During the EASOE campaign in the Arctic, all lidar measurements showed total depolarization value... more During the EASOE campaign in the Arctic, all lidar measurements showed total depolarization values for the Pinatubo stratospheric aerosol layer varying between 2% and 4%. If multiple 'scattering is negligible, the radiation backscattered by a polydispersion of spheres has the same polarization as the incident light. There is always some depolarization of a lidar return because of the anisotropy of the polarizability of the air molecules, but depolarization in the signal returned from aerosols indicates the presence of non-spherical particles. In this paper, we utilize scattering calculations for non-spherical particles to explore interpretations of lidar signals returned by Pinatubo aerosols. Taking into account several particle shapes and lognormal size distributions, calculations show that only slightly-to-moderate deformed spheres with equivalentmean-radius less then.0.2/•m yield depolarization values in the range given by the lidar measurements, if such particles form a substantial fraction of the population. The implications of particle non-sphericity on the retrieval of the aerosol size distribution are discussed.
Assay and Drug Development Technologies, Mar 1, 2013
We introduce a label-free technology based on digital holographic microscopy (DHM) with applicabi... more We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z 0-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC 50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.
ABSTRACT Based on an original numerical reconstruction algorithm (E. Cuche et al. Appl. Opt. 38, ... more ABSTRACT Based on an original numerical reconstruction algorithm (E. Cuche et al. Appl. Opt. 38, 6994 1999), we have developed a Digital Holographic Microscope (DHM), in a transmission mode, allowing to investigate noninvasively cellular structures and dynamics. DHM images of living cells in culture are presented. They represent the distribution of the optical path length over the cell, which contains information concerning both the cellular morphometry and the intracellular refractive index, and which has been measured with a sub-wavelength accuracy.
ABSTRACT Digital Holographic Microscopes (DHM) enables strictly noninvasive visualisation of unst... more ABSTRACT 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.
Reflection digital holographic microscopy (DHM) is a very powerful technique allowing measuring t... more Reflection digital holographic microscopy (DHM) is a very powerful technique allowing measuring topography with a sub-nanometer axial resolution from a single hologram acquisition. But as most of interferometer methods, the vertical range is limited to half the wavelength if numerical unwrapping procedure could not be applied (very high aspect ratio specimen). Nevertheless, it was already demonstrated that the use of dual-wavelength DHM allows increasing the vertical range up to several microns by saving the single wavelength resolution if conditions about phase noise are fulfilled (the higher the synthetic wavelength, the smaller the phase noise has to be). In this paper, we will demonstrate that the choice of a synthetic wavelength of about 17 microns allows measuring precisely a 4.463μm certified step. Furthermore, we will show the feasibility of a sub-nanometer resolution on a range higher than the synthetic wavelength by being able to map the dual-wavelength measurement on data acquired from a vertical scanning process, which precision is about 1 μm.
ABSTRACT Digital holographic microscopy allows measuring the complex wavefront reflected by or tr... more ABSTRACT Digital holographic microscopy allows measuring the complex wavefront reflected by or transmitted through a sample from a single hologram acquisition. In reflection configuration, assuming an homogeneous surface composed of a unique material, and excluding multiple reflections from underlying layers, the topography of the surface is proportional to the reconstructed phase and the topography can be retrieved with a so-called interferometric resolution. But, in presence of semi-transparent multilayers, the topography is no longer proportional to the reconstructed phase: multiple reflections from and between the layers interfaces modify the reflected wavefront. In this paper we demonstrate that using the complex wavefront information, i.e. not only the phase, acquired at several discrete wavelengths, we retrieve the topography by comparing the measured wavefront with the theoretical reflected wavefronts calculated from the model of the multilayers structure. The principle is demonstrated by comparing DHM and mechanical profilometer measurements for: staircase SiO2 on a Silicon substrate, hole dug by SIMS (Secondary Ion Mass Spectrometry) in a transparent multilayer films, and fluid (Tetraethylene-glycol) on a Si substrate.
A comparison of algorithms for depth-of-field extension in the particular field of digital hologr... more A comparison of algorithms for depth-of-field extension in the particular field of digital holographic microscopy is presented. Using the reconstruction properties of holography, a simple 3D reconstruction method from a single hologram is deducted.
