Systematic Evaluation of Chemically Distinct Tissue Optical Clearing Techniques in Murine Lymph Nodes.

The tissue optical clearing (TOC) technique could significantly improve the biomedical optical imaging depth, but most current investigations are limited to in vitro studies. For in vivo applications, the TOC method must provide a rapid treatment process, sufficient transparency, and safety for animals, which makes it more difficult. Recently developed innovative optical clearing methods for in vivo use show great potential for enhancing the contrast and resolution of laser speckle contrast imaging (LSCI) for blood flow monitoring. This paper gives an overview of recent progress in the use of TOC for vascular visualization with LSCI. First, the principle of TOC-induced improvement of LSCI and a quantitative analysis method for evaluating the improvement are described briefly. Second, the paper introduces transparent windows, including various skin windows and a cranial window, that permit LSCI to monitor dermal or cortical blood flow, respectively, with high resolution and contrast. Third, preliminary investigations of the safety of TOC demonstrate that the transparent skin window is switchable, which enables LSCI to repeatedly image blood flow. However, research on in vivo TOC is currently less advanced than that on in vitro TOC. Future work should focus on developing a highly effective, safe method and extending its applications.

Over the recent years, fluorescence microscopy has seen substantial progress, transitioning from primarily qualitative analysis to a profoundly quantitative methodology, yet limitations in imaging depth and data quality persist due to tissue opacity. Tissue optical clearing (TOC) methodologies were implemented to overcome this hurdle, enabling the imaging and quantitative analysis of large tissue samples such as entire murine organs. Further improvements, including expansion microscopy merge TOC benefits with improved imaging resolution, converting cells and tissues into tissue‒gel hybrids. Yet, its intricate protocols lead to a remarkable, ∼10‒20× tissue volume expansion, making the acquisition of even thin sections exceedingly challenging in terms of time, labor, and data handling. Here, we present a novel solution, Improved Segmentation by gEntle Expansion (ISEE), for both TOC and gentle (1.6‒1.7×) tissue expansion for significantly improved precision of fluorescent signal segmentation, while maintaining tolerable increase during data acquisition in everyday laboratory practice. When applied to thick ∼40‒200 µm tissue slices, ISEE renders them transparent and ultimately expanded within minutes, in a simple, one‐step process compatible with immunohistochemistry as well as with all major murine organs.

Tissue optical clearing technique provides a prospective solution for the application of advanced optical methods in life sciences. This chapter firstly gives a brief introduction to mechanisms of tissue optical clearing techniques, from the physical mechanism to chemical mechanism, which is the most important foundation to develop tissue optical clearing methods. During the past years, in vitro and in vivo tissue optical clearing methods were developed. In vitro tissue optical clearing techniques, including the solvent-based clearing methods and the hydrophilic reagents-based clearing methods, combined with labeling technique and advanced microscopy, can be applied to image 3D microstructure of tissue blocks or whole organs such as brain and spinal cord with high resolution. In vivo skin or skull optical clearing, promise various optical imaging techniques to detect cutaneous or cortical cell and vascular structure and function without surgical window.

The term "tissue optical clearing" (TOC) came into use at the end of the 20th century and is associated with the development of methods for controlling tissue scattering properties using the refractive index matching effect [...].

Three-Dimensional Cross-Sectional Light-Sheet Microscopy Imaging of the Inflamed Mouse Gut

Light Sheet Microscopy Optimized for Depth Penetration to Study Embryogenesis

To understand dynamic developmental processes, living tissues have to be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image, at cellular resolution, a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, to track cellular nuclei and to identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics.

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.

Animal development can be described as a complex, three- dimensional cellular system that changes dramatically across time as a consequence of cell proliferation, differentiation and movement. Using Drosophila embryogenesis as a model we are developing molecular, imaging and image analysis techniques to record an entire developmental system at cellular resolution. We image Drosophila embryos expressing fluorescent markers in toto using single plane illumination microscopy (SPIM). SPIM offers the unique ability to image large living biological specimens in their entirety by acquiring image stacks from multiple angles while also providing high temporal resolution necessary for following dynamic developmental processes. We have developed an image analysis pipeline that efficiently processes long-term time-lapse multi-view SPIM data by aligning the different angles with high precision for a single time point and propagating the alignment parameters throughout the time series. The registered views are fused using an approach that evaluates the image content in each view.

