Influence of shot noise on phase measurement accuracy in digital holographic microscopy.

Digital Holographic Microscopy (DHM) is a single shot interferometric technique, which provides quantitative phase images with subwavelength axial accuracy. A short hologram acquisition time (down to microseconds), allows DHM to offer a reduced sensitivity to vibrations, and real time observation is achievable thanks to present performances of personal computers and charge coupled devices (CCDs). Fast dynamic imaging at low-light level involves few photons, requiring proper camera settings (integration time and gain of the CCD; power of the light source) to minimize the influence of shot noise on the hologram when the highest phase accuracy is aimed. With simulated and experimental data, a systematic analysis of the fundamental shot noise influence on phase accuracy in DHM is presented.

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 1.2mum 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.

Total aberrations compensation in digital holographic microscopy with a reference conjugated hologram.

In this paper we present a new method to achieve quantitative phase contrast imaging in Digital Holographic Microscopy (DHM) that allows to compensate for phase aberrations and image distortion by recording of a single reference hologram.We demonstrate that in particular cases in which the studied specimen does not have abrupt edges, the specimen's hologram itself can be used as reference hologram. We show that image distortion and phase aberrations introduced by a lens ball used as microscope objective are completely suppressed with our method. Finally the concept of self-conjugated reference hologram is applied on a biological sample (Trypanosoma Brucei) to maintain a spatial phase noise level under 3 degrees.

Living specimen tomography by digital holographic microscopy: morphometry of testate amoeba.

This paper presents an optical diffraction tomography technique based on digital holographic microscopy. Quantitative 2-dimensional phase images are acquired for regularly-spaced angular positions of the specimen covering a total angle of pi, allowing to built 3-dimensional quantitative refractive index distributions by an inverse Radon transform. A 20x magnification allows a resolution better than 3 microm in all three dimensions, with accuracy better than 0.01 for the refractive index measurements. This technique is for the first time to our knowledge applied to living specimen (testate amoeba, Protista). Morphometric measurements are extracted from the tomographic reconstructions, showing that the commonly used method for testate amoeba biovolume evaluation leads to systematic under evaluations by about 50%.

Simultaneous cell morphometry and refractive index measurement with dual-wavelength digital holographic microscopy and dye-enhanced dispersion of perfusion medium.

Digital holographic microscopy (DHM) allows optical-path-difference (OPD) measurements with nanometric accuracy. OPD induced by transparent cells depends on both the refractive index (RI) of cells and their morphology. This Letter presents a dual-wavelength DHM that allows us to separately measure both the RI and the cellular thickness by exploiting an enhanced dispersion of the perfusion medium achieved by the utilization of an extracellular dye. The two wavelengths are chosen in the vicinity of the absorption peak of the dye, where the absorption is accompanied by a significant variation of the RI as a function of the wavelength.

Amplitude point-spread function measurement of high-NA microscope objectives by digital holographic microscopy.

We present here a three-dimensional evaluation of the amplitude point-spread function (APSF) of a microscope objective (MO), based on a single holographic acquisition of its pupil wavefront. The aberration function is extracted from this pupil measurements and then inserted in a scalar model of diffraction, allowing one to calculate the distribution of the complex wavefront propagated around the focal point. The accuracy of the results is compared with a direct measurement of the APSF with a second holographic system located in the image plane of the MO. Measurements on a 100 x 1.3 NA MO are presented.

Digital holographic microscopy applied to life sciences.

Digital holographic microscopy (DHM) is applied to life sciences applications and demonstrate its capability of real-time imaging and quantitative measurements of physiological parameters such as cell volume or mean cell hemoglobin concentration (MCHC) of erythrocyte cells. DHM has the advantage to be non-invasive (no phototoxicity, no contrast agents) and allows a high throughput measurements.

Submicrometer optical tomography by multiple-wavelength digital holographic microscopy.

We present a method for submicrometer tomographic imaging using multiple wavelengths in digital holographic microscopy. This method is based on the recording, at different wavelengths equally separated in the k domain, in off-axis geometry, of the interference between a reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A CCD camera records the holograms consecutively, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefront. Their relative phases are adjusted to be equal in a given plane of interest and the resulting complex wavefronts are summed. The result of this operation is a constructive addition of complex waves in the selected plane and destructive addition in the others. Tomography is thus obtained by the attenuation of the amplitude out of the plane of interest. Numerical variation of the plane of interest enables one to scan the object in depth. For the presented simulations and experiments, 20 wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices 725 nm thick.

Shot-noise influence on the reconstructed phase image signal-to-noise ratio in digital holographic microscopy.

In digital holographic microscopy, shot noise is an intrinsic part of the recording process with the digital camera. We present a study based on simulations and real measurements describing the shot-noise influence in the quality of the reconstructed phase images. Different configurations of the reference wave and the object wave intensities will be discussed, illustrating the detection limit and the coherent amplification of the object wave. The signal-to-noise ratio (SNR) calculation of the reconstructed phase images based on the decision statistical theory is derived from a model for image quality estimation proposed by Wagner and Brown [Phys. Med. Biol. 30, 489 (1985)]. It will be shown that a phase image with a SNR above 10 can be obtained with a mean intensity lower than 10 photons per pixel and per hologram coming from the observed object. Experimental measurements on a glass-chrome probe will be presented to illustrate the main results of the simulations.

Purely numerical compensation for microscope objective phase curvature in digital holographic microscopy: influence of digital phase mask position.

Introducing a microscope objective in an interferometric setup induces a phase curvature on the resulting wavefront. In digital holography, the compensation of this curvature is often done by introducing an identical curvature in the reference arm and the hologram is then processed using a plane wave in the reconstruction. This physical compensation can be avoided, and several numerical methods exist to retrieve phase contrast images in which the microscope curvature is compensated. Usually, a digital array of complex numbers is introduced in the reconstruction process to perform this curvature correction. Different corrections are discussed in terms of their influence on the reconstructed image size and location in space. The results are presented according to two different expressions of the Fresnel transform, the single Fourier transform and convolution approaches, used to propagate the reconstructed wavefront from the hologram plane to the final image plane.

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.

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.

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.

Time-domain optical coherence tomography with digital holographic microscopy.

We show that digital holography can be combined easily with optical coherence tomography approach. Varying the reference path length is the means used to acquire a series of holograms at different depths, providing after reconstruction images of slices at different depths in the specimen thanks to the short-coherence length of light source. A metallic object, covered by a 150-microm-thick onion cell, is imaged with high resolution. Applications in ophthalmology are shown: structures of the anterior eye, the cornea, and the iris, are studied on enucleated porcine eyes. Tomographic images of the iris border close to the pupil were obtained 165 microm underneath the eye surface.