Compensation of the inherent wave front curvature in digital holographic coherent microscopy for quantitative phase-contrast imaging
TL;DR: An approach is proposed for removing the wavefront curvature introduced by the microscope imaging objective in digital holography, which otherwise hinders the phase contrast imaging at reconstruction planes and it is shown that a correction effect can be obtained at all reconstruction planes.
Abstract: An approach is proposed for removing the wave front curvature introduced by the microscope imaging objective in digital holography, which otherwise hinders the phase contrast imaging at reconstruction planes. The unwanted curvature is compensated by evaluating a correcting wave front at the hologram plane with no need for knowledge of the optical parameters, focal length of the imaging lens, or distances in the setup. Most importantly it is shown that a correction effect can be obtained at all reconstruction planes. Three different methods have been applied to evaluate the correction wave front and the methods are discussed in detail. The proposed approach is demonstrated by applying digital holography as a method of coherent microscopy for imaging amplitude and phase contrast of microstructures.
Citations
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TL;DR: DHM is shown to be able to noninvasively and quantitatively detect changes in actin reticular formation, as well as actin distribution, in living unstained samples, and in agreement with conventional brightfield fluorescence microscopy a posteriori investigation.
Abstract: Previous investigations on mammalian cells have shown that microgravity, either that experienced in space, or simulated on earth, causes severe cellular modifications that compromise tissue determination and function. The aim of this study is to investigate, in real time, the morphological changes undergone by cells experiencing simulated microgravity by using digital holographic microscopy (DHM). DHM analysis of living mouse myoblasts (C2C12) is undertaken under simulated microgravity with a random positioning machine. The DHM analysis reveals cytoskeletal alterations similar to those previously reported with conventional methods, and in agreement with conventional brightfield fluorescence microscopy a posteriori investigation. Indeed, DHM is shown to be able to noninvasively and quantitatively detect changes in actin reticular formation, as well as actin distribution, in living unstained samples. Such results were previously only obtainable with the use of labeled probes in conjunction with conventional fluorescence microscopy, with all the classically described limitations in terms of bias, bleaching, and temporal resolution.
33 citations
TL;DR: An original method for determining the concentration profiles inside deformable objects in microconfined flows inside confined deformable bodies flowing in microchannels is presented.
Abstract: We investigate the use of a digital holographic microscope working with partially coherent spatial illumination to study concentration profiles inside confined deformable bodies flowing in microchannels. The studied phenomenon is rapidly changing in time and requires the recording of the complete holographic information for every frame. For this purpose, we implemented one of the classical methods of off-axis digital holography: the Fourier method. Digital holography allows one to numerically investigate a volume by refocusing the different planes of depth, allowing one to locate the objects under investigation in three dimensions. Furthermore, the phase is directly related to the refractive index, thus to the concentration inside the body. Based on simple symmetry assumptions, we present an original method for determining the concentration profiles inside deformable objects in microconfined flows. Details of the optical and numerical implementation, as well as exemplative experimental results are presented.
33 citations
TL;DR: The technique offers a whole-field and high-resolution wavefront reconstruction of unstained microstructures and phase maps of photoresist targets and human cheek cells are obtained to demonstrate the effectiveness of the method.
Abstract: A technique for phase microscopy using a phase diffuser and a reconstruction algorithm is proposed. A magnified specimen wavefront is projected on the diffuser plane that modulates the wavefront into a speckle field. The speckle patterns at axially displaced planes are sampled and used in an iterative phase retrieval algorithm based on a wave-propagation equation. The technique offers a whole-field and high-resolution wavefront reconstruction of unstained microstructures. Phase maps of photoresist targets and human cheek cells are obtained to demonstrate the effectiveness of our method.
30 citations
TL;DR: A simple and fast numerical approach to compensate for aberrations induced by objective phase curvature is proposed, based on the extraction of virtual background phase from reconstructed phase values using a line profile, followed by subtraction of the virtual Background phase from the reconstructed phase image.
Abstract: In this study, we proposed a simple and fast numerical approach to compensate for aberrations induced by objective phase curvature. This method is based on the extraction of virtual background phase from reconstructed phase values using a line profile, followed by subtraction of the virtual background phase from the reconstructed phase image. The performance and feasibility of the method were demonstrated by applying it to the phase imaging of polystyrene microspheres and red blood cells.
30 citations
TL;DR: In this article, the authors present results of imaging experiments using digital interference holography (DIH) and demonstrate an improvement of signal-to-noise ratio (SNR) with increasing number of holograms consistent with theoretical prediction.
Abstract: We present results of imaging experiments using digital interference holography (DIH). Calibration experiments using a resolution target demonstrate an improvement of signal-to-noise ratio (SNR) with increasing number of holograms consistent with theoretical prediction. Imaging experiments on retinal tissue reveal the topography of blood vessels as well as the optical thickness profile of the retinal layer. The SNR of tissue images is comparable to that of the resolution target, implying that the imaging system is operating close to theoretical optimum.
30 citations
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References
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TL;DR: A new method is proposed in which the distribution of complex amplitude at a plane is measured by phase-shifting interferometry and then Fresnel transformed by a digital computer, which can reconstruct an arbitrary cross section of a three-dimensional object with higher image quality and a wider viewing angle than from conventional digital holography using an off-axis configuration.
Abstract: A new method for three-dimensional image formation is proposed in which the distribution of complex amplitude at a plane is measured by phase-shifting interferometry and then Fresnel transformed by a digital computer. The method can reconstruct an arbitrary cross section of a three-dimensional object with higher image quality and a wider viewing angle than from conventional digital holography using an off-axis configuration. Basic principles and experimental verification are described.
1,813 citations
TL;DR: The principle of recording holograms directly on a CCD target is described and a real image of the object is reconstructed from the digitally sampled hologram by means of numerical methods.
Abstract: The principle of recording holograms directly on a CCD target is described. A real image of the object is reconstructed from the digitally sampled hologram by means of numerical methods.
1,444 citations
TL;DR: A new application of digital holography for phase-contrast imaging and optical metrology and an application to surface profilometry shows excellent agreement with contact-stylus probe measurements.
Abstract: 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.
1,202 citations
TL;DR: 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 to show that the transverse resolution is equal to the diffraction limit of the imaging system.
Abstract: 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.
1,174 citations
TL;DR: The principles and major applications of digital recording and numerical reconstruction of holograms (digital holography) are described, which are applied to measure shape and surface deformation of opaque bodies and refractive index fields within transparent media.
Abstract: This article describes the principles and major applications of digital recording and numerical reconstruction of holograms (digital holography). Digital holography became feasible since charged coupled devices (CCDs) with suitable numbers and sizes of pixels and computers with sufficient speed became available. The Fresnel or Fourier holograms are recorded directly by the CCD and stored digitally. No film material involving wet-chemical or other processing is necessary. The reconstruction of the wavefield, which is done optically by illumination of a hologram, is performed by numerical methods. The numerical reconstruction process is based on the Fresnel–Kirchhoff integral, which describes the diffraction of the reconstructing wave at the micro-structure of the hologram. In the numerical reconstruction process not only the intensity, but also the phase distribution of the stored wavefield can be computed from the digital hologram. This offers new possibilities for a variety of applications. Digital holography is applied to measure shape and surface deformation of opaque bodies and refractive index fields within transparent media. Further applications are imaging and microscopy, where it is advantageous to refocus the area under investigation by numerical methods.
1,171 citations