Digital Holographic Microscopy Research Papers (original) (raw)

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- Electron Microscopy, Microscopy, Multiphoton Microscopy, Fluorescence Microscopy

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In this manuscript, we introduce our present computer software algorithm dedicated to reconstruct a series of 2D images from one recorded 3D in-line hologram. The Fresnel-Kirchoff integral is implemented. Utilizing the symmetry of the free-space impulse response, we had speed-up the algorithm 4 times over its original speed. From one 1024¥1024 hologram input image, it requires 2 seconds to reconstruct one image layer at a certain depth (running over a 2GHz PC with 512MB RAM).

- by Ahmed Ayoub
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- Dhm, Digital Holographic Microscopy

We show that digital holography can be combined easily with optical coherence tomography approach. Varying the reference path length is the means used to acquire a series of holograms at different depths, providing after reconstruction images of slices at different depths in the specimen thanks to the shortcoherence length of light source. A metallic object, covered by a 150᎑m-thick onion cell, is imaged with high resolution. Applications in ophthalmology are shown: structures of the anterior eye, the cornea, and the iris, are studied on enucleated porcine eyes. Tomographic images of the iris border close to the pupil were obtained 165 m underneath the eye surface.

This paper describes an imaging microscopic technique based on heterodyne digital holography where subwavelength-sized gold colloids can be imaged in cell environments. Surface cellular receptors of 3T3 mouse fibroblasts are labeled with... more

- by Michael Atlan
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- Optics, Digital Holography, Microscopy, Nanoparticles

A detailed description of the digital microscopes, defining the working principle, instrumentation, the variety of the digital microscopes, their applications, advantages and limitations.

- by Zablon Faith
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- Digital Holographic Microscopy

Digital holographic microscopy enables a quantitative phase contrast metrology that is suitable for the investigation of reflective surfaces as well as for the marker-free analysis of living cells. The digital holographic feature of (subsequent) numerical focus adjustment makes possible applications for multifocus imaging. An overview of digital holographic microscopy methods is described. Applications of digital holographic microscopy are demonstrated by results obtained from livings cells and engineered surfaces.

- by Gert Von Bally
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- Mechanical Engineering, Algorithms, Holography, Applied Optics

A digital holographic microscope (DHM) has been employed in the retrieval and analysis of morphological images of bovine's sperm cells. Digital holography is a noncontact technique capable of investigating the shape of the sample without altering its characteristics and has been used for the first time in retrieving quantitative morphological information of sperm cells. Different spermatozoa have been analyzed by means of this technique allowing us to obtain 3-D images with precise topographical details and valuable information about morphological defects, provided with biological considerations. Moreover, by making use of a microfluidic system, the digital holographic technique has been employed to analyze unstained spermatozoa in their natural physiological surroundings. Detailed information on morphological images of spermatozoa acquired by DHM is expected to provide a better understanding of various reproductive pathways, which, in turn, can help in improving infertility management. This could constitute the basis of an alternative method for the zoothecnic industry aimed at the investigation of morphological features and the sorting of the motile sperm cells.

In-line digital holographic microscopy as a phase measurement tool for the inspection of micro-components is presented. Light diffracted by the micro-components interferes with the directly propagating beams to give the in-line digital hologram recorded by the CCD camera. The convolution method is used to calculate the diffractive propagation of the light in order to reconstruct the wavefront of the test specimen. A reference hologram without the test specimen is recorded for the phase reconstruction. Finally, the method is applied on a phase grating to test its refractive index of the coating material.

- by Weijuan Qu
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- Microscopy, Holography, Refractive Index, Convolution

The quantification of three-dimensional (3D) flow structures and particle dynamics is crucial for unveiling complex interactions in turbulent flows. This review summarizes recent advances in volumetric particle detection and 3D flow velocimetry involving holography. We introduce the fundamental principle of holography and discuss the debilitating depth-of-focus problem, along with methods that have been implemented to circumvent it. The focus of this review is on recent advances in the development of in-line digital holography in general, and digital holographic microscopy in particular. A mathematical background for the numerical reconstruction of digital holograms is followed by a summary of recently introduced 3D particle tracking and velocity measurement techniques. The review concludes with sample applications, including 3D velocity measurements that fully resolve the flow in the inner part of a turbulent boundary layer, the diffusion of oil droplets in high–Reynolds number tur...