This review delves into the rapidly evolving field of deep tissue imaging at cellular resolution, reviewing popular tissue clearing and staining methods in combination with light-sheet fluorescence microscopy (LSFM) including quantification and three-dimensional visualization tools, the field of applications and perspective, particularly with the focus on preclinical cancer research and drug development. The LSFM technique presented here allows an extremely fast optical sectioning for three-dimensional reconstruction of centimeter-sized tissue samples at cellular resolution. However, optical clearing methods are required to receive optical transparent tissue. Application of either tissue autofluorescence, in vivo fluorescence labeling, endogenous fluorescence or ex vivo whole-mount immunolabeling enables three-dimensional in situ visualization of morphological and functional features of unsectioned whole-mount tissue samples. This powerful and innovative imaging technique opens up a new dimension of tissue analysis providing detailed and comprehensive insights into biology. It enables the investigation of normal and pathological tissue features and disease progression and allows precise monitoring of potential therapeutic interventions in intact biological tissue on a cellular level.

The physical characteristics of obtainable diffraction-limited light sheets (thickness ∼µm) are keys to the determination of achievable imaging performance of light sheet fluorescence microscopy (LSFM) imaging modality. The beam shape and its characteristics are solely defined by the optical characteristics of the illumination arm, i.e.,by the optical characteristics of the optical components in the illumination arm and the beam profile of the incident optical beam. Typically, the illumination arm in LSFM is constituted by a cylindrical lens and a diffraction-limited spherical (converging) lens. The existing theoretical or analytical models are limited only to (i)an optical illumination arm with a single cylindrical lens (without a spherical lens) and (ii)a planar incident optical beam instead of the practically more relevant beam, for example, Gaussian. We report a complete and unique angular-spectrum-based theoretical formulation of beam-shaping, i.e.,combining cylindrical and spherical lenses, for LSFM that holds true for Gaussian as well as planar beams. Validation studies, both experiments and numerical simulation, were conducted. Results demonstrate that our model enables us to estimate the performance indices with better accuracy [spatial (axial) resolution (∼2.18%), imaging depth or admissible sample size/thickness (∼1.30%), field of views (FOVs) (∼39.15%), signal contrast ratio (CR) (∼8.65%), and SNR (∼2.47%)]. This report will be of significant impact on imaging (in general) and LSFM (specifically) and its technological advances.

<p>The light-sheet fluorescence microscopy is an excellent tool for the investigation of large three dimensional microscopy samples at the cellular level, however, the ability to resolve features is strongly affected by the presence of scattering and aberrations. These effects are two fold in light-sheet microscopy, as the illumination path providing the optical sectioning and the fluorescence detection path are both affected by the aberrations in different ways. To overcome these difficulties, we have developed hybrid adaptive optical and computational microscopy techniques to remove the effect of the aberrations in both the excitation and the fluorescence paths of these microscopes.</p>

Pulmonary fibrosis, characterized by excessive collagen deposition in the lungs, comprises a key and debilitating component of chronic lung diseases. Methods are lacking for the direct visualization of fibrillar collagen throughout the whole murine lung, a capability that would aid the understanding of lung fibrosis. We combined an optimized organ-level optical clearing (OC) approach with large-scale, label-free multiphoton microscopy (MPM) and second harmonic generation microscopy (SHGM) to reveal the complete network of fibrillar collagen in whole murine lungs. An innate inflammation-driven model based on repetitive poly(I:C) challenge was evaluated. Following OC, mosaic MPM/SHGM imaging with 3D reconstruction and whole organ quantitative analysis revealed significant differences in collagen deposition between PBS and poly(I:C) treated lungs. Airway specific analysis in whole lung acquisitions revealed significant sub-epithelial fibrosis evident throughout the proximal conductive and distal airways with higher collagen deposition in the poly(I:C) group vs PBS group. This study establishes a new, powerful approach based on OC and MPM/SHGM imaging for 3D analysis of lung fibrosis with macroscopic views of lung pathology based on microscopy and providing a new way to analyze the whole lung while avoiding regional sampling bias.

Cochlear implant imaging in the mouse and guinea pig using light-sheet microscopy

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.