Phase-Retrieved Tomography enables imaging of a Tumor Spheroid in Mesoscopy Regime

Optical tomographic imaging of biological specimen bases its reliability on the combination of both accurate experimental measures and advanced computational techniques. In general, due to high scattering and absorption in most of the tissues, multi view geometries are required to reduce diffuse halo and blurring in the reconstructions. Scanning processes are used to acquire the data but they inevitably introduces perturbation, negating the assumption of aligned measures. Here we propose an innovative, registration free, imaging protocol implemented to image a human tumor spheroid at mesoscopic regime. The technique relies on the calculation of autocorrelation sinogram and object autocorrelation, finalizing the tomographic reconstruction via a three dimensional Gerchberg Saxton algorithm that retrieves the missing phase information. Our method is conceptually simple and focuses on single image acquisition, regardless of the specimen position in the camera plane. We demonstrate increased deep resolution abilities, not achievable with the current approaches, rendering the data alignment process obsolete.Comment: 21 pages, 5 figure

Optical projection tomography as a tool for 3D imaging of hydrogels

An Optical Projection Tomography (OPT) system was developed and optimized to image 3D tissue engineered products based in hydrogels. We develop pre-reconstruction algorithms to get the best result from the reconstruction procedure, which include correction of the illumination and determination of sample center of rotation (CoR). Existing methods for CoR determination based on the detection of the maximum variance of reconstructed slices failed, so we develop a new CoR search method based in the detection of the variance sharpest local maximum. We show the capabilities of the system to give quantitative information of different types of hydrogels that may be useful in its characterization.The authors thank to Tekes, Finnish Cultural Foundation, CIMO, Jane and Aatos Erkko Foundation and EXTREMA COST Action MP1207 for supporting this work

Polarization-sensitive optical projection tomography for muscle fiber imaging

Optical projection tomography (OPT) is a tool used for three-dimensional imaging of millimeter-scale biological samples, with the advantage of exhibiting isotropic resolution typically in the micron range. OPT can be divided into two types: transmission OPT (tOPT) and emission OPT (eOPT). Compared with eOPT, tOPT discriminates different tissues based on their absorption coefficient, either intrinsic or after specific staining. However, it fails to distinguish muscle fibers whose absorption coefficients are similar to surrounding tissues. To circumvent this problem, in this article we demonstrate a polarization sensitive OPT system which improves the detection and 3D imaging of muscle fibers by using polarized light. We also developed image acquisition and processing protocols that, together with the system, enable the clear visualization of muscles. Experimental results show that the muscle fibers of diaphragm and stomach, difficult to be distinguished in regular tOPT, were clearly displayed in our system, proving its potential use. Moreover, polarization sensitive OPT was fused with tOPT to investigate the stomach tissue comprehensively. Future applications of polarization sensitive OPT could be imaging other fiberlike structures such as myocardium or other tissues presenting high optical anisotropy.This work is supported by the National Basic Research Program of China (973 Program) under Grant 2011CB707700, the National Natural Science Foundation of China under Grant No. 81227901, 61231004, 81501616, 81301346, 81527805 the Chinese Academy of Sciences Fellowship for Young Foreign Scientists under Grant No. 2010Y2GA03, 2013Y1GA0004, the Chinese Academy of Sciences Visiting Professorship for Senior International Scientists under Grant No. 2012T1G0036, 2013T1G0013, the Instrument Developing Project of the Chinese Academy of Sciences under Grant No. YZ201502, YZ201457 and the Strategic Priority Research Program (B) of Chinese Academy of Sciences (XDB02060010). A. Arranz acknowledges support from the Marie Curie Intra-European Fellowship program IEF-2010-275137. J.R. acknowledges support from EC FP7 IMI project PREDICT-TB, the EC FP7 CIG grant HIGH-THROUGHPUT TOMO, and the Spanish MINECO project grant FIS2013-41802-R MESO-IMAGING

Frame localisation optical projection tomography

Funder: EPSRC IPESAbstract: We present a tomographic reconstruction algorithm (flOPT), which is applied to Optical Projection Tomography (OPT) images, that is robust to mechanical jitter and systematic angular and spatial drift. OPT relies on precise mechanical rotation and is less mechanically stable than large-scale computer tomography (CT) scanning systems, leading to reconstruction artefacts. The algorithm uses multiple (5+) tracked fiducial beads to recover the sample pose and the image rays are then back-projected at each orientation. The quality of the image reconstruction using the proposed algorithm shows an improvement when compared to the Radon transform. Moreover, when adding a systematic spatial and angular mechanical drift, the reconstruction shows a significant improvement over the Radon transform