- by Jian Sheng
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- Engineering, Earth Sciences, Fluid Mechanics, Imaging

Better understanding of particle-particle and particle-fluid interactions requires accurate 3D measurements of particle distributions and motions. We introduce the application of in-line digital holographic microscopy as a viable tool for measuring distributions of dense micrometer ͑3.2 m͒ and submicrometer ͑0.75 m͒ particles in a liquid solution with large depths of 1-10 mm. By recording a magnified hologram, we obtain a depth of field of ϳ1000 times the object diameter and a reduced depth of focus of approximately 10 particle diameters, both representing substantial improvements compared to a conventional microscope and in-line holography. Quantitative information on depth of field, depth of focus, and axial resolution is provided. We demonstrate that digital holographic microscopy can resolve the locations of several thousand particles and can measure their motions and trajectories using cinematographic holography. A sample trajectory and detailed morphological information of a free-swimming copepod nauplius are presented.

- by Jian Sheng
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- Mechanical Engineering, Algorithms, Holography, Three Dimensional Imaging
- by Florian B Soulard
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- Digital Holography, Digital Holographic Microscopy

Three dimensional tracking of microrobots is demonstrated using stereo holographic projections. The method detects the lateral position of a microrobot in two orthogonal in-line holography images and triangulates to obtain the 3-D position in an observable volume of one cubic cm. The algorithm is capable of processing holograms at 25Hz on a desktop computer and has an accuracy of 24.7μm and 15.2μm in the two independent directions and 7.3μm in the shared direction of the two imaging planes. This is the first use of stereo holograms to track an object in real-time and does not rely on the computationally expensive process of holographic reconstruction.

- by Andrew Petruska and +1
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- Image Processing, Stereo Vision (Computer Vision), Microrobotics, Digital Holographic Microscopy

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.

- by Weijuan Qu
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- Microscopy, Holography, Spatial Frequency, Phase Unwrapping

A method is proposed for designing apodized apertures with a transmission profile which follows a curve defined using a cubic spline interpolation. The method is applied in digital holographic microscopy to perform digitally the apodization of the aperture of holograms recorded by a CCD camera. The transmission of the apodized aperture is entirely defined by four parameters which are adjusted iteratively to minimize intensity and phase fluctuations appearing in the images obtained by numerical reconstruction of the holograms. The performances of the method have been studied in the absence of experimental sources of noise using a computer generated hologram with which we demonstrate that the aperture apodization reduces the standard deviation of the reconstructed phase distribution from 1.6 nm to 0.15 nm. q

Microscopy by holographic means is attractive because it permits true three-dimensional (3D) visualization and 3D display of the objects. We investigate the necessary condition on the object size and spatial bandwidth for complete 3D microscopic imaging with phase-shifting digital holography with various common arrangements. The cases for which a Fresnel holographic arrangement is sufficient and those for which object magnification is necessary are defined. Limitations set by digital sensors are analyzed in the Wigner domain. The trade-offs between the various holographic arrangements in terms of conditions on the object size and bandwidth, recording conditions required for complete representation, and complexity are discussed.

Partial coherent light sources open up prospects for phase noise reduction in digital holographically reconstructed phase distributions by suppressing multiple reflections in the experimental setup. Thus, light emitting diodes (LEDs) are investigated for application in digital holographic microscopy. First, the spectral properties and the resulting coherence length of an LED are characterised. In addition, an analysis of dispersion effects and their influence on the hologram formation is carried out. The coherence length of LEDs in the range of a few micrometers restricts the maximum interference fringe number in off-axis holography for spatial phase shifting. Thus, the application of temporal phaseshifting-based digital holographic reconstruction techniques is compared to spatial phase-shiftingbased methods. It is demonstrated that LEDs are applicable for digital holographic microscopy in connection with both spatial and temporal phase-shifting-based techniques for reduction of noise in comparison to a laser-light-based experimental setup.

