Anca Marian

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.

Digital Holography Applied to Microscopy

We report on new developments of a technique called Digital Holographic Microscopy (DHM), for the numerical reconstruction of digital holograms taken in microscopy, which allows simultaneous amplitude and quantitative phase contrast imaging. The reconstruction method computes the propagation of the complex optical wavefront diffracted by the object and is used to determine the refractive index and/or shape of the object with accuracies in the nanometer range along the optical axis.. The method requires the acquisition of a single hologram. The technique comprises the recording of a digital hologram of the specimen by means of a standard CCD camera at the exit of a Mach-Zehnder or Michelson type interferometer. The quantitative nature of the reconstructed phase distribution has been demonstrated by an application to surface profilometry where step height differences of a few nanometers have been measured. Another application takes place in biology for transmission phase- contrast imaging of living cells in culture. The resolution for thickness measurements depends on the refractive index of the specimen and a resolution of approximately 30 nanometers in height, and about half of a micro in width, has been achieved for living neural cells in cultures by using a high numerical aperture.

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.

Point spread function model for microscopic image deconvolution in digital holographic microscopy

A simple model is proposed in order to evaluate the complex amplitude point spread function (intensity and phase) of a microscope objective. The model is based on the Fresnel diffraction theory and takes also into account the possible optical aberrations. Experimental evaluation of this amplitude point spread function has been carried out by using a holographic set-up and 60 nanometers gold spheres as punctual objects. The measured values are then compared with the theoretical predicted model.

Use of digital holographic microscopy in tomography

Digital Holographic Microscopy (DHM) provides three-dimensional (3D) images with a high vertical accuracy in the nanometer range and a diffracted limited transverse resolution. This paper focuses on 3 different tomographic applications based on DHM. First, we show that DHM can be combined with time gating: a series of holograms is acquired at different depths by varying the reference path length, providing after reconstruction images of slices at different depths in the specimen thanks to the short coherence length of the light source. Studies on enucleated porcine eyes will be presented. Secondly, we present a tomography based on the addition of several reconstructed wavefronts measured with DHM at different wavelengths. Each wavefront phase is individually adjusted to be equal in a given plane of interest, resulting in a constructive addition of complex waves in the selected plane and destructive addition in the others. Varying the plane of interest enables the scan of the object in depth. Thirdly, DHM is applied to perform optical diffraction tomography of a pollen grain: transmission phase images are acquired for different orientations of the rotating sample, then the 3D refractive index spatial distribution is computed by inverse radon transform. The presented works will exemplify the versatility of DHM, but above all its capability of providing quantitative tomographic data of biological specimen in a quick, reliable and non-invasive way.

Automatic procedure for aberrations compensation in digital holographic microscopy

Digital Holographic Microscopy (DHM) is a powerful imaging technique allowing, from a single amplitude image acquisition (hologram), the reconstruction of the entire complex wave front (amplitude and phase), reflected by or transmitted through an object. Because holography is an interferometric technique, the reconstructed phase leads to a sub-wavelength axial accuracy (below λ/100). Nevertheless, this accuracy is difficult to obtain from a single hologram. Indeed, the reconstruction process consisting to process the hologram with a digital reference wave (similar to classical holographic reconstruction) seems to need a-priori knowledge about the physical values of the setup. Furthermore, the introduction of a microscope objective (MO), used to improve the lateral resolution, introduces a wave front curvature in the object wave front. Finally, the optics of the set-up can introduce different aberrations that decrease the quality and the accuracy of the phase images. We propose here an automatic procedure allowing the adjustment of the physical values and the compensation for the phase aberrations. The method is based on the extraction of reconstructed phase values, along line profiles, located on or around the sample, in assumed to be flat area, and which serve as reference surfaces. The phase reconstruction parameters are then automatically adjusted by applying curve-fitting procedures on the extracted phase profiles. An example of a mirror and a USAF test target recorded with high order aberrations (introduced by a thick tilted plate placed in the set-up) shows that our procedure reduces the phase standard deviation from 45 degrees to 5 degrees.

Point spread function models for digital holographic microscopy

Few models, based on the diffraction theory, are proposed in order to evaluate the point spread function of different microscope objectives used in a digital holographic microscope. Because in holography the phase information is essential, a 3D amplitude point spread function (APSF), modulus and phase, is necessary, in order to properly deconvolute the 3D images obtained. Scalar Debye theory, paraxial approximation and vectorial Debye theory are used to solve the diffraction problem and the theoretical predicted 3D APSFs obtained with these models are compared.