We propose and experimentally demonstrate a system in which off-axis digital holographic microscopy is realized using a broadband illumination source. Single-shot holographic measurements are enabled, while the coherence noise is removed thanks to the broad bandwidth of the illuminating source. The proposed digital holographic camera is portable and can be attached to the camera port of a conventional optical microscope. This camera is capable of obtaining the complex wavefront i.e the intensity and phase information of the light transmitted or reflected from a sample. A combination of a thick transmission volume grating recorded holographically into thick photosensitive glass and thin transmission phase gratings recorded holographically into thin photopolymers, spatially filters the beam of light containing the sample information in two dimensions through diffraction. This filtered beam creates the reference arm of the interferometer. The untouched transmitted beam creates the sample arm of the interferometer. The spatial filtering performed by the combination of gratings above reduces the alignment spatial sensitivity which is an advantage over conventional spatial filtering done by pinholes. Besides, using a second thin grating, we introduce a desired coherence plane tilt in the reference beam which is sufficient to create high-visibility interference over the entire field of view in off-axis configuration. Full-field off-axis interferograms are thus created from which the phase information can be extracted.
In digital holographic microscopy (DHM), the long coherence length of laser light causes parasitic interferences due to
multiple reflections in and by optical components in the optical path of the microscope and thus degrades the image
quality. The parasitic effects are greatly reduced by using a short coherence length light. The main drawback of using a
short coherence light source in an off-axis digital holographic microscope, is the reduction of the interference fringe
contrast occurring in the field of view. Previously, we introduced a volume diffractive optical element (VDOE) placed
in the reference arm of a DHM to correct the coherence plane tilt so as to obtain a high interference contrast throughout
the field of view . Here, we experimentally quantify the spatial and temporal phase noise in the extracted phase image
caused by non-homogeneities and scattering of the VDOE element itself. The results over five VDOEs show that the
temporal phase noise is unchanged and a slight increase (up to 20%) is observed in the spatial phase noise. These results
show that even with a low coherence source, a full field of view can be obtained with an off-axis DHM thanks to the
VDOE without introducing significant additional phase noise.
In off-axis digital holographic microscopy, short coherence length of the source results in an unwanted reduced field of
view. A diffractive optical element (DOE) which combines two high efficiency transmission volume phase gratings
holographically recorded into a thin photopolymer, is proposed to manipulate the coherence plane tilt of beam containing
a plurality of wavelengths simultaneously. The DOE extends the interference pattern between object and reference
beams in digital holographic microscope (DHM) over the whole physical beam overlap area. We experimentally
demonstrate full field imaging in a commercial, two colors (685 nm and 794 nm) reflection digital holographic
microscope (DHM). The synthetic wavelength created by the two colors extends the unambiguous depth range of the
DHM from 0.39μm to 2.49μm .
We present dual-wavelength Digital Holographic Microscopy (DHM) measurements on a certified 8.9 nm high
Chromium thin step sample and demonstrate sub-nanometer axial accuracy. We introduce a modified DHM
Reference Calibrated Hologram (RCH) reconstruction algorithm taking into account amplitude contributions.
By combining this with a temporal averaging procedure and a specific dual-wavelength DHM arrangement, it
is shown that specimen topography can be measured with an accuracy, defined as the axial standard deviation,
reduced to at least 0.9 nm. Indeed, it is reported that averaging each of the two wavefronts recorded with real-time
dual-wavelength DHM can provide up to 30% spatial noise reduction for the given configuration, thanks to
their non-correlated nature.
We report on advanced dual-wavelength digital holographic microscopy (DHM) methods, enabling single-acquisition
real-time micron-range measurements while maintaining single-wavelength interferometric resolution in the nanometer
regime. In top of the unique real-time capability of our technique, it is shown that axial resolution can be
further increased compared to single-wavelength operation thanks to the uncorrelated nature of both recorded
wavefronts. It is experimentally demonstrated that DHM topographic investigation within 3 decades measurement
range can be achieved with our arrangement, opening new applications possibilities for this interferometric
technique.
In optics, optical elements are used to transform, to filter or to process physical wavefronts in order to magnify
images, compensate for aberration or to suppress unwanted diffracted order for example. Because digital
holography provides numerical wavefronts, we developed a digital optics, involving numerical elements such as
numerical lenses and pinholes, to mimic numerically what is usually done physically, with the advantage to be
able to define any shape for these elements and to place them everywhere without obstruction problems. We
demonstrate that automatic and non-automatic procedures allow diffracted order or parasitic interferences filtering,
compensation for aberration and image distortion, and control of position and magnification of reconstructed
wavefront. We apply this digital optics to compensate for chromatic aberration in multi-wavelength holography
in order to have perfect superposition between wavefronts reconstructed from digital hologram recorded with
different wavelengths. This has a great importance for synthetic wavelength digital holography or tomographic
digital holography that use multiple wavelengths.