- by Gert Von Bally and +1
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- Optical physics, Optics and lasers, Digital Holographic Microscopy, Spectral Properties

The application of a single cube beam splitter (SCBS) microscope to micro-optics characterization is presented. The SCBS in the optical path, with a small angle between the optical axis and its central semireflecting layer, not only gives off-axis digital holograms but also provides dual-channel imaging. It is a unique and easy way to perform uniformity inspection across the entire microlens array. Experimental results on physical spherical phase compensation, single lens characterization, dual-channel imaging, and uniformity inspection are provided to demonstrate the unique properties of SCBS microscopy.

- by Oi Choo Chee
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- Mechanical Engineering, Surface Roughness, Refractive Index, Applied Optics

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.

- by Christian Depeursinge
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- Optics, Optical physics, Trypanosoma brucei, Digital Holographic Microscopy

A new optical configuration for digital holographic microscopy is presented. Digital off-axis holograms are recorded by use of a single cube beam splitter in a nonconventional configuration to both split and combine a diverging spherical wavefront as it emerges from a single point source. Both the amplitude and the phase can then be reconstructed, yielding intensity and phase images with improved resolution. The novelty of the proposed configuration is its simplicity, minimal number of optical elements, insensitivity to vibration, and its inherent capability to compensate for the phase curvature that results from the illuminating wavefront in the case of microscopic samples.

- by Kallol Bhattacharya
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- Mechanical Engineering, Microscopy, Holography, Optical devices

An optical configuration for parallel two-step phase-shifting digital holographic microscopy (DHM) based on a grating pair is proposed for the purpose of real-time phase microscopy. Orthogonally circularly polarized object and reference waves are diffracted twice by a pair of gratings, and two parallel copies for each beams come into being. Combined with polarization elements, parallel two-step phase-shifting holograms are obtained. Based on the proposed configuration, two schemes of DHM, i.e., slightly off-axis and on-axis DHM, have been implemented. The slightly off-axis DHM suppresses the dc term by subtracting the two phase-shifting holograms from each other, thus the requirement on the off-axis angle and sampling power of the CCD camera is reduced greatly. The on-axis DHM has the least requirement on the resolving power of the CCD camera, while it requires that the reference wave is premeasured and its intensity is no less than 2 times the maximal intensity of the object wave.

I n this work an experimental optical system of digital holographic microscopy is implemented to measure thicknesses of thin films. The three-dimensional surface reconstruction was achieved as a phase contrast image obtained through computational processing of digital holograms. These images allow quantitative measurements of thin films thicknesses. This technique was applied to measure silica depositions bioactivated with particles of wollastonite produced by means of sol-gel technique. The depositions were made through dip-coating method with different velocities and using 10% p/p of wollastonite particles dispersed into the silica sol.

- by Claudia Garcia
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- Silica, Surface Reconstruction, Digital Holographic Microscopy, Phase Contrast

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.

- by Etienne Cuche
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- Digital Holography, 3-D Imaging, Refractive Index, Spatial Distribution

We demonstrate the use of digital holographic microscopy (DHM) as a metrological tool in micro-optics testing. Measurement principles are compared with those performed with Twyman-Green, Mach-Zehnder, and white-light interferometers. Measurements performed on refractive microlenses with reflection DHM are compared with measurements performed with standard interferometers. Key features of DHM such as digital focusing, measurement of shape differences with respect to a perfect model, surface roughness measurements, and optical performance evaluation are discussed. The capability of imaging nonspherical lenses without any modification of the optomechanical setup is a key advantage of DHM compared with conventional measurement tools and is demonstrated on a cylindrical microlens and a square lens array.

- by Tristan Colomb and +2
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- Mechanical Engineering, Surface Roughness, Performance Evaluation, Applied

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Telecentric architecture is proposed for circumventing, by the pure-optical method, the residual parabolic phase distortion inherent to standard configuration of digital holographic microscopy. This optical circumvention produces several important advantages. One is that there is no need for computer compensation of the parabolic phase during the phase map recovering procedure. The other is that in off-axis configuration, the spatial frequency useful domain is enlarged. The validity of the method is demonstrated by performing quantitative measurement of depth differences with high axial resolution.

