Correction for specimen movement and rotation errors for in-vivo Optical Projection Tomography.
ABSTRACT: The application of optical projection tomography to in-vivo experiments is limited by specimen movement during the acquisition. We present a set of mathematical correction methods applied to the acquired data stacks to correct for movement in both directions of the image plane. These methods have been applied to correct experimental data taken from in-vivo optical projection tomography experiments in Caenorhabditis elegans. Successful reconstructions for both fluorescence and white light (absorption) measurements are shown. Since no difference between movement of the animal and movement of the rotation axis is made, this approach at the same time removes artifacts due to mechanical drifts and errors in the assumed center of rotation.
Project description:Volumetric imaging allows global understanding of three-dimensional (3D) complex systems. Light-sheet fluorescence microscopy and optical projection tomography have been reported to image 3D volumes with high resolutions and at high speeds. Such methods, however, usually rely on fluorescent labels for chemical targeting, which could perturb the biological functionality in living systems. We demonstrate Bessel-beam-based stimulated Raman projection (SRP) microscopy and tomography for label-free volumetric chemical imaging. Our SRP microscope enables fast quantitation of chemicals in a 3D volume through a two-dimensional lateral scan. Furthermore, combining SRP and sample rotation, we demonstrate the SRP tomography that can reconstruct the 3D distribution of chemical compositions with optical spatial resolution at a higher speed than the Gaussian-beam-based stimulated Raman scattering sectioning imaging can. We explore the potential of our SRP technology by mapping polymer particles in 3D volumes and lipid droplets in adipose cells.
Project description:We describe the implementation of an OPT plate to perform optical projection tomography (OPT) on a commercial wide-field inverted microscope, using our open-source hardware and software. The OPT plate includes a tilt adjustment for alignment and a stepper motor for sample rotation as required by standard projection tomography. Depending on magnification requirements, three methods of performing OPT are detailed using this adaptor plate: a conventional direct OPT method requiring only the addition of a limiting aperture behind the objective lens; an external optical-relay method allowing conventional OPT to be performed at magnifications >4x; a remote focal scanning and region-of-interest method for improved spatial resolution OPT (up to ~1.6 ?m). All three methods use the microscope's existing incoherent light source (i.e. arc-lamp) and all of its inherent functionality is maintained for day-to-day use. OPT acquisitions are performed on in vivo zebrafish embryos to demonstrate the implementations' viability.
Project description:Gradient projection rotation (GPR) is an openly available and promising tool for factor and component rotation. We compare GPR toward the Varimax criterion in principal component analysis to the built-in Varimax procedure in SPSS. In a simulation study, we tested whether GPR-Varimax yielded multiple local solutions by creating population simple structure with a single optimum and with two optima, a global and a local one (double-optimum condition). The other conditions comprised the number of components (k = 3, 6, 9, and 12), the number of variables per component (m/k = 4, 6, and 8), the number of iterations per rotation (i = 25 and 250), and whether loadings were Kaiser normalized before rotation or not. GPR-Varimax was conducted with unrotated and multiple (q = 1, 10, 50, and 100) random start loadings. We found equal results for GPR-Varimax and SPSS-Varimax in most conditions. The few very small differences in favor of SPSS-Varimax were eliminated when Kaiser-normalized loadings and 250 iterations per rotation were used. Selecting the best solution out of multiple random starts in GPR-Varimax increased proximity to population components in the double-optimum condition with Kaiser normalized loadings, for which GPR-Varimax recovered population structure better than SPSS-Varimax. We also included an empirical example and found that GPR-Varimax and SPSS-Varimax yielded highly similar solutions for orthogonal simple structure in a real data set. We suggest that GPR-Varimax can be used as an alternative to Varimax rotation in SPSS. Users of GPR-Varimax should allow for at least 250 iterations, normalize loadings before rotation, and select the best solution from at least 10 random starts to ensure optimal results.
Project description:Optical imaging through biological samples is compromised by tissue scattering and currently various approaches aim to overcome this limitation. In this paper we demonstrate that an all optical technique, based on non-linear upconversion of infrared ultrashort laser pulses and on multiple view acquisition, allows the reduction of scattering effects in tomographic imaging. This technique, namely Time-Gated Optical Projection Tomography (TGOPT), is used to reconstruct three dimensionally the internal structure of adult zebrafish without staining or clearing agents. This method extends the use of Optical Projection Tomography to optically diffusive samples yielding reconstructions with reduced artifacts, increased contrast and improved resolution with respect to those obtained with non-gated techniques. The paper shows that TGOPT is particularly suited for imaging the skeletal system and nervous structures of adult zebrafish.
