method for the correction of the phase aberrations is presented. We present a detailed description of the reconstruction procedure and show that the transverse resolution is equal to the diffraction limit of the imaging system.

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.

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

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.

Spatial filtering for zero-order and twin-image elimination in digital off-axis holography.

Off-axis holograms recorded with a CCD camera are numerically reconstructed with a calculation of scalar diffraction in the Fresnel approximation. We show that the zero order of diffraction and the twin image can be digitally eliminated by means of filtering their associated spatial frequencies in the computed Fourier transform of the hologram. We show that this operation enhances the contrast of the reconstructed images and reduces the noise produced by parasitic reflections reaching the hologram plane with an incidence angle other than that of the object wave.

Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy

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.

Cell refractive index tomography by digital holographic microscopy

For what we believe to be the first time, digital holographic microscopy is applied to perform optical diffraction tomography of a pollen grain. Transmission phase images with nanometric axial accuracy are numerically reconstructed from holograms acquired for different orientations of the rotating sample; then the three-dimensional refractive index spatial distribution is computed by inverse radon transform. A precision of 0.01 for the refractive index estimation and a spatial resolution in the micrometer range are demonstrated.

Real-time dual-wavelength digital holographic microscopy with a single hologram acquisition.

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 1.2μm 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.

Automatic procedure for aberration compensation in digital holographic microscopy and applications to specimen shape compensation

We present a procedure that compensates for phase aberrations in digital holographic microscopy by computing a polynomial phase mask directly from the hologram. The phase-mask parameters are computed automatically without knowledge of physical values such as wave vectors, focal lengths, or distances. This method enables one to reconstruct correct and accurate phase distributions, even in the presence of strong and high-order aberrations. Examples of applications are shown for microlens imaging and for compensating for the deformations associated with a tilted thick plate. Finally we show that this method allows compensation for the curvature of the specimen, revealing its surface defects and roughness. Examples of applications are shown for microlenses and metallic sphere imaging.

Numerical parametric lens for shifting, magnification and complete aberration compensation in digital holographic microscopy

The concept of numerical parametric lenses (NPL) is introduced to achieve wavefront reconstruction in digital holography. It is shown that operations usually performed by optical components and described in ray geometrical optics, such as image shifting, magnification, and especially complete aberration compensation (phase aberrations and image distortion), can be mimicked by numerical computation of a NPL. Furthermore, we demonstrate that automatic one-dimensional or two-dimensional fitting procedures allow adjustment of the NPL parameters as expressed in terms of standard or Zernike polynomial coefficients. These coefficients can provide a quantitative evaluation of the aberrations generated by the specimen. Demonstration is given of the reconstruction of the topology of a microlens.

Characterization of microlenses by digital holographic microscopy

We demonstrate the use of digital holographic microscopy (DHM) as a metrological tool in micro-optics testing. Measurement principles are compared with those performed with Twyman-Green, Mach-Zehnder, and white-light interferometers. Measurements performed on refractive microlenses with reflection DHM are compared with measurements performed with standard interferometers. Key features of DHM such as digital focusing, measurement of shape differences with respect to a perfect model, surface roughness measurements, and optical performance evaluation are discussed. The capability of imaging nonspherical lenses without any modification of the optomechanical setup is a key advantage of DHM compared with conventional measurement tools and is demonstrated on a cylindrical microlens and a square lens array.