Many applications in non-destructive testing at a microscopic level and in live cell imaging require automated focusing due to unstable environmental conditions, moving specimen or the limited depth of field of the applied optical imaging systems. Digital holography permits the recording and the numerical reconstruction of optical wave fields in amplitude and phase. This enables imaging of multiple focal planes from a single recorded hologram without mechanical realignment. The combination of numerical refocusing with image sharpness quantification algorithms yields subsequent autofocusing. With calibrated optical imaging systems this feature can be used also to determine the position and axial displacements of a sample. In order to show the application potential of digital holographic autofocusing in microscopy the method and results from investigations on several amplitude and phase objects are reviewed. This includes a demonstration of the reliability of automated refocusing, multi-focus quantitative phase contrast imaging of suspended cells, refocusing of quantitative phase contrast images during the analysis of the temporal dependency of cell spreading on surfaces and the quantification of toxin mediated morphological cell alterations during long-term observations. It is also shown for the example of sedimenting red blood cells that the method can be applied for minimally-invasive tracking of multiple particles. Finally, the usage of numerical autofocus for quantitative migration analysis of arbitrary shaped cells in a threedimensional collagen matrix is demonstrated.

We propose and test a focus plane determination method that computes the digital refocus distance of an object investigated by digital holographic microscopy working in transmission. For this purpose we analyze the integrated amplitude modulus as a function of the digital holographic reconstruction distance. It is shown that when the focus distance is reached, the integrated amplitude is minimum for pure amplitude object and maximum for pure phase object. After a theoretical analysis, the method is demonstrated on actual digital holograms for the refocusing of pure amplitude and of pure phase microscopic samples.

- by Cedric Schockaert
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- Optics, Digital Holography, Holographic Interferometry, Theoretical Analysis

We investigate a digital holographic microscope that permits us to modify the spatial coherence state of the sample illumination by changing the spot size of a laser beam on a rotating ground glass. Out-offocus planes are refocused by digital holographic reconstruction with numerical implementation of the Kirchhoff-Fresnel integral. The partial coherence nature of the illumination reduces the coherent artifact noise with respect to fully coherent illumination. The investigated configuration allows the spatial coherence state to be changed without modifying the illumination level of the sample. The effect of the coherence state on the digital holographic reconstruction is theoretically and experimentally evaluated. We also show how multiple reflection interferences are limited by the use of reduced spatial coherent illumination.

- by Christophe Minetti and +1
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- Mechanical Engineering, Image Reconstruction, Holography, Applied

- by Weijuan Qu
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- Microscopy, Holography, Spatial Frequency, Phase Unwrapping

This paper presents Digital Holographic Microscopy (DHM) quantitative measurements of transparent high aspect-ratio microstructures. Our experiment was performed using a digital holographic microscope in transmission configuration with a 60x magnification 1.3 NA oil immersion microscope objective, with a diode laser source at 664 nm. We used a calculation model based on the use of two immersion liquids for the experiment, the first one to resolve the phase jumps by using a refractive index liquid close to the sample index, in combination with a second one to retrieve the sample topology from the optical path length information. Such a model makes absolute topographic measurements of high aspect ratio transparent samples achievable by DHM. The model is then applied to measure 25 and 50 m transparent micro-corner cubes arrays, which exhibit up to 1:1,4 aspect ratio with theoretical slopes up to about 55 degrees. Thanks to our phase measurement precision down to 1°, we found possible to measure accurately the slopes of each face of the microstructures under investigation, and this with a good theoretical agreement.

- by Yves Emery
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- Microstructure, Digital Holography, Microscopy, Holography

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 threedimensional 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.

- by Tristan Colomb and +1
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- Algorithms, Optics, Quantum Physics, Microscopy

We present a theory to extend the classical Abbe resolution limit by introducing a spatially varying phase into the illumination beam of a phase imaging system. It allows measuring lateral and axial distance differences between point sources to a higher accuracy than intensity imaging alone. Various proposals for experimental realization are debated. Concretely, the phase of point scatterers' interference is experimentally visualized by high numerical aperture (NA = 0.93) digital holographic microscopy combined with angular scanning. Proof-ofprinciple measurements are presented by using sub-wavelength nanometric holes on an opaque metallic film. In this manner, Rayleighs classical two-point resolution condition can be rebuilt. With different illumination phases, enhanced bandpass information content is demonstrated, and its spatial resolution is theoretically shown to be potentially signal-to-noise ratio limited. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