Project description:An error in tomographic reconstruction parameters can result considerable artifacts in the reconstructed image, particularly in micro-computed tomography and nano-computed tomography. This study involved designing an automatic method for efficiently correcting errors resulting from incorrectly determined rotational axes and projection angles. In this method, errors are corrected by minimizing the "total variation" of a reconstructed image, and minimization is accomplished by using the gradient descent method. Compared with two previous methods, the proposed method achieved the best reconstruction results.
Project description:Shadowgraphic projection artifacts from superficial vasculature interfere with the visualization of deeper vascular networks in optical coherence tomography angiography (OCT-A). We developed a novel algorithm to remove this artifact by resolving the ambiguity between in situ and projected flow signals. The algorithm identifies voxels with in situ flow as those where intensity-normalized decorrelation values are higher than all shallower voxels in the same axial scan line. This "projection-resolved" (PR) algorithm effectively suppressed the projection artifact on both en face and cross-sectional angiograms and enhanced depth resolution of vascular networks. In the human macula, the enhanced angiograms show three distinct vascular plexuses in the inner retina and no vessels in the outer retina. We demonstrate that PR OCT-A cleanly removes flow projection from the normally avascular outer retinal slab while preserving the density and continuity of the intermediate and deep retinal capillary plexuses.
Project description:We describe a versatile optical projection tomography system for rapid three-dimensional imaging of microscopic specimens in vivo. Our tomographic setup eliminates the in xy and z strongly asymmetric resolution, resulting from optical sectioning in conventional confocal microscopy. It allows for robust, high resolution fluorescence as well as absorption imaging of live transparent invertebrate animals such as C. elegans. This system offers considerable advantages over currently available methods when imaging dynamic developmental processes and animal ageing; it permits monitoring of spatio-temporal gene expression and anatomical alterations with single-cell resolution, it utilizes both fluorescence and absorption as a source of contrast, and is easily adaptable for a range of small model organisms.
Project description:Pulsars are rotating neutron stars emitting lighthouse-like beams. Owing to their unique properties, pulsars are a unique astrophysical tool to test general relativity, inform on matter in extreme conditions, and probe galactic magnetic fields. Understanding pulsar physics and emission mechanisms is critical to these applications. Here we show that mechanical-optical rotation in the pulsar magnetosphere affects polarisation in a way which is indiscernible from Faraday rotation in the interstellar medium for typical GHz observations frequency, but which can be distinguished in the sub-GHz band. Besides being essential to correct for possible systematic errors in interstellar magnetic field estimates, this result offers a unique means to determine the rotation direction of pulsars, providing additional constraints on magnetospheric physics. With the ongoing development of sub-GHz observation capabilities, our finding promises discoveries, such as the spatial distribution of pulsars rotation directions, which could exhibit potentially interesting, but presently invisible, correlations or features.
Project description:We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions.
Project description:X-ray 3D tomographic techniques are powerful tools for investigating the morphology and internal structures of specimens. A common strategy for obtaining 3D tomography is to capture a series of 2D projections from different X-ray illumination angles of specimens mounted on a finely calibrated rotational stage. However, the reconstruction quality of 3D tomography relies on the precision and stability of the rotational stage, i.e. the accurate alignment of the 2D projections in the correct three-dimensional positions. This is a crucial problem for nano-tomographic techniques due to the non-negligible mechanical imperfection of the rotational stages at the nanometer level which significantly degrades the spatial resolution of reconstructed 3-D tomography. Even when using an X-ray micro-CT with a highly stabilized rotational stage, thermal effects caused by the CT system are not negligible and may cause sample drift. Here, we propose a markerless image auto-alignment algorithm based on an iterative method. This algorithm reduces the traditional projection matching method into two simplified matching problems and it is much faster and more reliable than traditional methods. This algorithm can greatly decrease hardware requirements for both nano-tomography and data processing and can be easily applied to other tomographic techniques, such as X-ray micro-CT and electron tomography.