In Digital holography Microscopes (DHM) implemented in the so-called "off axis" configuration, th... more In Digital holography Microscopes (DHM) implemented in the so-called "off axis" configuration, the object and reference wave fronts are not co-planar but form an angle of a few degrees. This results into two main drawbacks. First, the contrast of the interference is not uniform spatially when the light source has low coherence. The interference contrast is optimal along a line, but decreases when moving away from it, resulting in a lower image quality. Second, the non-coplanarity between the coherence plane of both wavefronts impacts the coherence vertical scanning measurement mode: when the optical path difference between the signal and the reference beam is changed, the region of maximum interference contrast shifts laterally in the plane of the objective. This results in more complex calculations to extract the topography of the sample and requires scanning over a much larger vertical range, leading to a longer measurement time. We have previously shown that by placing a volume diffractive optical element (VDOE) in the reference arm, the wavefront can be made coplanar with the object wavefront and the image plane of the microscope objective, resulting in a uniform and optimal interferogram. In this paper, we demonstrate a vertical scanning speed improvement by an order of magnitude. Noise in the phase and intensity images caused by scattering and non-uniform diffraction in the VDOE is analyzed quantitatively. Five VDOEs were fabricated with an identical procedure. We observe that VDOEs introduce a small intensity nonuniformity in the reference beam which results in a 20% noise increase in the extracted phase image as compared to the noise in extracted phase image when the VDOE is removed. However, the VDOE has no impact on the temporal noise measured from extracted phase images.
We demonstrate the potential of spatial light modulators for the spectral control of a broadband ... more We demonstrate the potential of spatial light modulators for the spectral control of a broadband source in digital holographic microscopy. Used in a 'pulse-shaping' geometry, the spatial light modulator provides a versatile control over the bandwidth and wavelength of the light source. The control of these properties enables adaptation to various experimental conditions. As a first application, we show that the source bandwidth can be adapted to the off-axis geometry to provide quantitative phase imaging over the whole field of view. As a second application, we generate sequences of appropriate wavelengths for a hierarchical optical phase unwrapping algorithm, which enables the measurement of the topography of high-aspect ratio structures without phase ambiguity. Examples are given with step heights up to 50 µm.
We investigated the question of how the perception of threedimensional information reconstructed ... more We investigated the question of how the perception of threedimensional information reconstructed numerically from digital holograms of real-world objects, and presented on conventional displays, depends on motion and stereoscopic presentation. Perceived depth in an adjustable random pattern stereogram was matched to the depth in hologram reconstructions. The objects in holograms were a microscopic biological cell and a macroscopic metal coil. For control, we used real physical objects in additional to hologram reconstructions of real objects. Stereoscopic presentation increased perceived depth substantially in comparison to nonstereoscopic presentation. When stereoscopic cues were weak or absent e.g. because of blur, motion increased perceived depth considerably. However, when stereoscopic cues were strong, the effect of motion was small. In conclusion, for the maximization of perceived three-dimensional information of holograms on conventional displays, it seems highly beneficial to use the combination of motion and stereoscopic presentation.
Digital holographic microscopy (DHM) is applied to life sciences applications and demonstrate its... more 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.
The limited depth-of-field is a main drawback of microscopy that prevents from observing, for exa... more The limited depth-of-field is a main drawback of microscopy that prevents from observing, for example, thick semi-transparent objects with all their features in focus. Several algorithms have been developed during the past years to fuse images having various planes of focus and thus obtain a completely focused image with virtually extended depth-of-field. We present a comparison of several of these methods in the particular field of digital holographic microscopy, taking advantage of some of the main properties of holography. We especially study the extended depth-of-field for phase images reconstructed from the hologram of a biological specimen. A criterion of spatial measurement on the object is considered, completed with a visual criterion. The step of distance taken into account to build the stack of images is less than the instrument depth-of-field. Then, preserving the distance of focus associated with each pixel of the image, a three-dimensional representation is presented after automatic detection of the object. The limits of such a method of extraction of 3D information are discussed.