- by M. Fatih Toy
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- Biomedical Engineering, Microscopy, Scanning Electron Microscopy, Super resolution

We present a method for submicrometer tomographic imaging using multiple wavelengths in digital holographic microscopy. This method is based on the recording, at different wavelengths equally separated in the k domain, in off-axis geometry, of the interference between a reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A CCD camera records the holograms consecutively, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefront. Their relative phases are adjusted to be equal in a given plane of interest and the resulting complex wavefronts are summed. The result of this operation is a constructive addition of complex waves in the selected plane and destructive addition in the others. Tomography is thus obtained by the attenuation of the amplitude out of the plane of interest. Numerical variation of the plane of interest enables one to scan the object in depth. For the presented simulations and experiments, 20 wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices 725 nm thick.

- by Christian Depeursinge
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- Mechanical Engineering, Microscopy, Optical Tomography, Tomography

In this paper, processing methods of Fourier optics implemented in a digital holographic microscopy system are presented. The proposed methodology is based on the possibility of the digital holography in carrying out the whole reconstruction of the recorded wave front and consequently, the determination of the phase and intensity distribution in any arbitrary plane located between the object and the recording plane. In this way, in digital holographic microscopy the field produced by the objective lens can be reconstructed along its propagation, allowing the reconstruction of the back focal plane of the lens, so that the complex amplitudes of the Fraunhofer diffraction, or equivalently the Fourier transform, of the light distribution across the object can be known. The manipulation of Fourier transform plane makes possible the design of digital methods of optical processing and image analysis. The proposed method has a great practical utility and represents a powerful tool in image analysis and data processing. The theoretical aspects of the method are presented, and its validity has been demonstrated using computer generated holograms and images simulations of microscopic objects.

- by Laszlo Sajo-Bohus
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- Optics, Digital Holography, Image Analysis, Optical physics

Digital holographic microscopes using two orthogonal polarized reference waves provides real-time polariscopes. Birefringence induced by internal stress is imaged in optical fibers and in fused silica substrates, where lines are written... more

- by Yves Bellouard
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- Fused Silica Etching, Low Energy Buildngs, Real Time, Optical fiber

In digital holographic microscopy, shot noise is an intrinsic part of the recording process with the digital camera. We present a study based on simulations and real measurements describing the shot-noise influence in the quality of the reconstructed phase images. Different configurations of the reference wave and the object wave intensities will be discussed, illustrating the detection limit and the coherent amplification of the object wave. The signal-to-noise ratio (SNR) calculation of the reconstructed phase images based on the decision statistical theory is derived from a model for image quality estimation proposed by Wagner and Brown [Phys. Med. Biol. 30, 489 (1985)]. It will be shown that a phase image with a SNR above 10 can be obtained with a mean intensity lower than 10 photons per pixel and per hologram coming from the observed object. Experimental measurements on a glass-chrome probe will be presented to illustrate the main results of the simulations.

- by Etienne Cuche
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- Mechanical Engineering, Image Quality, Holography, Applied

We present a digital holographic microscope that permits one to image polarization state. This technique results from the coupling of digital holographic microscopy and polarization digital holography. The interference between two orthogonally polarized reference waves and the wave transmitted by a microscopic sample, magnified by a microscope objective, is recorded on a CCD camera. The off-axis geometry permits one to reconstruct separately from this single hologram two wavefronts that are used to image the object-wave Jones vector. We applied this technique to image the birefringence of a bent fiber. To evaluate the precision of the phase-difference measurement, 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.

- by Etienne Cuche
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- Mechanical Engineering, Digital Holography, Applied, Applied Optics

We report on a method to achieve real-time dual-wavelength digital holographic microscopy with a single hologram acquisition. By recording both interferograms from two laser sources at different wavelengths in only one spatially-multiplexed digital hologram, we are able to independently propagate and apply numerical corrections on both wavefronts in order to obtain a beat-wavelength phase map of the specimen. This beat-wavelength being up to 10-100 times larger than the original wavelengths, we are in a situation where the 2π phase ambiguity of conventional DHM is removed and the phase measurement range of the technique is extended up to several tens of microns in height. The unique capability to perform such an operation with a single acquisition unables real-time dual-wavelength DHM measurements. Results on a moving micro-mirror are presented in this paper. We think that such a real-time dual-wavelength method represents a strong improvement to the current DHM state-of-the-art, and that it opens a whole new field of applications for this technique.

