The flagellum is vital for eukaryotic cell survival. We propose method utilizing digital holographic microscopy (DHM) advantages, such as high-speed imaging and precise axial localization, to develop a versatile approach for four-dimensional tracking (X, Y, Z, time) of eukaryotic microorganisms' bodies and flagella, addressing challenges faced by existing methods. We reconstructed for the first time the shape of a 200 nm diameter Chrysochromulina simplex flagellum and measured mouse sperm flagella over time, capturing approximately 800 points. Our technique opens new avenues for studying flagella's roles in cellular functions and survival strategies, offering high-speed and precise 3D tracking at the nanoscale.
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.
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.
Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.
Digital holography microscopy (DHM) is an optical microscopy technique which allows recording non-invasively the
phase shift induced by living cells with nanometric sensitivity. Here, we exploit the phase signal as an indicator of dry
mass (related to the protein concentration). This parameter allows monitoring the protein production rate and its
evolution during the cell cycle.
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.
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.
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.
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.
In this paper, we present a transmission polarization digital holographic microscope (Pol-DHM) that allows for imaging
the state of polarization of a wave front with the acquisition of a single hologram. This apparatus records, using a CCD
camera, the interference in off-axis geometry between two orthogonally polarized reference waves and an object wave
transmitted through a microscopic sample and magnified by a microscope objective. Since the reference waves have
orthogonal polarizations, they do not interfere and the hologram results in two different fringes patterns, which can be
separately filtered in the spatial Fourier domain and reconstructed separately to compute the amplitude and phase of two
wave fronts. These four images allow computing the polarization ellipse azimuth and the phase difference associated
with the polarization state of the object wave. The method is illustrated by imaging the strain induced in a
polymethylmethacrylate (PMMA) sample and in a bended optical fiber.
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.
Femtosecond laser irradiation has various noticeable effects on fused silica. It can locally increase the index of refraction and modify the material chemical selectivity. Regions that have been exposed to the laser are etched hundred fold faster than unexposed regions. These effects are of practical importance from an application point-of-view and open new opportunities for the development of integrated photonics devices that combine structural and optical functions.
Various observations reported in the literature indicate that those effects are potentially related to a combination of both structural changes and the presence of internal stress. In this paper, we present further investigations on the effect of femtosecond laser irradiation on fused silica substrate (a-SiO2). In particular, we use nanoindentation and holography-based birefringence measurements, coupled with direct SEM observations on chemically etched specimens to characterize the effect of various laser parameters such as power, scanning speed and irradiation pattern. We show evidence of an interface between two different etching regimes that may be related to the presence of two different material phases induced by the laser irradiation.
In this paper, we present a modified transmission digital holographic microscope that can be used to image the state of polarization. The resulting device, called polarization digital holographic microscope (Pol-DHM), records in off-axis geometry the interference between two orthogonally polarized reference waves and the object wave transmitted by a microscopic sample and magnified by a microscope objective. A CCD camera records the resulting hologram. Using a single hologram, we reconstruct separately the amplitude and phase of two wave fronts, which are used to represent the object wave's state of polarization, represented by the azimuth and the phase difference associated to the polarization ellipse. The proposed method is illustrated with two applications. The first application is to use a 10-times magnification microscope objective to measure the birefringence induced by internal stresses in transparent materials such as a bended optical fiber. The second application is to use a 20-times microscope objective to image the state of polarization of a thin concrete sample and reveals birefringent properties of the different aggregates found in the concrete.
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.
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).
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 present a method to image Jones vector by use of digital holography. We show that with unique hologram acquisition, our method permits to image and to calculate Jones vector and therefore polarization parameters. The idea is to use two reference waves polarized perpendicularly that interfere with object wave and to reconstruct separately the two wave fronts. The precision and limitations of the method are evaluated here using a numerical model.
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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