ABSTRACT The use of numerical procedure at a level never reach so far in optical imaging enables ... more ABSTRACT The use of numerical procedure at a level never reach so far in optical imaging enables DHM to overcome two cumbersome alignment procedures of interferometric microscopes: (1) fine focus of the sample, and (2) sample tilt adjustment. In addition the technology enables to retrieve the full information from a single hologram acquisition. This enables very short acquisition time, smaller than typical propagation frequencies of ambient vibrations. These instruments can be operated without vibration insulation tables. Combined with the simplicity of the opto-mechanical design and the absence of moving parts, DHM are new measurement systems with a nanometer scale resolution ready to take place in production lines. DHM can be used to characterized shapes and surface roughness of a large variety of samples and their use for industrial inspection should definitely spread during the next years
We have developed a digital holographic microscope (DHM), in a transmission mode, adapted to the ... more We have developed a digital holographic microscope (DHM), in a transmission mode, adapted to the quantitative study of cellular dynamics. Living cells in culture are optically probed by measuring the phase shift they produce on the transmitted wave front. The high temporal stability of the phase signal, equivalent to λ/1800, and the low acquisition time (~20μs) enable to monitor cellular dynamics processes. An experimental procedure allowing to calculate both the integral refractive index and the cellular thickness (morphometry) from the measured phase shift is presented. Specifically, the method has been applied to study the dynamics of neurons in culture during a hypotonic stress. Such stress produces a paradoxical decrease of the phase which can be entirely resolved by applying the methodological approach described in this article; indeed the method allows to determine independently the thickness and the integral refractive index of cells.
... Frédéric Montfort a, Yves Emery a, Eduardo Solanas a, Etienne Cuche a, Nicolas Aspert a, Pier... more ... Frédéric Montfort a, Yves Emery a, Eduardo Solanas a, Etienne Cuche a, Nicolas Aspert a, Pierre Marquet a, Claude Joris a, Jonas Kühn b, Christian Depeursing b ABSTRACT ... A hologram is recorded from the ball surface and the complex wave front is then reconstructed. ...
ABSTRACT Digital holography has proven its ability to acquire high accuracy full field 3D data wi... more ABSTRACT Digital holography has proven its ability to acquire high accuracy full field 3D data with one single image acquisition. This means that in principle this technique offers the chance to perform 3D serial inspection processes, as well. However, one limitation in digital holography is its limited ability to measure rough surfaces. In the presence of rough surfaces, the magnification of the image has to be increased to capture the required phase information on each camera pixel. However, this leads to significant reduction of inspection speed. If low magnification is selected, the rough surface produces speckles which cannot be treated properly by digital holography algorithms. In this paper, we describe the extension of digital holography to rough surface applications using speckle interferometry technique. This technique is capable of fast inspection of rough surfaces with sub-micrometer accuracy. The principle of this approach is shown and a practical application for 3D surface inspection of wafer cutting processes is given.
During the EASOE campaign in the Arctic, all lidar measurements showed total depolarization value... more During the EASOE campaign in the Arctic, all lidar measurements showed total depolarization values for the Pinatubo stratospheric aerosol layer varying between 2% and 4%. If multiple 'scattering is negligible, the radiation backscattered by a polydispersion of spheres has the same polarization as the incident light. There is always some depolarization of a lidar return because of the anisotropy of the polarizability of the air molecules, but depolarization in the signal returned from aerosols indicates the presence of non-spherical particles. In this paper, we utilize scattering calculations for non-spherical particles to explore interpretations of lidar signals returned by Pinatubo aerosols. Taking into account several particle shapes and lognormal size distributions, calculations show that only slightly-to-moderate deformed spheres with equivalentmean-radius less then.0.2/•m yield depolarization values in the range given by the lidar measurements, if such particles form a substantial fraction of the population. The implications of particle non-sphericity on the retrieval of the aerosol size distribution are discussed.
Assay and Drug Development Technologies, Mar 1, 2013
We introduce a label-free technology based on digital holographic microscopy (DHM) with applicabi... more We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z 0-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC 50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.
ABSTRACT Based on an original numerical reconstruction algorithm (E. Cuche et al. Appl. Opt. 38, ... more ABSTRACT Based on an original numerical reconstruction algorithm (E. Cuche et al. Appl. Opt. 38, 6994 1999), we have developed a Digital Holographic Microscope (DHM), in a transmission mode, allowing to investigate noninvasively cellular structures and dynamics. DHM images of living cells in culture are presented. They represent the distribution of the optical path length over the cell, which contains information concerning both the cellular morphometry and the intracellular refractive index, and which has been measured with a sub-wavelength accuracy.
ABSTRACT Digital Holographic Microscopes (DHM) enables strictly noninvasive visualisation of unst... more ABSTRACT 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.