- by Yves Emery
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- Digital Holography, Real Time, Optical physics, Phase measurement

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.

- by Christian Depeursinge
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- Digital Holography, Microscopy, Computer Aided Design, Phase Retrieval

The main microscopic systems for industrial inspection are optical microscopes, including confocal scanning instruments, Scanning Electron Microscopes (SEM), Atomic Force Microscopes (AFM), and a few interferometers. With the recent technological advances, the demand for nanometer scale resolution of full wafers of micro lenses, biochips, Micro Electro-Mechanical and Opto-Mechanical Systems (MEMS and MOEMS), and of large quantities of individual samples increases. The use of current systems in industrial environments is limited either by their limited resolution (optical microscopes), or by their strong sensitivity to vibrations due to their long measurement time associated with the scanning or phase shifting mechanisms, or by the lengthy measurement protocols (SEM), or by the difficulty arising from the precise automated positioning and focalization of the sample (white light interferometers). An ideal system should offer high measurement rates, robustness, ease of use, non contact measurement, no specific preparation of the samples simultaneously with nanometer scale resolution. We have developed Digital Holographic Microscopes (DHM), presently used in production environments This paper describe the principles of the technology and show up the potential of DHM for industrial application in with an interferometer resolution. Ease of use, associated the use of numerical procedure to a level never reach so fair in microscopy with, is demonstrated on two features: focusing and sample tilt adjustment.

- by Etienne Cuche
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- Mechanism Design, Surface Roughness, Optical Imaging, FRINGE
- by Tristan Colomb
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- Digital Holography, Digital Holographic Microscopy

The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTR inh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTRdefective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific smallinterfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

- by Frédéric Becq
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- Water, Microscopy, Biological Sciences, Cell line

Digital Holographic Microscopy (DHM) allows quantitative multi-focus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms and a calibrated imaging system enables the determination of axial sample displacements. The evaluation of quantitative DHM phase contrast images permits also an effective detection of lateral object movements. Thus, data for 3D tracking is provided. Partially coherent light sources and multi-wavelength techniques open up prospects for an increased phase resolution in DHM by reduction of parasitic interference effects due to multiple reflections within the measurement setup. For this purpose, the utilization of light emitting diodes (LEDs) as well as the generation of short coherence properties by tunable laser light has been investigated for application in DHM. Results from investigations on sedimenting erythrocytes in suspension demonstrate that DHM enables (automated) quantitative dynamic 3D tracking of multiple cells without mechanical focus adjustment. Furthermore, it is shown that LEDs and multi-wavelength techniques enhance the axial resolution in inspection of reflective surfaces and quantitative digital holographic cell imaging.

We address the problem of zero-order-free image reconstruction in digital holographic microscopy. We show how the goal can be achieved by confining the object-wave modulation to one quadrant of the frequency domain, and by maintaining a reference-wave intensity higher than that of the object. The proposed technique is nonlinear, noniterative, and leads to exact reconstruction in the absence of noise. We also provide experimental results on holograms of yew pollen grains to validate the theoretical results.

A dual mode microscope is developed to study morphological evolution of mouse myoblast cells under simulated microgravity in real time. Microscope operates in Digital Holographic Microscopy (DHM) and widefield epifluorescence microscopy modes in a time sequential basis. DHM provides information on real time cellular morphology. EGFP transfected actin filaments in mouse myoblast cells function as the reporter for the fluorescence microscopy mode. Experimental setup is fixed in the RPM to observe microgravity induced dynamic changes in live cells. Initial results revealed two different modifications. Disorganized structures become visible in the formed lamellipodias, and proteins accumulate in the perinuclear region.

- by M. Fatih Toy
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- Microscopy, Fluorescence Microscopy, Cytoskeleton, Cell morphology