Diffuse optical tomography of the brain: effects of inaccurate baseline optical parameters and refinements using learned post-processing.

Diffuse optical tomography (DOT) uses near-infrared light to image spatially varying optical parameters in biological tissues. In functional brain imaging, DOT uses a perturbation model to estimate the changes in optical parameters, corresponding to changes in measured data due to brain activity. The perturbation model typically uses approximate baseline optical parameters of the different brain compartments, since the actual baseline optical parameters are unknown.

Multispectral imaging for characterizing autofluorescent tissues.

Selective Plane Illumination Microscopy (SPIM) has become an emerging technology since its first application for 3D in-vivo imaging of the development of a living organism. An extensive number of works have been published, improving both the speed of acquisition and the resolution of the systems. Furthermore, multispectral imaging allows the effective separation of overlapping signals associated with different fluorophores from the spectrum over the whole field-of-view of the analyzed sample.

Development and testing of a sedation protocol for Neocaridina davidi.

Neocaridina davidi, a small freshwater shrimp native to Asia, specifically China, Japan, Korea, and Vietnam, possesses remarkable resistance to poor water quality and offers various advantages over other invertebrate species to examine crucial issues in neuroscience and other related areas. These advantages include robustness, ease of maintenance, and transparency, making them useful for in vivo studies with optical imaging techniques. Despite its suitability for research purposes, particularly in the fields of imaging and fluorescent techniques, the lack of attention given to this species has resulted in the absence of a robust and replicable sedation protocol for immobilization and safe manipulation.

Single Plane Illumination Microscopy for Microfluidic Device Imaging.

Three-dimensional imaging of live processes at a cellular level is a challenging task. It requires high-speed acquisition capabilities, low phototoxicity, and low mechanical disturbances. Three-dimensional imaging in microfluidic devices poses additional challenges as a deep penetration of the light source is required, along with a stationary setting, so the flows are not perturbed.

Non-invasive visualization of amyloid-beta deposits in Alzheimer amyloidosis mice using magnetic resonance imaging and fluorescence molecular tomography.

Abnormal cerebral accumulation of amyloid-beta peptide (Aβ) is a major hallmark of Alzheimer's disease. Non-invasive monitoring of Aβ deposits enables assessing the disease burden in patients and animal models mimicking aspects of the human disease as well as evaluating the efficacy of Aβ-modulating therapies. Previous assessments of plaque load have been predominantly based on macroscopic fluorescence reflectance imaging (FRI) and confocal or two-photon microscopy using Aβ-specific imaging agents.

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease.

Three-dimensional (3D) optical imaging techniques can expand our knowledge about physiological and pathological processes that cannot be fully understood with 2D approaches. Standard diagnostic tests frequently are not sufficient to unequivocally determine the presence of a pathological condition. Whole-organ optical imaging requires tissue transparency, which can be achieved by using tissue clearing procedures enabling deeper image acquisition and therefore making possible the analysis of large-scale biological tissue samples.

Development of an Inverted Epifluorescence Microscope for Long-Term Monitoring of Bacteria in Multiplexed Microfluidic Devices.

Developing more efficient methods for antibiotic susceptibility testing is a pressing issue in novel drug development as bacterial resistance to antibiotics becomes increasingly common. Microfluidic devices have been demonstrated to be powerful platforms that allow researchers to perform multiplexed antibiotic testing. However, the level of multiplexing within microdevices is limited, evidencing the need of creating simple, low-cost and high-resolution imaging systems that can be integrated in antibiotic development pipelines.

Multimodal imaging combining time-domain near-infrared optical tomography and continuous-wave fluorescence molecular tomography.

Fluorescence molecular tomography (FMT) emerges as a powerful non-invasive imaging tool with the ability to resolve fluorescence signals from sources located deep in living tissues. Yet, the accuracy of FMT reconstruction depends on the deviation of the assumed optical properties from the actual values. In this work, we improved the accuracy of the initial optical properties required for FMT using a new-generation time-domain (TD) near-infrared optical tomography (NIROT) system, which effectively decouples scattering and absorption coefficients.

Smart Toolkit for Fluorescence Tomography: Simulation, Reconstruction, and Validation.

Objective: Fluorescence molecular tomography (FMT) can provide valuable molecular information by mapping the bio-distribution of fluorescent reporter molecules in the intact organism. Various prototype FMT systems have been introduced during the past decade. However, none of them has evolved as a standard tool for routine biomedical research.

Mutations in and genes cause congenital heart defects by tissue-specific perturbation of Wnt/β-catenin signaling.

Bcl9 and Pygopus (Pygo) are obligate Wnt/β-catenin cofactors in , yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, β-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the β-catenin-BCL9-Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators.

Demonstrating Improved Multiple Transport-Mean-Free-Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics.

Optical microscopy constitutes, one of the most fundamental paradigms for the understanding of complex biological mechanisms in the whole-organism and live-tissue context. Novel imaging techniques such as light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) combined with phase-retrieval algorithms (PRT) can produce highly resolved 3D images in multiple transport-mean-free-path scales. Our study aims to exemplify the microscopic capabilities of LSFM when imaging protein dynamics in Caenorhabditis elegans and the distribution of necrotic cells in cancer cell spheroids.

Optical projection tomography via phase retrieval algorithms.

