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.
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.
Digital Holographic Microscopes (DHMs) have unique features especially relevant for static and dynamical MEMS characterization. They provide both 3D topography with interferometric resolution and intensity image in a single acquisition at camera rate, without any lateral or vertical scanning. In this presentation, DHM is operated in conjunction with a laser pulsed stroboscopic module providing synchronization of camera, laser pulses, and MEMS excitation signal up to 25 MHz. Three methods for DHM analysis of resonant frequencies are presented with concrete examples. The first method, gives the more general, precise and complete information. Sine wave excitation with increasing (or decreasing) frequencies is used. For each frequency, laser pulses are used to “freeze” the movement of the MEMS. Using the stroboscopic synchronization, each period of the excitation signal is sampled at high resolution, and the topography of the MEMS can be measured at each of those samples points. As implemented, the change of frequency is a continuous sweep: quantitative values in term of displacement amplitude and Bode diagrams can be measured for nonlinear resonances as well. The second method uses sine wave excitation with increasing (or decreasing) frequencies. For each frequency, the optical signal is integrated over an entire number of periods of the MEMS. At resonance, constructive and destructive interference build up on the intensity images. It enables fast frequency scan over large ranges. But it provides neither quantitative values of displacement amplitude, nor Bode diagrams. The third method is to measure the system response to an impulse or chirp excitation signal for instance, and to make a Fourier analysis of this response to determine resonant frequencies. This method is less sensitive as it spreads the excitation energy in many frequencies.
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.
Digital holography has proven its ability to acquire high accuracy full field 3D data with one single image acquisition. This means that in principle this technique offers the chance to perform 3D serial inspection processes, as well. However, one limitation in digital holography is its limited ability to measure rough surfaces. In the presence of rough surfaces, the magnification of the image has to be increased to capture the required phase information on each camera pixel. However, this leads to significant reduction of inspection speed. If low magnification is selected, the rough surface produces speckles which cannot be treated properly by digital holography algorithms. In this paper, we describe the extension of digital holography to rough surface applications using speckle interferometry technique. This technique is capable of fast inspection of rough surfaces with sub-micrometer accuracy. The principle of this approach is shown and a practical application for 3D surface inspection of wafer cutting processes is given.
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 fast high-roughness and non-contact surface measurements by digital holographic microscopy (DHM).
By using single- and dual-wavelength operation modes, coupled with advanced image stitching and non-measured
points management methods, the technique enables two-dimensional roughness measurements up to the micrometer
(N6). The sample is mechanically scanned over a surface up to 5 × 0.3 mm2 with 17 holograms each acquired
in less than 500 μs, the corresponding phase images stitched together by software, and therefore providing multiple
profiles measurement in the ISO definition in less than 30 s. The approach is validated by inspection of
several different roughness standards and our technique is demonstrated to be in agreement with two existing
well-known techniques in the field.
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.
KEYWORDS: Digital holography, Holograms, 3D displays, Liquid crystal on silicon, Cameras, 3D image reconstruction, Holography, Spatial light modulators, Visualization, Optoelectronics
In digital holography, holograms are usually optically captured and then two-dimensional slices of the reconstruction
volume are reconstructed by computer and displayed on a two-dimensional display. When the recording is
of a three-dimensional scene then such two-dimensional display becomes restrictive. We outline our progress on
capturing larger ranges of perspectives of three-dimensional scenes, and our progress on four approaches to better
visualise this three-dimensional information encoded in the digital holograms. The research has been performed
within a European Commission funded research project dedicated the capture, processing, transmission, and
display of real-world 3D and 4D scenes using digital holography.
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.
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 two stroboscopic
modes with standard cameras. The first one enables precise characterization up to excitation frequencies of 100 kHz
with recovery cycle of 10% simply by triggering properly the camera. The second one uses a pulsed source 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 illustrate the two stroboscopic modes with an example of a high frequency micro mirror.
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.
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.
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.
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 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.
Based on an original numerical reconstruction algorithm (E. Cuche et al. Appl. Opt. 38, 6994 1999), we have developed a Digital Holographic Microscope (DHM), in a transmission mode, allowing to investigate noninvasively cellular structures and dynamics. DHM images of living cells in culture are presented. They represent the distribution of the optical path length over the cell, which contains information concerning both the cellular morphometry and the intracellular refractive index, and which has been measured with a sub-wavelength accuracy.
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.
There have been several studies of the potential accuracy of LIDAR measurements of sound velocity in the ocean by measuring the spectral shift of the backscattered Brillouin lines. However, due to technical limitations, such systems have not previously been experimentally demonstrated. Measurement of the Brillouin shift as a function of depth in the ocean requires a stabilized, narrow linewidth, pulsed laser, and a high-resolution spectroscopic technique. We have used a scanning Fabry-Perot to obtain the first frequency resolved measurements of Brillouin scattering in water using a pulsed laser; these results will be presented here. But for the practical application of measuring Brillouin shifts as a function of depth in the ocean a non- scanning spectroscopic technique is required to measure the small frequency shifts; the edge technique meets this requirement. Using it in conjunction with the edges of absorption lines in the molecular spectra of I2 and/or Br2, avoids the limitations associated with use of a Fabry-Perot etalon; specifically, its small solid angle of acceptance and its vulnerability in noisy environments. This new approach will be briefly described.
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