Reflection digital holographic microscopy (DHM) is a very powerful technique allowing measuring t... more Reflection digital holographic microscopy (DHM) is a very powerful technique allowing measuring topography with a sub-nanometer axial resolution from a single hologram acquisition. But as most of interferometer methods, the vertical range is limited to half the wavelength if numerical unwrapping procedure could not be applied (very high aspect ratio specimen). Nevertheless, it was already demonstrated that the use of dual-wavelength DHM allows increasing the vertical range up to several microns by saving the single wavelength resolution if conditions about phase noise are fulfilled (the higher the synthetic wavelength, the smaller the phase noise has to be). In this paper, we will demonstrate that the choice of a synthetic wavelength of about 17 microns allows measuring precisely a 4.463μm certified step. Furthermore, we will show the feasibility of a sub-nanometer resolution on a range higher than the synthetic wavelength by being able to map the dual-wavelength measurement on data acquired from a vertical scanning process, which precision is about 1 μm.
ABSTRACT Digital holographic microscopy allows measuring the complex wavefront reflected by or tr... more ABSTRACT Digital holographic microscopy allows measuring the complex wavefront reflected by or transmitted through a sample from a single hologram acquisition. In reflection configuration, assuming an homogeneous surface composed of a unique material, and excluding multiple reflections from underlying layers, the topography of the surface is proportional to the reconstructed phase and the topography can be retrieved with a so-called interferometric resolution. But, in presence of semi-transparent multilayers, the topography is no longer proportional to the reconstructed phase: multiple reflections from and between the layers interfaces modify the reflected wavefront. In this paper we demonstrate that using the complex wavefront information, i.e. not only the phase, acquired at several discrete wavelengths, we retrieve the topography by comparing the measured wavefront with the theoretical reflected wavefronts calculated from the model of the multilayers structure. The principle is demonstrated by comparing DHM and mechanical profilometer measurements for: staircase SiO2 on a Silicon substrate, hole dug by SIMS (Secondary Ion Mass Spectrometry) in a transparent multilayer films, and fluid (Tetraethylene-glycol) on a Si substrate.
A comparison of algorithms for depth-of-field extension in the particular field of digital hologr... more A comparison of algorithms for depth-of-field extension in the particular field of digital holographic microscopy is presented. Using the reconstruction properties of holography, a simple 3D reconstruction method from a single hologram is deducted.
In Digital holography Microscopes (DHM) implemented in the so-called "off axis" configuration, th... more In Digital holography Microscopes (DHM) implemented in the so-called "off axis" configuration, the object and reference wave fronts are not co-planar but form an angle of a few degrees. This results into two main drawbacks. First, the contrast of the interference is not uniform spatially when the light source has low coherence. The interference contrast is optimal along a line, but decreases when moving away from it, resulting in a lower image quality. Second, the non-coplanarity between the coherence plane of both wavefronts impacts the coherence vertical scanning measurement mode: when the optical path difference between the signal and the reference beam is changed, the region of maximum interference contrast shifts laterally in the plane of the objective. This results in more complex calculations to extract the topography of the sample and requires scanning over a much larger vertical range, leading to a longer measurement time. We have previously shown that by placing a volume diffractive optical element (VDOE) in the reference arm, the wavefront can be made coplanar with the object wavefront and the image plane of the microscope objective, resulting in a uniform and optimal interferogram. In this paper, we demonstrate a vertical scanning speed improvement by an order of magnitude. Noise in the phase and intensity images caused by scattering and non-uniform diffraction in the VDOE is analyzed quantitatively. Five VDOEs were fabricated with an identical procedure. We observe that VDOEs introduce a small intensity nonuniformity in the reference beam which results in a 20% noise increase in the extracted phase image as compared to the noise in extracted phase image when the VDOE is removed. However, the VDOE has no impact on the temporal noise measured from extracted phase images.
We demonstrate the potential of spatial light modulators for the spectral control of a broadband ... more We demonstrate the potential of spatial light modulators for the spectral control of a broadband source in digital holographic microscopy. Used in a 'pulse-shaping' geometry, the spatial light modulator provides a versatile control over the bandwidth and wavelength of the light source. The control of these properties enables adaptation to various experimental conditions. As a first application, we show that the source bandwidth can be adapted to the off-axis geometry to provide quantitative phase imaging over the whole field of view. As a second application, we generate sequences of appropriate wavelengths for a hierarchical optical phase unwrapping algorithm, which enables the measurement of the topography of high-aspect ratio structures without phase ambiguity. Examples are given with step heights up to 50 µm.