We describe a computational method for accurate, quantitative tomographic reconstructions in Optical Projection Tomography, based on phase retrieval algorithms. Our method overcomes limitations imposed by light scattering in opaque tissue samples under the memory effect regime, as well as reduces artifacts due to mechanical movements, misalignments or vibrations. We make use of Gerchberg-Saxton algorithms, calculating first the autocorrelation of the object and then retrieving the associated phase under four numerically simulated measurement conditions.

Phase-Retrieved Tomography enables Mesoscopic imaging of Opaque Tumor Spheroids.

We present a new Phase-Retrieved Tomography (PRT) method to radically improve mesoscopic imaging at regimes beyond one transport mean-free-path and achieve high resolution, uniformly throughout the volume of opaque samples. The method exploits multi-view acquisition in a hybrid Selective Plane Illumination Microscope (SPIM) and Optical Projection Tomography (OPT) setup and a three-dimensional Gerchberg-Saxton phase-retrieval algorithm applied in 3D through the autocorrelation sinogram. We have successfully applied this innovative protocol to image optically dense 3D cell cultures in the form of tumor spheroids, highly versatile models to study cancer behavior and response to chemotherapy.

Looking inside the heart: a see-through view of the vascular tree.

The ability to acquire 3D images of the heart and its vasculature at cellular resolution facilitates a more detailed study of many heart diseases. Here, we describe a novel technique to image in 3D the heart vasculature by combining the CUBIC clearing protocol combined with administration of fluorescent-labeled lectin. The use of these techniques in combination with elective lane llumination icroscopy (SPIM) made it possible to obtain high resolution 3D images of the cardiac vascular tree.

Antigen Availability and DOCK2-Driven Motility Govern CD4 T Cell Interactions with Dendritic Cells In Vivo.

Parenchymal migration of naive CD4 T cells in lymph nodes (LNs) is mediated by the Rac activator DOCK2 and PI3Kγ and is widely assumed to facilitate efficient screening of dendritic cells (DCs) presenting peptide-MHCs (pMHCs). Yet how CD4 T cell motility, DC density, and pMHC levels interdependently regulate such interactions has not been comprehensively examined. Using intravital imaging of reactive LNs in DC-immunized mice, we show that pMHC levels determined the occurrence and timing of stable CD4 T cell-DC interactions.

Optimized CUBIC protocol for three-dimensional imaging of chicken embryos at single-cell resolution.

The CUBIC tissue-clearing protocol has been optimized to produce translucent immunostained whole chicken embryos and embryo brains. When combined with multispectral light-sheet microscopy, the validated protocol presented here provides a rapid, inexpensive and reliable method for acquiring accurate histological images that preserve three-dimensional structural relationships with single-cell resolution in whole early-stage chicken embryos and in the whole brains of late-stage embryos.

Fluorescence Diffusion in the Presence of Optically Clear Tissues in a Mouse Head Model.

Diffuse Optical Tomography commonly neglects or assumes as insignificant the presence of optically clear regions in biological tissues, estimating their contribution as a small perturbation to light transport. The inaccuracy introduced by this practice is examined in detail in the context of a complete, based on realistic geometry, virtual fluorescence Diffuse Optical Tomography experiment where a mouse head is imaged in the presence of cerebral spinal fluid. Despite the small thickness of such layer, we point out that an error is introduced when neglecting it from the model with possibly reduction in the accuracy of the reconstruction and localization of the fluorescence distribution within the brain.

pMHC affinity controls duration of CD8+ T cell-DC interactions and imprints timing of effector differentiation versus expansion.

During adaptive immune responses, CD8 T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity.

Stripe artifact elimination based on nonsubsampled contourlet transform for light sheet fluorescence microscopy.

Stripe artifacts, caused by high-absorption or high-scattering structures in the illumination light path, are a common drawback in both unidirectional and multidirectional light sheet fluorescence microscopy (LSFM), significantly deteriorating image quality. To circumvent this problem, we present an effective multidirectional stripe remover (MDSR) method based on nonsubsampled contourlet transform (NSCT), which can be used for both unidirectional and multidirectional LSFM. In MDSR, a fast Fourier transform (FFT) filter is designed in the NSCT domain to shrink the stripe components and eliminate the noise.

3D imaging in CUBIC-cleared mouse heart tissue: going deeper.

The ability to acquire high resolution 3D images of the heart enables to study heart diseases more in detail. In this work, the CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) clearing protocol was optimized for thick mouse heart sections to enhance the penetration depth of the confocal microscope lasers into the tissue. In addition, the optimized CUBIC clearing of the heart enhances antibody penetration into the tissue by a factor of five.

Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography.

Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities.

Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.

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.

Fluorescent Molecular Tomography for In Vivo Imaging of Mouse Atherosclerosis.

Optical imaging technologies such as fluorescence molecular tomography (FMT) are gaining great relevance in cardiovascular research. The main reason is the increased number of available fluorescent agents, especially those termed "activatable probes," which remain quenched under baseline conditions and are fluorescent when a specific enzymatic activity is present. A major characteristic of FMT is the possibility of obtaining quantitative data of fluorescence signal distribution in a noninvasive fashion and using nonionizing radiation, making FMT an invaluable tool for longitudinal studies with biomedical applications.