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 2&pgr; 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.
The study of the internal structures of specimens has a great importance in life and materials sciences. The principle of
optical diffraction tomography (ODT) consists in recording the complex wave diffracted by an object, while changing
the k vector of the illuminating wave. This way, the frequency domain of the specimen is scanned, allowing
reconstructing the scattering potential of the sample in the spatial domain. This work presents a method for sub-micron
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, of the interference between an off-axis reference
wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A charged
coupled device (CCD) camera records consecutively the holograms, which are then numerically reconstructed following
the convolution formulation to obtain each corresponding complex object wavefronts. 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 a destructive one 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 to scan the object in depth. For the presented simulations and experiments, twenty wavelengths are used in the
480-700 nm range. The result is a sectioning of the object in slices of 725 nm thick.
Process engineering and failure analysis of MEMS and MOEMS require static and dynamical characterization of both
their in-plane and out of plane response to an excitation. A remarkable characteristic of Digital Holography Microscopes
(DHM) is the extremely short acquisition time required to grab the whole information necessary to provide 3D optical
topography of the sample: a unique frame grab, without any vertical or lateral scan provides the information over the
full field of view. First, it ensures DHM measurements to be insensitive to vibrations. Second, it opens the door to fast
dynamical characterization of micro-systems. For periodic movement analysis, DHM can operate in stroboscopic mode
with standard cameras. It enables precise characterization up to excitation frequencies of 100 kHz with recovery cycle
of 10% simply by triggering properly the camera. Pulsed sources can be used for investigation of higher excitation
frequencies. For non periodic movement analysis fast acquisition cameras and postponed treatment are used. DHM are
therefore unique and very efficient tool for dynamical characterization of in-plane and out-of-plane response.
In this paper we show the basics of the technology and illustrate process engineering and failure analysis using DHM
with an example of in and out of plane characterization of movements of a variable capacitor using the stroboscopic
mode of acquisition.
Digital Holographic Microscopes (DHM) allows the capture of all the information necessary to provide 3D phase measurements with a nanometer vertical resolution in a single image acquisition. DHM images provide measurements of the surface topography which can be used for surface analysis, roughness measurements for example. In this paper we present roughness measurements on micro-balls of different sizes for which numerical procedures are applied for form factor and waviness removal. DHM thus permits quantitative measurements of the roughness on a 2 dimensional area allowing enlarged information compared to common profilometers. Mean roughness of 5 to 30 nm are measured and compared to values obtained by a profilometer.
KEYWORDS: Signal to noise ratio, Holograms, Digital holography, Holography, 3D image reconstruction, Microscopy, Neurons, Optical simulations, Statistical analysis, Wavefronts
In digital holographic microscopy, shot noise is an intrinsic part of the recording process with a digital camera. We present a study based on simulations describing how shot noise influences the quality of the reconstructed phase images under different beams intensities configurations. A model for image quality estimation based on the decision statistical theory proposed by Wagner and Brown (R.F. Wagner and D.G. Brown, "Unified SNR Analysis of Medical Imaging-Systems," Phys. Med. Biol. 30, 489-518 (1985)) will be presented. A simulation will depict the variations of the reconstructed phase images SNR for different distributions of the total intensity between the reference and the object beam.
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.
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.
In this paper we present a method for 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
sample and magnified by a microscope objective. A couple charged device (CCD) camera records consecutively
the resulting holograms, which are then numerically reconstructed to obtain their resulting wavefront. Those
wavefronts are then summed. The result of this operation is a constructive addition of complex waves in the
selected plane and destructive addition in the others. Varying the plane of interest enables the scan the object
in depth.
For the presented simulations and experiments, twenty wavelengths are used in the 480-700 nm range. An object
consisting of irregularly stairs with heights of 375, 525, 975, 1200 and 1275 nm is reconstructed. Its lateral
dimensions are 250 × 250 microns. The results show clearly a 3D imaging technique with axial resolution under
the micron.
