One of the directions of development in quantitative phase imaging is to provide the capability to reconstruct the phase or preferably refractive index (RI) distribution within thick, highly scattering samples. This direction coincides with current trends in biology, where three-dimensional (3D) organoids are currently replacing standard 2D cultures as more physiological models for tissue growth and organ formation in a dish. The biological complexity of these 3D structures makes the imaging and RI reconstruction particularly challenging, and thus calibration as well as validation structures are important and sought-after tools in instrumentation development. For this reason, in this work, we present the full preparation and measurement procedure for organoid phantoms printed with two-photon polymerization along with the method to obtain the ground truth of the object structure independently of RI reconstruction errors and artifacts.
Bortezomib is one of the most researched proteasome inhibitor drug in cancer cell research. Studying its effects, measuring and monitoring treatment response and effectiveness is a widely developing area in cancer research. The introduction of non-invasive measurement tools into the this research is a very important and desirable development, as it is a promising alternative to existing chemical tests. In our work we presented multimodal methodology connecting multiple non-invasive and label-free techniques to study effects of bortezomib on RPMI8226 cells. We connected digital holographic microscopy and holographic tomography with chemical specificity from Raman micro spectroscopy and we showed that treatment with bortezomib caused decrease of RI in the cells and their nucleolus and that changes in chemical compositions after treatment indicate cell apoptosis.
KEYWORDS: Microfluidics, Refractive index, Tomography, Statistical analysis, Holography, Biological research, Lab on a chip, 3D metrology, Imaging systems
Holographic tomography (HT) is a label-free, high-resolution and non-invasive method that retrieves 3D refractive index (RI) information about analysed biological specimens. The most common measurement scenario includes culturing and analysing cells directly in a Petri dish. However, it does not mimic the in vivo conditions unlike the microfluidic approach. Thus, in our work, we have focused on the development of a measurement configuration that is dedicated to analysis of cell dynamics in a lab-on-chip. It includes a fast HT system, a new ultra-thin microfluidic chip that allows for long term monitoring in controlled environment, a stitching algorithm that allows to combine single fields of view (FoV) into a synthetic field of view in three dimensions and the full volume RI analysis of internal cellular organelles during measurements. This setup provides the ability to track changes occurring in individual cell organelles as well as getting statistically valuable data. In experimental verification, A549-type and MeWo cells were cultured under microfluidic conditions in the chip and put under observation using HT.
The results of quantitative, label-free measurements of SHSY-5Y cells under Low-Level Laser Therapy treatment are presented. The time-lapse investigations performed by means of digital holographic microscopy are focused on dry mass density changes after light exposure.
Limited angle optical diffraction tomography (ODT) is a 3D quantitative phase imaging method that allows to retrieve information about 3D refractive index (RI) distribution of live, unlabeled biosamples. The main limitation of this method is that its common transmission configuration results in very low axial resolution. On the other hand, optical coherence tomography (OCT), working in its most popular reflection configuration retrieves information about the gradient of the RI of investigated samples. However, the results are of qualitative nature. Moreover, due to low numerical aperture of the objective lens typically used in OCT systems, the resolution is high in the axial direction and relatively low in transverse direction. From the point of view of K-space filling, these two imaging modalities are complementary. Here we present a method of combining ODT projections with OCT scans. The combined technique, called optical coherence diffraction tomography (OCDT) operates in transflective mode, where ODT is captured in transmission and OCT in reflection. Theory behind conversion of OCT scans into ODT projections is given. With the use of numerical simulations we show what enhancement can be obtained when OCT and ODT data are combined directly. Also, experimental verification is presented.
A desktop tomography system, based on laser-interaction with a gas puff target, which results in efficient plasma formation emitting in the soft X-ray (SXR, λ = 0.1 - 10 nm) region, was developed at IOE-WAT (Warsaw, Poland). The system, coupled with an ellipsoidal condenser and a Fresnel zone-plate and working in the “water window” spectral range (λ = 2.3 - 4.4 nm) at the quasi-monochromatic He-like nitrogen spectral line (λ=2.88nm), allows acquiring images approaching a resolution of few microns. The development of such setup offers the possibility to obtain a reconstruction of three-dimensional images in a laboratory environment, without the involvement of large “photon facilities”. Details about the system and its optimization as well as some imaged samples will be presented and discussed.
