Novel fluorescent tools and techniques for 3D imaging of the cleared brain

Background: To better understand the complexity of the brain and how it becomes impaired under different pathological states, a considerably large number of brains would be needed for imaging to generate highly detailed maps in 3D. Chemical probes can offer a readily scalable labelling method that is robust, easy to use with the quick operation, and feasible for human tissue where genetic viral and toxin tracers are inappropriate. The drawbacks of immunostaining methods have spurred interest in developing alternative strategies to visualize the optically transparent brain, especially from fixed archived samples or human autopsies that are not optimally fixed. Purpose: We envision AIE-based probes and techniques as robust tools when paired with clearing methods for visualizing the human brain. This thesis aims to develop alternative strategies to tissue labelling using novel AIE-based fluorescent chemical probes and methods that offer easy operation, high brightness, photostability and contrast suitable for 3D visualization of neurons and nerve fibers in mouse brains. Paper I: The novel water-soluble silver-ion sensitive AIE probe TPE-4TA achieved by tetrazole-Ag+ coordination, allowed for the development of a new fluorescent silver (silver-AIE) method to visualize separated proteins following sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Compared with conventional silver nitrate stains, silver-AIE not only offers sensitive fluorogenic detection of proteins, but it is quantifiable, easy to use, has a broad linear dynamic range and a great contrast which rivals the popular commercial stain, SYPRO Ruby. Study II describes how to troubleshoot the fluorescent silver gel stain, alternative steps for rapid staining and techniques to carry out the procedure correctly to avoid suboptimal results. Paper II: We report a novel fluorescent silver stain for fixed mouse brain tissue compatible with multiplexed immunofluorescence imaging in paraffin sections. The Ag+-specific aggregation-induced emission (AIE) strategy outperforms the chromogenic detection employed by many conventional silver staining protocols to visualize neurites and fiber tracts in paraffin sections or passive Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / in situ-hybridization-compatible Tissue hYdrogel (CLARITY) -cleared tissue. This enables imaging using standard fluorescent widefield or optical sectioning microscopies. Not only does our method uses less hazardous reagents, but the highly sensitive TPE-4TA also uses silver nitrate concentrations up to two million-fold lower than the standard Yamamoto-Hirano’s modification of the Bielschowsky stain. Paper III: Development of the novel near-infrared AIE fluorescent probe PM-ML with D-π-A (donor-pi-acceptor) structure for the selective staining of myelinated fibers in the teased sciatic nerves, mouse brain cryosections and ClearT-cleared mouse brain tissue for 3D fluorescent imaging. We envision PM-ML as a potential tool for studying demyelination and evaluated its selectivity, photostability and signal-to-background (SBR) ratio which outperformed common commercial fluorescent myelin staining dyes

Raman Imaging in Cell Membranes, Lipid-Rich Organelles, and Lipid Bilayers

Raman-based optical imaging is a promising analytical tool for noninvasive, label-free chemical imaging of lipid bilayers and cellular membranes. Imaging using spontaneous Raman scattering suffers from a low intensity that hinders its use in some cellular applications. However, developments in coherent Raman imaging, surface-enhanced Raman imaging, and tip-enhanced Raman imaging have enabled video-rate imaging, excellent detection limits, and nanometer spatial resolution, respectively. After a brief introduction to these commonly used Raman imaging techniques for cell membrane studies, this review discusses selected applications of these modalities for chemical imaging of membrane proteins and lipids. Finally, recent developments in chemical tags for Raman imaging and their applications in the analysis of selected cell membrane components are summarized. Ongoing developments toward improving the temporal and spatial resolution of Raman imaging and small-molecule tags with strong Raman scattering cross sections continue to expand the utility of Raman imaging for diverse cell membrane studies

Novel imaging tools for investigating the role of immune signalling in the brain

Abstract not availableJonathan Henry W. Jacobsen, Lindsay M. Parker, Arun V. Everest-Dass, Erik P. Schartner, Georgios Tsiminis, Vasiliki Staikopoulos, Mark R. Hutchinson, Sanam Mustaf

Contribution of Intravital Neuroimaging to Study Animal Models of Multiple Sclerosis

Multiple sclerosis (MS) is a complex and long-lasting neurodegenerative disease of the central nervous system (CNS), characterized by the loss of myelin within the white matter and cortical fbers, axonopathy, and infammatory responses leading to consequent sensory-motor and cognitive defcits of patients. While complete resolution of the disease is not yet a reality, partial tissue repair has been observed in patients which ofers hope for therapeutic strategies. To address the molecular and cellular events of the pathomechanisms, a variety of animal models have been developed to investigate distinct aspects of MS disease. Recent advances of multiscale intravital imaging facilitated the direct in vivo analysis of MS in the animal models with perspective of clinical transfer to patients. This review gives an overview of MS animal models, focusing on the current imaging modalities at the microscopic and macroscopic levels and emphasizing the importance of multimodal approaches to improve our understanding of the disease and minimize the use of animals

Single Cell Optical Imaging and Spectroscopy

In his 1665 treatise, Micrographia, Robert Hooke described the many observations he had made using a microscope, including compartment-like structures in cork samples that he termed “cells

