The study of the movements of microscopic phase objects, as biological cells, is of high interest in the scientific community. Digital Holographic Microscopy (DHM) is a technique widely used for analysis of phase objects. It can record, in one shot, the sample complex field and then the complex field is refocused in several planes for the 3D sample reconstruction. However, when in the view field there are several cells at different distances from the hologram plane, the correct location of each cell is critical for the analysis of the sample. There are diverse approaches to find the optimal focusing distance of a phase object; however, most of them depend on the input of the cell location in the view field as parameter. This condition restrings their application to cells in movement. We present the analysis of moving phase objects, using an alternative focusing criterion based on the analysis of different sized windows. With this criterion, it is possible to create a depth map of the objects in the sample, and at the same time finds its location in the view field and discriminate them from the background. The depth map is segmented with the clustering K-means method and each cluster is analyzed to determine the optimal object focusing distance. Then an extended focus image of the sample is created and displayed for the user. The method can detect autonomously when a new cell enters in the view field and calculates its focusing distance. The resulting images present all the cells in the sample well focused, and can be used for counting or tracking purposes. We present simulated and experimental results.
Acousto-optic (AO) modulation of light is used to extract both temporal and spectral information of diffusive media such as biological tissue, where they provide measures of blood flow and oxygen saturation of hemoglobin, respectively. The temporal information is extracted from the width of the power spectrum of the light intensity, whereas the spectral information is calculated from the spatial decay of the cross correlation between the light intensity and the generated ultrasonic signal. The ultrasonic signal is a coded phase modulated signal with a narrow autocorrelation, enabling localization of the measurement volume. Two different liquid phantoms are used, with similar scattering but different absorption properties. The difference in absorption calculated with the AO signal is compared to calculations based on the modified Beer Lambert law. As the same AO signal is used to extract both modalities, it might be used to extract hemodynamic related changes in the brain for diagnostic and functional assessment.
We propose a new portable imaging configuration that can double the field of view (FOV) of existing off-axis interferometric imaging setups, including broadband off-axis interferometers. This configuration is attached at the output port of the off-axis interferometer and optically creates a multiplexed interferogram on the digital camera, which is composed of two off-axis interferograms with straight fringes at orthogonal directions. Each of these interferograms contains a different FOV of the imaged sample. Due to the separation of these two FOVs in the spatial-frequency domain, they can be fully reconstructed separately, while obtaining two complex wavefronts from the sample at once. Since the optically multiplexed off-axis interferogram is recorded by the camera in a single exposure, fast dynamics can be recorded with a doubled imaging area. We used this technique for quantitative phase microscopy of biological samples with extended FOV. We demonstrate attaching the proposed module to a diffractive phase microscopy interferometer, illuminated by a broadband light source. The biological samples used for the experimental demonstrations include microscopic diatom shells, cancer cells, and flowing blood cells.
We review new and efficient algorithms, lately presented by us, for rapid reconstruction of quantitative phase maps from off-axis digital interferograms. These algorithms improve the conventional Fourier-based algorithm by using the Fourier transforms and the phase unwrapping process more efficiently, and thus decrease the calculation complexity required for extracting the sample phase map from the recorded interferograms. Using the new algorithms, on a standard personal computer without using the graphic processing-unit programming or parallel computing, we were able to speed up the processing and reach frame rates of up to 45 frames per second for one megapixel off-axis interferograms. These capabilities allow real-time visualization, calculation and data extraction for dynamic samples and processes, inspected by off-axis digital holography. Specific applications include biological cell imaging without labeling and real-time nondestructive testing.
We present a new approach of optically multiplexing several off-axis interferograms on the same digital camera, each of which encodes a different field of view of the sample. Since the fringes of these interferograms are in different directions, as obtained experimentally by the optical system, we are able to double or even triple the amount of information that can be acquired in a single camera exposure, with the same number of camera pixels, while sharing the camera dynamic range. We show that this method can partially solve the problem of limited off-axis interferometric field of view due to low-coherence illumination. Our experimental demonstrations include quantitative phase imaging of microscopic diatom shells, fast swimming sperm cells and microorganisms, and contracting cardiomyocytes.
