Advantages of digital holographic microscopy for real-time full field absolute phase imaging

Different interferometric techniques were developed last decade to obtain full field, quantitative, and absolute phase imaging, such as phase-shifting, Fourier phase microscopy, Hilbert phase microscopy or digital holographic microscopy (DHM). Although, these techniques are very similar, DHM combines several advantages. In contrast, to phase shifting, DHM is indeed capable of single-shot hologram recording allowing a real-time absolute phase imaging. On the other hand, unlike to Fourier phase or Hilbert phase microscopy, DHM does not require to record in focus images of the specimen on the digital detector (CCD or CMOS camera), because a numerical focalization adjustment can be performed by a numerical wavefront propagation. Consequently, the depth of view of high NA microscope objectives is numerically extended. For example, two different biological cells, floating at different depths in a liquid, can be focalized numerically from the same digital hologram. Moreover, the numerical propagation associated to digital optics and automatic fitting procedures, permits vibrations insensitive full- field phase imaging and the complete compensation for a priori any image distortion or/and phase aberrations introduced for example by imperfections of holders or perfusion chamber. Examples of real-time full-field phase images of biological cells have been demonstrated.

Numerical aberrations compensation and polarization imaging in digital holographic microscopy

Tristan Colomb

In this thesis, we describe a method for the numerical reconstruction of the complete wavefront properties from a single digital hologram: the amplitude, the phase and the polarization state. For this purpose, we present the principle of digital holographic microscopy (DHM) and the numerical reconstruction process which consists of propagating numerically a wavefront from the hologram plane to the reconstruction plane. We then define the different parameters of a Numerical Parametric Lens (NPL) introduced in the reconstruction plane that should be precisely adjusted to achieve a correct reconstruction. We demonstrate that automatic procedures not only allow to adjust these parameters, but in addition, to completely compensate for the phase aberrations. The method consists in computing directly from the hologram a NPL defined by standard or Zernike polynomials without prior knowledge of physical setup values (microscope objective focal length, distance between the object and the objective...). This method enables to reconstruct correct and accurate phase distributions, even in the presence of strong and high order aberrations. Furthermore, we show that this method allows to compensate for the curvature of specimen. The NPL parameters obtained by Zernike polynomial fit give quantitative measurements of micro-optics aberrations and the reconstructed images reveal their surface defects and roughness. Examples with micro-lenses and a metallic sphere are presented. Then, this NPL is introduced in the hologram plane and allows, as a system of optical lenses, numerical magnification, complete aberration compensation in DHM (correction of image distortions and phase aberrations) and shifting. This NPL can be automatically computed by polynomial fit, but it can also be defined by a calibration method called Reference Conjugated Hologram (RCH). We demonstrate the power of the method by the reconstruction of non-aberrated wavefronts from holograms recorded specifically with high orders aberrations introduced by a tilted thick plate, or by a cylindrical lens or by a lens ball used instead of the microscope objective. Finally, we present a modified digital holographic microscope permitting the reconstruction of the polarization state of a wavefront. The principle consists in using two reference waves polarized orthogonally that interfere with an object wave. Then, the two wavefronts are reconstructed separately from the same hologram and are processed to image the polarization state in terms of Jones vector components. Simulated and experimental data are compared to a theoretical model in order to evaluate the precision limit of the method for different polarization states of the object wave. We apply this technique to image the birefringence and the dichroism induced in a stressed polymethylmethacrylate sample (PMMA), in a bent optical fiber and in a thin concrete specimen. To evaluate the precision of the phase difference measurement in DHM design, the birefringence induced by internal stress in an optical fiber is measured and compared to the birefringence profile captured by a standard method, which had been developed to obtain high-resolution birefringence profiles of optical fibers. A 6 degrees phase difference resolution is obtained, comparable with standard imaging polariscope, but with the advantage of a single acquisition allowing real-time reconstruction.

EPFL2006

Digital Holography Microscopy (DHM): Fast and robust systems for industrial inspection with interferometer resolution

Nicolas Aspert, Florian Charrière, Tristan Colomb, Etienne Cuche, Christian Depeursinge, Jonas Kühn, Pierre Marquet, Frédéric Montfort

With the recent technological advances, there is an increasing need for measurement systems providing interferometer resolution for inspection of large quantities of individual samples in manufacturing environments.. Such applications require high measurement rates, robustness, ease of use, and non-contact systems. We show here that Digital Holographic Microscopy (DIM), a new method that implements digitally the principle of holography, is particularly well suited for such industrial applications. With the present computers power and the developments of digital cameras, holograms can be numerically interpreted within a tenth of second to provide simultaneously: the phase information, which reveals object surface with vertical resolution at the nanometer scale along the optical axis, and intensity images, as obtained by conventional optical microscope. The strength of DHM lies in particular on the use of the so-called off-axis configuration, which enables to capture the whole information by a single image acquisition, i.e. typically during a few ten of microseconds. These extremely short acquisition times make DHM systems insensitive to vibrations. These instruments can operate without vibration insulation means, making them a cost effective solution not only for R&D, but also especially for an implementation on production lines. Numerous application examples are presented in this paper such as shape and surface characterization of high aspect ratio micro-optics, surface nanostructures, and surface roughness.

SPIE2005

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

Nicolas Aspert, Florian Charrière, Tristan Colomb, Christian Depeursinge, Jonas Kühn, Pierre Marquet

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.

2006

Numerical aberrations compensation and polarization imaging in digital holographic microscopy

Tristan Colomb

EPFL2006