Structural and molecular interrogation of intact biological systems

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease

Prototype Nerve-Specific Near-Infrared Fluorophores

Nerve preservation is an important issue during most surgery because accidental transection or injury results in significant morbidity, including numbness, pain, weakness, or paralysis. Currently, nerves are still identified only by gross appearance and anatomical location during surgery, without intraoperative image guidance. Near-infrared (NIR) fluorescent light, in the wavelength range of 650-900 nm, has the potential to provide high-resolution, high-sensitivity, and real-time avoidance of nerve damage, but only if nerve-specific NIR fluorophores can be developed. In this study, we evaluated a series of Oxazine derivatives to highlight various peripheral nerve structures in small and large animals. Among the targeted fluorophores, Oxazine 4 has peak emission near into the NIR, which provided nerve-targeted signal in the brachial plexus and sciatic nerve for up to 12 h after a single intravenous injection. In addition, recurrent laryngeal nerves were successfully identified and highlighted in real time in swine, which could be preserved during the course of thyroid resection. Although optical properties of these agents are not yet optimal, chemical structure analysis provides a basis for improving these prototype nerve-specific NIR fluorophores even further

Label-free polarisation-resolved optical imaging of biological samples

Myelin is a biological structure present in all the gnathostomata. It is a highly- ordered structure, in which many lipid-enriched and densely compacted phospho- lipid bilayers are rolled up in a cylindrical symmetry around a subgroup of axons. The myelin sheath increases the electrical transverse resistance and reduces the ca- pacitance making the saltatory conduction of action potentials possible and therefore leading to a critically improved performance in terms of nervous impulse conduc- tion speeds and travel lengths. Myelin pathologies are a large group of neurological diseases that often result in death or disability. In order to investigate the main causes of myelin damage and its temporal progression many microscopy techniques are currently employed, such as electron microscopy and histochemistry or fluorescence imaging. However, electron microscopy and histochemistry imaging require complex sample prepara- tion and are therefore unsuitable for live imaging. Fluorescence imaging, as well as its derivatives, confocal and two-photon imaging, relies on the use of fluorescent probes to generate the image contrast but fluorophores and the associated sample processing, when applicable to living specimens, might nonetheless modify the bi- ological properties of the target molecule and perturb the whole biological process under investigation; moreover, fluorescent immunostaining still requires the fixation of the cells. Coherent anti-Stokes Raman Scattering (CARS) microscopy, on the other hand, is a powerful and innovative imaging modality that permits the study of liv- ing specimens with excellent chemical contrast and spatial resolution and without the confounding and often tedious use of chemical or biological probes. This is par- ticularly important in clinical settings, where the patient biopsy must be explanted in order to stain the tissue. In these cases it may be useful to resort to a set of label-free microscopy techniques. Among these, CARS microscopy is an ideal tool to investigate myelin morphology and structure, thanks to its abundance of CH2 bonds. The chemical selectivity of CARS microscopy is based on the properties of the contrast-generating CARS process. This is a nonlinear process in which the energy difference of a pair of incoming photons (\u201cpump\u201d and \u201cStokes\u201d) matches the energy of one of the vibrational modes of a molecular bond of interest. This vibrational excited state is coherently probed by a third photon (\u201cprobe\u201d) and anti-Stokes radi- ation is emitted. In this thesis I shall discuss the development of a multimodal nonlinear opti- cal setup implementing CARS microscopy together with general Four-Wave Mix- ing, Second Harmonic Generation and Sum Frequency Generation microscopies. Moreover, I shall present a novel polarisation-resolved imaging scheme based on the CARS process, which I named Rotating-Polarisation (RP) CARS microscopy and implemented in the same setup. This technique, using a freely-rotating pump-and- probe-beam-polarisation plane, exploits the CARS polarisation-dependent rules in order to probe the degree of anisotropy of the chemical-bond spatial orientations inside the excitation point-spread function and their average orientation, allowing at the same time the acquisition of large-field-of-view images with minimal polarisa- tion distortions. I shall show that RP-CARS is an ideal tool to investigate the highly- ordered structure of myelinated nervous fibres thanks to the strong anisotropy and symmetry properties of the myelin molecular architecture. I shall also demonstrate that this technique allows the fully label-free assessment of the myelin health status both in a chemical model of myelin damage (lysophos- phatidylcholine-exposed mouse nerve) and in a genetic model (twitcher mouse) of a human leukodystrophy (Krabbe disease) while giving useful insights into the pathogenic mechanisms underlying the demyelination process. I shall also discuss the promises of this technique for applications in optical tractography of the nerve fibres in the central nervous system and for the investigation of the effects of ageing on the peripheral nervous system. Moreover, I shall demonstrate by means of numer- ical simulations that RP-CARS microscopy is extremely robust against the presence of scatterers (such as lipid vesicles, commonly found in the peripheral nervous sys- tem). Finally, I shall discuss the results of the exploitation of my multimodal setup in a different area at the boundary of biophysics and nanomedicine: the observation of the internalization of different kinds of nanoparticles (boron-nitride nanotubes, barium-titanate nanoparticles and barium-titanate-core/gold-shell nanoparticles) by cultured cells and the demonstration of the nanopatterned nature of a structure built with two-photon lithography

Photoacoustic brain imaging: from microscopic to macroscopic scales

Human brain mapping has become one of the most exciting contemporary research areas, with major breakthroughs expected in the coming decades. Modern brain imaging techniques have allowed neuroscientists to gather a wealth of anatomic and functional information about the brain. Among these techniques, by virtue of its rich optical absorption contrast, high spatial and temporal resolutions, and deep penetration, photoacoustic tomography (PAT) has attracted more and more attention, and is playing an increasingly important role in brain studies. In particular, PAT complements other brain imaging modalities by providing high-resolution functional and metabolic imaging. More importantly, PAT’s unique scalability enables scrutinizing the brain at both microscopic and macroscopic scales, using the same imaging contrast. In this review, we present the state-of-the-art PAT techniques for brain imaging, summarize representative neuroscience applications, outline the technical challenges in translating PAT to human brain imaging, and envision potential technological deliverables