Enhancing the performance of digital holographic microscopy (original) (raw)
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Numerical aberrations compensation and polarization imaging in digital holographic microscopy
2006
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 permit-ii
Optics Express, 2006
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.
Automatic procedure for aberrations compensation in digital holographic microscopy
Optical Micro- and Nanometrology in Microsystems Technology, 2006
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 setup 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 setup) shows that our procedure reduces the phase standard deviation from 45 degrees to 5 degrees.
Automatic procedure for aberrations compensation in digital holographic microscopy
Optical Micro- and Nanometrology in Microsystems Technology, 2006
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.
Physical phase compensation in digital holographic microscopy
2009
In digital holographic microscopy (DHM), using microscope objective for sample imaging may introduce additional spherical phase curvature. It can be physically compensated by introducing a same phase curvature in the reference beam. A theoretical analysis of the wavefront interefence between the reference beam and object beam is provided to indicate the physical phase compensation. The spatial frequency spectra of the hologram are involved for the judgement of the physical phase compensation status. Different DHM setups are presented in order to fulfill the physical compensation of the introduced spherical phase. In the DHM setups based on the Michelson interferometric configuration, an adjustable lens is used to perform the quasi physical phase compensation during the hologram recording. In the common-path DHM setups, digital off-axis holograms are recorded by using a single cube beam splitter in a non-conventional configuration so as to both split and combine a diverging spherical wavefront emerging from a microscope objective. A simple plane numerical reference wavefront is used for the reconstruction and the correct quantitative phase map of the test object is obtained after phase unwrapping. Its simplicity of the presented setups make it easy to be well aligned and with lower cost.
Digital Holographic Microscopy (DHM)
Imaging & Microscopy, 2006
Digital Holographic Microscopes (DHM) enables strictly noninvasive visualisation of unstained transparent and partially reflective specimens, in real time, by providing simultaneously amplitude and phase changes of a light wave transmitted or reflected. They are used for characterisation of samples at the nanometer scale, for quality control on production line, and for dynamical analysis of biological specimen and micro systems. more than 15 reconstructions per second for 512 x 512 pixels holograms with a standard personal computer.
Quasi-physical phase compensation in digital holographic microscopy
Journal of The Optical Society of America A-optics Image Science and Vision, 2009
In digital holographic microscopy, if an optical setup is well aligned, the phase curvature introduced by the microscope objective (MO) together with the illuminating wave to the object wave is a spherical phase curvature. It can be physically compensated by introducing the same spherical phase curvature in the reference beam. Digital holographic microscopy setups based on the Michelson interferometric configuration with MO and an adjustable lens are presented, which can well perform the quasi-physical phase compensation during the hologram recording. In the reflection mode, the adjustable lens serves as both the condensing lens and the compensation lens. When the spatial frequency spectra of the hologram become a point spectrum, one can see that the phase curvature introduced by imaging is quasi-physically compensated. A simple plane numerical reference wavefront used for the reconstruction can give the correct quantitative phase map of the test object. A theoretical analysis and experimental demonstration are given. The simplicity of the presented setup makes it easy to align it well at lower cost.
Quasi-physical phase compensation in digital holographic microscopy: errata
Journal of The Optical Society of America A-optics Image Science and Vision, 2010
In digital holographic microscopy, if an optical setup is well aligned, the phase curvature introduced by the microscope objective (MO) together with the illuminating wave to the object wave is a spherical phase curvature. It can be physically compensated by introducing the same spherical phase curvature in the reference beam. Digital holographic microscopy setups based on the Michelson interferometric configuration with MO and an adjustable lens are presented, which can well perform the quasi-physical phase compensation during the hologram recording. In the reflection mode, the adjustable lens serves as both the condensing lens and the compensation lens. When the spatial frequency spectra of the hologram become a point spectrum, one can see that the phase curvature introduced by imaging is quasi-physically compensated. A simple plane numerical reference wavefront used for the reconstruction can give the correct quantitative phase map of the test object. A theoretical analysis and experimental demonstration are given. The simplicity of the presented setup makes it easy to align it well at lower cost.