Optical tomography provides three-dimensional data of the measured specimen, while quantitative phase
imaging enables measuring the induced phase-shifts. Combining those two technologies makes possible to
get three-dimensional refractive index reconstruction. This can be achieved by introducing a scan in the
measurement process, which can be done in several ways. We present and compare results of tomographic
measurements, taken either in angle-scanning or wavelength-scanning approach, respectively in transmission
or in reflection microscopy, in the framework of digital holographic microscopy.
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.
Different interferometric techniques were developed last decade to obtain full field, quantitative, and absolute
phase imaging, such as phase-shifting, Fourier phase microscopy, Hilbert phase microscopy or digital holographic
microscopy (DHM). Although, these techniques are very similar, DHM combines several advantages. In contrast,
to phase shifting, DHM is indeed capable of single-shot hologram recording allowing a real-time absolute phase
imaging. On the other hand, unlike to Fourier phase or Hilbert phase microscopy, DHM does not require to record
in focus images of the specimen on the digital detector (CCD or CMOS camera), because a numerical focalization
adjustment can be performed by a numerical wavefront propagation. Consequently, the depth of view of high NA
microscope objectives is numerically extended. For example, two different biological cells, floating at different
depths in a liquid, can be focalized numerically from the same digital hologram. Moreover, the numerical
propagation associated to digital optics and automatic fitting procedures, permits vibrations insensitive full-
field phase imaging and the complete compensation for a priori any image distortion or/and phase aberrations
introduced for example by imperfections of holders or perfusion chamber. Examples of real-time full-field phase
images of biological cells have been demonstrated.
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.
Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram,
quantitative phase image of living cell with interferometric accuracy (Marquet et al., 2005). Specifically the
optical phase shift induced by the specimen on the transmitted wave front can be regarded as a powerful
endogenous contrast agent, depending on both the thickness and the refractive index of the sample. We have
recently proposed (Rappaz et al., 2005) a new and efficient decoupling procedure allowing to directly obtain
separate measurements of the thickness and the integral refractive index of a given living cell. Consequently, it
has been possible, for the first time to our knowledge, to accurately measure (with a precision of 0.0003) the
mean refractive index of living erythrocytes.. On the other hand, the cellular thickness measurements allow to
calculate the volume and shape of erythrocytes. In addition, DHM, thanks to its subwavelength phase shift
measurements, was found to yield an efficient tool to assess erythrocyte cell membrane fluctuations (ECMF).
Typically, ECMF characterized by an amplitude within the range of 45 nm were observed.
KEYWORDS: Refractive index, Digital holography, Holograms, Microscopy, Holography, Signal processing, Holographic interferometry, Digital recording, Digital imaging, Neurons
Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram,
quantitative phase image of living cell with interferometric accuracy. Specifically, the optical phase shift induced
by the specimen on the transmitted wave front can be regarded as a powerful endogenous contrast agent,
depending on both the thickness and the refractive index of the sample. We discuss some approaches allowing to
directly obtain separate measurements of the thickness and the refractive index (RI) of a given living cell from
the phase signal.
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.
Digital holographic microscopy (DHM) is an interferometric technique, providing quantitative mapping of the phase
shift induced by semi-transparent microscopic specimens, such as cells, with sub-wavelength resolution along the optical
axis. Thanks to actual PCs and CCDs, DHM provides nowadays cost-effective instruments for real-time measurements at
very high acquisition rates, with sub-micron transverse resolution. However, DHM phase images do not reveal the threedimensional
(3D) internal distribution of refractive index, but a phase shift resulting from a mean refractive index (RI)
integrated over the cellular thickness. Standard optical diffraction tomography (ODT) techniques can be efficiently
applied to reveal internal structures and to measure 3D RI spatial distributions, by recording 2D DHM phase data for
different sample orientations or illumination beam direction, in order to fill up entirely the Ewald sphere in the Fourier
space. The 3D refractive index can then be reconstructed, even in the direct space with backpropagation algorithms or
from the Fourier space with inverse Fourier transform. The presented technique opens wide perspectives in 3D cell
imaging: the DHM-based micro-tomography furnishes invaluable data on the cell components optical properties,
potentially leading to information about organelles intracellular distribution. Results obtained on biological specimens
will be presented. Morphometric measurements can be extracted from the tomographic data, by detection based on the
refractive index contrast within the 3D reconstructions. Results and perspectives about sub-cellular organelles
identification inside the cell will also be exposed.
Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram,
quantitative phase image of living cell with interferometric accuracy. Specifically the optical phase shift induced
by the specimen on the transmitted wave front can be regarded as a powerful endogenous contrast agent,
depending on both the thickness and the refractive index of the sample. We have recently proposed a new and
efficient decoupling procedure allowing to directly obtain separate measurements of the thickness and the
integral refractive index of a given living cell. Consequently, it has been possible to accurately measure (with a
precision of 0.0003) the mean refractive index and the volume of living erythrocytes. Here, application of this
decoupling procedure on erythrocyte allows to measure a refractive index of 1.40 and a mean volume of about
106 μm3.
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.
In this paper, Digital Holographic Microscopy (DHM) is presented as a powerful tool for quality control of microoptical
components. It will be shown that not only the single-shot full field-of-view nanometer axial resolution makes
DHM an ideal solution for such samples, but the DHM numerical wavefront correction formalism is perfectly adapted
to provide advanced features like aberration coefficients, radius of curvature or optical surfaces roughness
measurements. Both transmission and reflection configurations can be used depending of the micro-components under
investigation. A transparent high aspect-ratio micro-components investigation procedure is also exposed in order to
unable phase unwrapping. Each feature is illustrated with typical examples, ranging from a wide variety of micro-lenses
(aspherical, cylindrical, squared) to cornercube micro-structures or diffractive elements.
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.
This paper present a novel approach to perform the tomography of biological specimen based on Digital Holographic Microscopy (DHM). A hologram results from the interference between a reference wave and an object wave reflected from or transmitted through a sample. In the hologram, both amplitude and phase of the field transmitted through the object are registered. In DHM, the object field is recovered when the hologram is processed by a digitally computed replica of the reference wave, allowing quantitative measurement of both phase and amplitude. Phase measurements provide high accuracy optical path length measurements across the specimen along the optical axis. To proceed to a tomographic reconstruction of the refractive index of the sample based on this quantitative phase measurement, such 2-dimentionnal data must be recorded for different sample orientations covering an angle of 180° to cover all the object spatial frequencies in the reciprocal space. The representation of the data in function of the angle is known as a sinogram. The 3-dimentionnal refractive index can then be reconstructed from the sinograms by a filtered backprojection algorithm. In our system, the specimen is inserted in a glass micropipette to permit its rotation. To our knowledge, a quantitative tomography of the refractive index of a pollen cell with a resolution in the micron range is presented for the first time.
KEYWORDS: Signal to noise ratio, Holograms, 3D image reconstruction, Digital holography, Optical simulations, Holography, Neurons, Microscopy, Image quality, Digital cameras
We present a study based on simulations describing how shot noise, an intrinsic part of the recording process with a
digital camera, influences the quality of the reconstructed phase images under different beams intensities configurations.
The ability of digital holographic microscopy (DHM) to provide both amplitude and phase images of a specimen makes it a convenient tool for cells analysis and recognition. Practical applications are demonstrated on pollen particles.
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.
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