We review the interferometric double-imaging area (IDIA) technique,1 a new holographic principle and an optical setup
for doubling the field of view of interferometric imaging setups, and obtaining wider off-axis interference areas with
low-coherence light sources. The method enables measuring larger samples that cannot fit into one interferometric field
of view without decreasing the microscope magnification or performing scanning, while losing in the camera frame rate.
The new principle was implemented using a modified off-axis τ interferometer, which is compact, portable, and easy to
construct and align even with low-coherence light sources. We demonstrate using the proposed technique for imaging
the quantitative phase maps of a transparent microscopic test target and live neurons.
We review new algorithms that have been presented by us lately1 for fast reconstruction and phase unwrapping of sample wave-fronts recorded using off-axis digital holographic imaging. These algorithms enable reconstruction and phase
unwrapping of sample wave-fronts up to 16 times faster than the conventional Fourier-based reconstruction algorithm.
The algorithms exploit the compression properties of holographic imaging for decreasing the calculation complexity
required for extracting the sample wave-front from the recorded interferogram. Using the presented algorithms, we were
able to reconstruct, for the first time, 1 Mega pixels off-axis interferograms in more than 30 frames per second using a
standard single-core personal computer on a Matlab-Labview interface, without using a graphic processing-unit
programming or parallel computing. This computational speedup is important for real-time visualization, calculation and
data extraction for dynamic samples and processes that are evaluated using off-axis digital holography such as biological
cell imaging and real-time nondestructive testing.
Interferometric phase microscopy (IPM) is a quantitative optical imaging method for capturing the phase profiles of thin
samples. While being an invaluable tool for biological and medical research, most IPM setups are unfriendly for
inexperienced users, and have limited field of view (FOV). To overcome the limited FOV problem, it is possible to scan
the sample and record a wider FOV. However, dynamic samples might move by the time the scan is over. Here, we
review our previously published work presenting a new quantitative imaging technique, referred to as interferometry
with tripled-imaging area (ITIA), which is capable of capturing three off-axis interferometric fields of view in a single
camera exposure, thus tripling the acquired information without the need to scan the sample, without decreasing the
image resolution, and without changing the system magnification. Our experimental demonstrations were done by using
an inverted transmission microscope illuminated by a Helium-Neon laser. The sample is projected onto the image plane
at the output of the microscope, where the ITIA module is placed. Various biological and non-biological samples were
imaged.
We present our recent advances in the development of compact, highly portable and inexpensive wide-field interferometric modules. By a smart design of the interferometric system, including the usage of low-coherence illumination sources and common-path off-axis geometry of the interferometers, spatial and temporal noise levels of the resulting quantitative thickness profile can be sub-nanometric, while processing the phase profile in real time. In addition, due to novel experimentally-implemented multiplexing methods, we can capture low-coherence off-axis interferograms with significantly extended field of view and in faster acquisition rates. Using these techniques, we quantitatively imaged rapid dynamics of live biological cells including sperm cells and unicellular microorganisms. Then, we demonstrated dynamic profiling during lithography processes of microscopic elements, with thicknesses that may vary from several nanometers to hundreds of microns. Finally, we present new algorithms for fast reconstruction (including digital phase unwrapping) of off-axis interferograms, which allow real-time processing in more than video rate on regular single-core computers.