Helical Optical Projection Tomography

A new technique termed Helical Optical Projection Tomography (hOPT) has been developed with the aim to overcome some of the limitations of current 3D optical imaging techniques. hOPT is based on Optical Projection Tomography (OPT) with the major difference that there is a translation of the sample in the vertical direction during the image acquisition process, requiring a new approach to image reconstruction. Contrary to OPT, hOPT makes possible to obtain 3D-optical images of intact long samples without imposing limits on the sample length. This has been tested using hOPT to image long murine tissue samples such as spinal cords and large intestines. Moreover, 3D-reconstructed images of the colon of DSS-treated mice, a model for Inflammatory Bowel Disease, allowed the identification of the structural alterations. Finally, the geometry of the hOPT device facilitates the addition of a Selective Plane Illumination Microscopy (SPIM) arm, providing the possibility of delivering high resolution images of selected areas together with complete volumetric informationThis work was partially supported by EC FP7 collaborative grant FMT-XCT and the Bill and Melinda Gates foundation. A.A. wishes to acknowledge support from Marie Curie IEF-2010-275137. J.R. wishes to acknowledge support from EC FP7 IMI project PREDICT-TB, and the EC FP7 CIG grant HIGH-THROUGHPUT TOMO. D.D., S.Z. and J.T. acknowledge support from the National Basic Research Program of China (973 Program) under Grant 2011CB707700, the Fellowship for Young International Scientists of the Chinese Academy of Sciences under Grant 2010Y2GA03, the National Natural Science Foundation of China under Grant 81101084 and Instrument Developing Project of the Chinese Academy of Sciences under Grant No. YZ201164Publicad

Optical projection tomography for rapid whole mouse brain imaging

In recent years, three-dimensional mesoscopic imaging has gained significant importance in life sciences for fundamental studies at the whole-organ level. In this manuscript, we present an optical projection tomography (OPT) method designed for imaging of the intact mouse brain. The system features an isotropic resolution of ~50 μm and an acquisition time of four to eight minutes, using a 3-day optimized clearing protocol. Imaging of the brain autofluorescence in 3D reveals details of the neuroanatomy, while the use of fluorescent labels displays the vascular network and amyloid deposition in 5xFAD mice, an important model of Alzheimer’s disease (AD). Finally, the OPT images are compared with histological slices

Optical projection tomography for whole organ imaging

In the past twenty years, far-reaching studies of molecular and cellular processes have reached a milestone in their maturation, and the knowledge from these studies was ready to apply at higher organizational levels. At that time, rodent models were long established. However, methods were inappropriate to image a whole rodent organ, such as the mouse brain, which drove the emergence of a new range of imaging techniques, later gathered under the name mesoscopy. Mesoscopic techniques filled a gap between classical microscopy and medical imaging techniques, such as magnetic resonance imaging, and X-ray computed tomography. They allow the acquisition of centimeter-sized samples. In this thesis, we focus on one of these mesoscopic imaging techniques called optical projection tomography, or OPT, and its potential application to Alzheimer's disease (AD) research. We review the fundamentals of OPT and describe the filtered back-projection algorithm, which is the primary tomographic reconstruction method of this technique. We also go through the implementation of OPT for whole mouse brain imaging, including sample preparation. We show that OPT is suitable to image the whole brain anatomy based on endogenous fluorescence, and the whole neural vasculature as well as amyloid plaques (a hallmark of AD) with adequate fluorescent markers. Then, we dwell on the characterization of OPT instruments. We give some insights on the instrument point spread function and discuss the influence of the number of projections on the quality of the reconstructed image. Afterward, we illustrate the application of OPT to study amyloidosis progression in a preliminary cross-sectional study, where we have used supervised learning to quantify the amyloid plaque load. In this study, we show that OPT can be used to quantify amyloidosis in whole mouse brains and that comparison between individuals of different age can be performed. Imaging of a whole mouse brain is unquestionably necessary. At this scale though, it has some constraints. We present the limitations of OPT, and we share how we think they can be circumvented by combining this modality with another microscopy technique, namely structured illumination microscopy. We see that this other microscopy technique has the potential to produce high-resolution zooms in selected regions of interest based on a prior OPT acquisition. The results presented in this work have led to the duplication of our OPT instrument in Lund University, and we hope they will help to foster advances in OPT and broaden its range of application. We also hope that this work will contribute to making OPT more accessible and user-friendly