Limited angle holographic tomography (LAHT) is currently the most common tool in biomedical applications of 3D quantitative phase imaging. It uses the refractive index (RI) as contrast agent for a single cell or tissue analysis and provides highly accurate RI values in the full measurement volume. Recently several new systems have been built in laboratories and new devices have been released into the market. All of them apply algorithms and processing paths which significantly influence correctness of the results. In our work we perform study of the selected LAHT systems and compare their 3D metrological features and other functional parameters.
Limited angle optical diffraction tomography is a technique that allows for non-destructive and quantitative analysis of biological samples directly from Petri dishes and microscope slides. Recently, a new reconstruction method, called Generalized Total Variation Iterative Constraint (GTVIC), was proposed. It is an iterative modular technique that calculates the reconstruction without the errors that are otherwise present due to limited angular range of projections. Unlike other similar techniques, GTVIC is dedicated to biological structures that have non-piecewise constant refractive index distribution. However, GTVIC is sensitive to local deviations of the immersion refractive index from its nominal value. Such deviations are present due to e.g. dust or cellular debris in the measurement volume. Therefore, we propose a method for preprocessing of a sinogram that numerically removes unwanted objects from the measurement volume without affecting the investigated object and its diffraction pattern. The described method is found useful in any tomographic algorithm that utilizes support constraint in the reconstruction process.
In the paper the advantages of two different microscopic techniques, namely digital holographic microscopy (DHM) and Confocal Laser Scanning Microscopy (CLSM) have been combined with the aim to investigate HeLa cell culture in terms of statistical analysis of area of subcellular structures of HeLa cells and related to them dry mass estimation. To assure the proper statistical representation of the cells both measurements comprised of multiple fields of view (FoVs), stitched together to form two FoVs with overlapping regions. The results suggest a strong linear correlation of nucleoli dry mass to their projection area, a result that is promising in terms of its biological relevance.
Limited-angle optical diffraction tomography (LAODT) is a powerful tool for measuring 3D refractive index distribution in biological microsamples. However, when thick objects are measured, reconstructions are erroneous due to diffraction errors even in the case when tomographic reconstruction algorithms take into account this phenomenon. We propose a hardware-based solution which allows to change a focal plane position with a liquid tunable lens in LAODT system. For each illumination angle, projections with different focal plane positions are recorded, and thus diffraction errors in the neighborhood of these planes are minimized. In this paper, we describe a method for processing data from a varifocal tomography setup that utilizes a Generalized Total Variation Iterative Constraint algorithm.
In this paper a new, hardware-based solution for extending the depth of field in holographic tomography is presented. The solution is based on a 4f system and an electric, focus-tunable lens, which provides fast, motion-free defocusing of the plane conjugate with the camera, which acquires holograms. The optimum parameters for the required axial scanning are provided for a specific model of a commercially available tunable lens. Then, the quality of the system equipped with the designed module is analyzed and the reconstruction of a standard object (microsphere) scanned by the 4f-based defocusing system is presented. Finally, the result of the increased depth of field in the measurement domain is demonstrated with a reconstruction of a mouse fibroblast cell.
We demonstrate an active, holographic tomography system, working with limited angle of projections, realized by optical-only, diffraction-based beam steering. The system created for this purpose is a Mach–Zehnder interferometer modified to serve as a digital holographic microscope with a high numerical aperture illumination module and a spatial light modulator (SLM). Such a solution is fast and robust. Apart from providing an elegant solution to viewing angle shifting, it also adds new capabilities of the holographic microscope system. SLM, being an active optical element, allows wavefront correction in order to improve measurement accuracy. Integrated phase data captured with different illumination scenarios within a highly limited angular range are processed by a new tomographic reconstruction algorithm based on the compressed sensing technique: total variation minimization, which is applied here to reconstruct nonpiecewise constant samples. Finally, the accuracy of full measurement and the proposed processing path is tested for a calibrated three-dimensional micro-object as well as a biological object—C2C12 myoblast cell.