Interferometric phase microscopy (IPM) enables to obtain quantitative optical thickness profiles of transparent samples, including live cells in-vitro, and track them in time with sub-nanometer accuracy without any external labeling, contact or force application on the sample. The optical thickness measured by IPM is a multiplication between the cell integral refractive index differences and its physical thickness. Based on the time-dependent optical thickness profile, one can generate the optical thickness fluctuation map. For biological cells that are adhered to the surface, the variance of the physical thickness fluctuations in time is inversely proportional to the spring factor indicating on cell stiffness, where softer cells are expected fluctuating more than more rigid cells. For homogenous refractive index cells, such as red blood cells, we can calculate a map indicating on the cell stiffness per each spatial point on the cell. Therefore, it is possible to obtain novel diagnosis and monitoring tools for diseases changing the morphology and the mechanical properties of these cells such as malaria, certain types of anaemia and thalassemia. For cells with a complex refractive-index structure, such as cancer cells, decoupling refractive index and physical thickness is not possible in single-exposure mode. In these cases, we measure a closely related parameter, under the assumption that the refractive index does not change much within less than a second of measurement. Using these techniques, we lately found that cancer cells fluctuate significantly more than healthy cells, and that metastatic cancer cells fluctuate significantly more than primary cancer cells.
We present a compact, highly portable and inexpensive interferometric module for obtaining spatial interferograms of
microscopic biological samples, without the strict stability and the highly-coherent illumination that are usually required
for interferometric microscopy setups. The module is built using off-the-shelf optical elements and can easily operate
with low-coherence illumination, while being positioned in the output of a conventional transmission microscope. The
interferograms are processed into the quantitative amplitude and phase profiles of the sample. Based on the phase profile,
the optical thickness or optical path delay profile of the sample is obtained with temporal and spatial stabilities, at the
order of 0.2-0.3 nm. We show several configurations of this interferometer that are suitable for both on-axis and off-axis
holographic geometries, and present various experimental results, including imaging live cells in a non-contact label-free
manner and transparent elements with nano-scale thickness. Since the interferometer can be connected to the output of a
transmission microscope and operate in a simple way, without involvement of an expert user with a knowledge in optics
and without complicated alignment prior to every experiment, and still obtain a remarkably high accuracy, we believe
that this new setup will make interferometric phase microscopy more accessible and affordable for biologists and
clinicians, while significantly broadening its range of applications.
We propose a low-coherence spectral-domain phase microscopy (SDPM) system for accurate quantitative phase measurements in red blood cells (RBCs) for the prognosis and monitoring of disease conditions that affect the visco-elastic properties of RBCs. Using the system, we performed time-recordings of cell membrane fluctuations, and compared the nano-scale fluctuation dynamics of healthy and glutaraldehyde-treated RBCs. Glutaraldehyde-treated RBCs possess lower amplitudes of fluctuations, reflecting an increased membrane stiffness. To demonstrate the ability of our system to measure fluctuations of lower amplitudes than those measured by the commonly used holographic phase microscopy techniques, we also constructed wide-field digital interferometry (WFDI) system and compared the performances of both systems. Due to its common-path geometry, the optical-path-delay stability of SDPM was found to be less than 0.3 nm in liquid environment, at least three times better than WFDI under the same conditions. In addition, due to the compactness of SDPM and its inexpensive and robust design, the system possesses a high potential for clinical applications.
We demonstrate the use of a low-coherence spectral-domain phase microscopy (SDPM) system for accurate quantitative
phase measurements in red blood cells (RBCs) for the prognosis and monitoring of disease conditions that affect the
visco-elastic properties of RBCs. Using the system, we performed time-recordings of cell membrane fluctuations, and
compared the nano-scale fluctuation dynamics of healthy and glutaraldehyde-treated RBCs. Glutaraldehyde-treated
RBCs possess a lower amplitude of fluctuations reflecting an increased membrane stiffness. To demonstrate the ability
of our system to measure fluctuations of lower amplitudes than those measured by the commonly used holographic phase
microscopy techniques, we also constructed a wide-field digital interferometric microscope and compared the
performances of the two systems. Due to its common-path geometry, the optical-path-delay stability of SDPM was found
to be less than 0.3nm in liquid environment, at least three times better than in holographic phase microscopy under the
same conditions. In addition, due to the compactness of SDPM and its inexpensive and robust design, the system
possesses a high potential for clinical applications.
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