We investigated the question of how the perception of threedimensional information reconstructed ... more We investigated the question of how the perception of threedimensional information reconstructed numerically from digital holograms of real-world objects, and presented on conventional displays, depends on motion and stereoscopic presentation. Perceived depth in an adjustable random pattern stereogram was matched to the depth in hologram reconstructions. The objects in holograms were a microscopic biological cell and a macroscopic metal coil. For control, we used real physical objects in additional to hologram reconstructions of real objects. Stereoscopic presentation increased perceived depth substantially in comparison to nonstereoscopic presentation. When stereoscopic cues were weak or absent e.g. because of blur, motion increased perceived depth considerably. However, when stereoscopic cues were strong, the effect of motion was small. In conclusion, for the maximization of perceived three-dimensional information of holograms on conventional displays, it seems highly beneficial to use the combination of motion and stereoscopic presentation.
Digital holographic microscopy (DHM) is applied to life sciences applications and demonstrate its... more 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.
The limited depth-of-field is a main drawback of microscopy that prevents from observing, for exa... more The limited depth-of-field is a main drawback of microscopy that prevents from observing, for example, thick semi-transparent objects with all their features in focus. Several algorithms have been developed during the past years to fuse images having various planes of focus and thus obtain a completely focused image with virtually extended depth-of-field. We present a comparison of several of these methods in the particular field of digital holographic microscopy, taking advantage of some of the main properties of holography. We especially study the extended depth-of-field for phase images reconstructed from the hologram of a biological specimen. A criterion of spatial measurement on the object is considered, completed with a visual criterion. The step of distance taken into account to build the stack of images is less than the instrument depth-of-field. Then, preserving the distance of focus associated with each pixel of the image, a three-dimensional representation is presented after automatic detection of the object. The limits of such a method of extraction of 3D information are discussed.
ABSTRACT The use of numerical procedure at a level never reach so far in optical imaging enables ... more ABSTRACT The use of numerical procedure at a level never reach so far in optical imaging enables DHM to overcome two cumbersome alignment procedures of interferometric microscopes: (1) fine focus of the sample, and (2) sample tilt adjustment. In addition the technology enables to retrieve the full information from a single hologram acquisition. This enables very short acquisition time, smaller than typical propagation frequencies of ambient vibrations. These instruments can be operated without vibration insulation tables. Combined with the simplicity of the opto-mechanical design and the absence of moving parts, DHM are new measurement systems with a nanometer scale resolution ready to take place in production lines. DHM can be used to characterized shapes and surface roughness of a large variety of samples and their use for industrial inspection should definitely spread during the next years
We have developed a digital holographic microscope (DHM), in a transmission mode, adapted to the ... more We have developed a digital holographic microscope (DHM), in a transmission mode, adapted to the quantitative study of cellular dynamics. Living cells in culture are optically probed by measuring the phase shift they produce on the transmitted wave front. The high temporal stability of the phase signal, equivalent to λ/1800, and the low acquisition time (~20μs) enable to monitor cellular dynamics processes. An experimental procedure allowing to calculate both the integral refractive index and the cellular thickness (morphometry) from the measured phase shift is presented. Specifically, the method has been applied to study the dynamics of neurons in culture during a hypotonic stress. Such stress produces a paradoxical decrease of the phase which can be entirely resolved by applying the methodological approach described in this article; indeed the method allows to determine independently the thickness and the integral refractive index of cells.
... Frédéric Montfort a, Yves Emery a, Eduardo Solanas a, Etienne Cuche a, Nicolas Aspert a, Pier... more ... Frédéric Montfort a, Yves Emery a, Eduardo Solanas a, Etienne Cuche a, Nicolas Aspert a, Pierre Marquet a, Claude Joris a, Jonas Kühn b, Christian Depeursing b ABSTRACT ... A hologram is recorded from the ball surface and the complex wave front is then reconstructed. ...
ABSTRACT Digital holography has proven its ability to acquire high accuracy full field 3D data wi... more ABSTRACT Digital holography has proven its ability to acquire high accuracy full field 3D data with one single image acquisition. This means that in principle this technique offers the chance to perform 3D serial inspection processes, as well. However, one limitation in digital holography is its limited ability to measure rough surfaces. In the presence of rough surfaces, the magnification of the image has to be increased to capture the required phase information on each camera pixel. However, this leads to significant reduction of inspection speed. If low magnification is selected, the rough surface produces speckles which cannot be treated properly by digital holography algorithms. In this paper, we describe the extension of digital holography to rough surface applications using speckle interferometry technique. This technique is capable of fast inspection of rough surfaces with sub-micrometer accuracy. The principle of this approach is shown and a practical application for 3D surface inspection of wafer cutting processes is given.
Uploads
Papers by Yves Emery