Standard tomographic algorithms applied to optical limited-angle tomography result in the reconstructions that have highly anisotropic resolution and thus special algorithms are developed. State of the art approaches utilize the Total Variation (TV) minimization technique. These methods give very good results but are applicable to piecewise constant structures only. In this paper, we propose a novel algorithm for 3D limited-angle tomography – Total Variation Iterative Constraint method (TVIC) which enhances the applicability of the TV regularization to non-piecewise constant samples, like biological cells. This approach consists of two parts. First, the TV minimization is used as a strong regularizer to create a sharp-edged image converted to a 3D binary mask which is then iteratively applied in the tomographic reconstruction as a constraint in the object domain. In the present work we test the method on a synthetic object designed to mimic basic structures of a living cell. For simplicity, the test reconstructions were performed within the straight-line propagation model (SIRT3D solver from the ASTRA Tomography Toolbox), but the strategy is general enough to supplement any algorithm for tomographic reconstruction that supports arbitrary geometries of plane-wave projection acquisition. This includes optical diffraction tomography solvers. The obtained reconstructions present resolution uniformity and general shape accuracy expected from the TV regularization based solvers, but keeping the smooth internal structures of the object at the same time. Comparison between three different patterns of object illumination arrangement show very small impact of the projection acquisition geometry on the image quality.
The case of diffraction tomography with limited angle of projections is discussed from the algorithmic and experimental points of view. To reconstruct a three-dimensional distribution of refractive index of a micro-object under study, we use a hybrid approach based on the simultaneous algebraic reconstruction technique (SART) enhanced by a compressed sensing reconstruction technique. It enables us to apply the standard computed tomography algorithms (which assume that the rays are traveling in straight lines through the object) for phase data obtained by means of digital holography. We present the results of analysis of a phantom and real objects obtained by applying SART with anisotropic total variation (ATV) minimization. The real data are acquired from an experimental setup based on a Mach–Zehnder interferometer configuration. Also, it is proven that in the case of simulated data, the limited number of projections captured in a limited angular range can be compensated by a higher number of iterations of the algorithm. We also show that the SART + ATV method applied for experimental data gives better results than the data replenishment algorithm.
In the paper we demonstrate a holographic tomography system with limited angle of projections, realized by optical– only, diffraction-based beam steering. The system created for this purpose is a Mach-Zehnder interferometer modified to serve as a digital holographic microscope with high Numerical Aperture illumination module and a Spatial Light Modulator. Such solution is fast and robust. Apart from providing an elegant solution to the viewing angle shifting, it also adds new capabilities of the holographic microscope system. SLM, being an active optical element, allows wavefront correction in order to improve measurement accuracy. Integrated phase data captured with different scenarios within a highly limited angular range are processed by a new tomographic reconstruction algorithm based on the compressed sensing technique: total variation minimization, which is applied to non-piecewise constant samples. Finally, the accuracy of full measurement and processing path proposed is tested for a calibrated 3D microobject.
In the paper the case of diffraction tomography with limited angle of projections is discussed from the experimental and algorithmic point of views. To reconstruct a 3D distribution of refractive index of an object under study, we use the hybrid approach, which enables to apply the standard Computer Tomography algorithms for phase data obtained by digital holography. We present the results of applying Simultaneous Algebraic Reconstruction Technique together with Anisotropic Total Variation minimization (SART+ATV) on both a phantom object and real data acquired from an experimental setup based on a Mach-Zehnder interferometer configuration. Also, the analysis of the influence of the limited number of projections within a limited angular range is presented. We prove that in the case of simulated data, the limited number of projections captured in a limited angular range can be compensated by higher number of iterations of the algorithm. We also show that SART+ATV method applied for experimental data gives better results than the popular Data Replenishment algorithm.
The paper presents the last data regarding new elements based on photonic crystal fibers such as the low-loss patch
cord with a single mode fiber, the fused coupler, the asymmetric coupler for an active fiber power pump. Their
fundamental optical characteristics including wavelength depending loss as a coupling ratio are presented in this paper
as well as their inner structure (cross section) obtained by SEM. However, the use of SEM for the investigation of the
inner element structure is destructive, thus in the last part of the paper we present the tomographic in-line
determination of geometry and refractive index distribution changes along the investigated photonic structure.
The analysis of different approaches to the photonic crystal fiber data capture with a sufficient optical resolution is
given. The data obtained from the Mach-Zehnder interferometer with different laser sources as well as from the in-line
digital holographic setup are presented and compared. The further enhancement required for the digital in-line
holography is discussed.
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