Digital Holographic Microscopes (DHM) enables recording the whole information necessary to provide real time nanometric vertical displacement measurements with a single image acquisition. The use of fast acquisition camera or stroboscopic acquisition mode makes these new systems ideal tools for investigating the topography and dynamical behavior of MEMS and MOEMS. This is illustrated by the investigation of resonant frequencies of a dual axis micromirror.
This enables the definition of the linear, non-linear, and modal resonance zones of its dynamical response.
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.
Digital holographic Microscopy (DHM) is an imaging modality reconstructing the wavefront in a numerical form,
directly from a single digitalized hologram. It brings quantitative data derived simultaneously from the amplitude and
phase of the complex reconstructed wavefront diffracted by the object and it is used to determine the refractive index
and/or shape of the object with accuracy in the nanometer range along the optical axis. DHM comprises a microscope
objective to adapt the sampling capacity of the camera to the information content of the hologram.
This paper illustrates some of the possibilities offered by DHM for micro-optics quality control. Actual results obtained
by DHM, yielding an axial precision up to 3.7 nm, will be compared with measurements performed with interferometers
by SUSS MicroOptics SA and with the profiles measured with a mechanical scanning probe instrument (Alpha step 200
from Tencor Instrument). Two different micro-lenses arrays where tested: a quartz refractive lenses array (observed with
transmission DHM) and a Silicon refractive lens array (observed with reflection DHM).
With the recent technological advances, there is an increasing need for measurement systems providing interferometer resolution for inspection of large quantities of individual samples in manufacturing environments.. Such applications require high measurement rates, robustness, ease of use, and non-contact systems.
We show here that Digital Holographic Microscopy (DHM), a new method that implements digitally the principle of holography, is particularly well suited for such industrial applications. With the present computers power and the developments of digital cameras, holograms can be numerically interpreted within a tenth of second to provide simultaneously: the phase information, which reveals object surface with vertical resolution at the nanometer scale along the optical axis, and intensity images, as obtained by conventional optical microscope.
The strength of DHM lies in particular on the use of the so-called off-axis configuration, which enables to capture the whole information by a single image acquisition, i.e. typically during a few ten of microseconds. These extremely short acquisition times make DHM systems insensitive to vibrations. These instruments can operate without vibration insulation means, making them a cost effective solution not only for R&D, but also especially for an implementation on production lines.
Numerous application examples are presented in this paper such as shape and surface characterization of high aspect ratio micro-optics, surface nanostructures, and surface roughness.
We report on a method called Digital Holographic Microscopy (DHM) for the numerical reconstruction of digital holograms taken with a microscope. It allows for simultaneous amplitude and quantitative phase contrast imaging. The reconstruction method computes the propagation of the complex optical wavefront diffracted by the object and is used to determine the refractive index and/or shape of the object with an accuracy in the nanometer range along the optical axis. A single hologram is needed for reconstruction. The method requires the adjustment of several reconstruction parameters. The adjustment is performed automatically by using a suitable algorithm. The method has been applied to the measurement of several integrated optics devices, MOEMS, and integrated micro-optical components: microlenses.
The aim of Optical Digital Holography, applied to cells and tissue imaging, is to provide an accurate 3D imaging of biologic materials, down to the microscopic scale. The method has been developed to yield a very precise determination of cells and tissues morphology. Targeted accuracies are in the sub-micron range and allow for the observation of very small movements and deformations, produced, in particular, by depolarization of excitable cells and their metabolic activities. Direct imaging of tissue structures by the newly developed digital holography is deemed to offer unique investigation means in biology and medicine and attractive diagnostic capabilities.
We report on new developments of a technique called Digital Holographic Microscopy (DHM), for the numerical reconstruction of digital holograms taken in microscopy, which allows simultaneous amplitude and quantitative phase contrast imaging. The reconstruction method computes the propagation of the complex optical wavefront diffracted by the object and is used to determine the refractive index and/or shape of the object with accuracies in the nanometer range along the optical axis.. The method requires the acquisition of a single hologram. The technique comprises the recording of a digital hologram of the specimen by means of a standard CCD camera at the exit of a Mach-Zehnder or Michelson type interferometer. The quantitative nature of the reconstructed phase distribution has been demonstrated by an application to surface profilometry where step height differences of a few nanometers have been measured. Another application takes place in biology for transmission phase- contrast imaging of living cells in culture. The resolution for thickness measurements depends on the refractive index of the specimen and a resolution of approximately 30 nanometers in height, and about half of a micro in width, has been achieved for living neural cells in cultures by using a high numerical aperture.
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