Deposits of amyloid-β (Aβ) in the brains of rodents can be analysed by invasive intravital microscopy on a submillimetre scale, or via whole-brain images from modalities lacking the resolution or molecular specificity to accurately characterize Aβ pathologies. Here we show that large-field multifocal illumination fluorescence microscopy and panoramic volumetric multispectral optoacoustic tomography can be combined to longitudinally assess Aβ deposits in transgenic mouse models of Alzheimer's disease. We used fluorescent Aβ-targeted probes (the luminescent conjugated oligothiophene HS-169 and the oxazine-derivative AOI987) to transcranially detect Aβ deposits in the cortex of APP/PS1 and arcAβ mice with single-plaque resolution (8 μm) and across the whole brain (including the hippocampus and the thalamus, which are inaccessible by conventional intravital microscopy) at sub-150 μm resolutions. Two-photon microscopy, light-sheet microscopy and immunohistochemistry of brain-tissue sections confirmed the specificity and regional distributions of the deposits. High-resolution multiscale optical and optoacoustic imaging of Aβ deposits across the entire brain in rodents thus facilitates the in vivo study of Aβ accumulation by brain region and by animal age and strain.

The abnormal deposition of fibrillar beta-amyloid (Aβ) deposits in the brain is one of the major histopathological hallmarks of Alzheimer’s disease (AD). Here, we characterized curcumin-derivative CRANAD-2 for multi-spectral optoacoustic tomography and fluorescence imaging of brain Aβ deposits in the arcAβ mouse model of AD cerebral amyloidosis. CRANAD-2 showed a specific and quantitative detection of Aβ fibrils in vitro, even in complex mixtures, and it is capable of distinguishing between monomeric and fibrillar forms of Aβ. In vivo epi-fluorescence microscopy and optoacoustic tomography after intravenous CRANAD-2 administration demonstrated higher cortical retention in arcAβ compared to non-transgenic littermate mice. Immunohistochemistry showed co-localization of CRANAD-2 and Aβ deposits in arcAβ mouse brain sections, thus verifying the specificity of the probe. In conclusion, we demonstrate suitability of CRANAD-2 for optical detection of Aβ deposits in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting Aβ deposits.

Liquid–liquid phase separation of polymer and protein solutions is central in many areas of biology and material sciences. Here, an experimental and theoretical framework is provided to investigate the thermodynamics and kinetics of liquid–liquid phase separation in volumes comparable to cells. The strategy leverages droplet microfluidics to accurately measure the volume of the dense phase generated by liquid–liquid phase separation of solutions confined in micro‐sized compartments. It is shown that the measurement of the volume fraction of the dense phase at different temperatures allows the evaluation of the binodal lines that determine the coexistence region of the two phases in the temperature‐concentration phase diagram. By applying a thermodynamic model of phase separation in finite volumes, it is further shown that the platform can predict and validate kinetic barriers associated with the formation of a dense droplet in a parent dilute phase, therefore connecting thermodynamics and kinetics of liquid–liquid phase separation.

The size distribution and the rheological properties of dispersions of biological colloids are relevant quality attributes for a variety of industrial applications, including pharmaceutical, food, and cosmetic products. For instance, the biophysical properties of monoclonal antibodies and therapeutic proteins, which represent an important class of drugs in the pharmaceutical market, are important for their safety and efficacy. In this work, we apply a microfluidic diffusion platform to analyze protein sizes and interactions in high-concentration antibody solutions directly in the liquid state with minimal perturbation of the sample. We show that this method provides size distributions in a size range scaling from a few angstroms to hundreds of nanometers. The detection sensitivity of the technique is independent of the particle size, and the method provides number-average distributions, enabling the simultaneous detection of both monomeric species and soluble aggregates. We further show that the same platform can be applied to measure viscosity–scaling effects in crowded environments by probing the Brownian motion of several tracers with different sizes. Such tracers experience a shift from the microviscosity to the macroviscosity of the sample at a critical probe size that is equal to the characteristic dimension of the main components of the dispersions. The technique simultaneously provides quantitative measurement of the microrheological properties and the macroviscosity of the sample, as well as information on the characteristic size of the components of the solution. Overall, these methods represent attractive tools in the context of the analysis of sizes and interactions of proteins in both diluted and high-concentration solutions during development, manufacturing, and formulation.

The abnormal deposition of fibrillar beta-amyloid (Ab) deposits in the brain is one of the major histopathological hallmarks of Alzheimers disease (AD). Here we characterize curcumin-derivative CRANAD-2 for multi-spectral optoacoustic tomography (MSOT) and fluorescence imaging of brain Ab; deposits in the arcAb; mouse model of AD cerebral amyloidosis. CRANAD-2 shows a specific and quantitative detection of Ab; fibrils in vitro, even in complex mixtures, and it is capable to distinguish between monomeric and fibrillar forms of Ab;. In vivo epifluorescence and MSOT after intravenous CRANAD-2 administration demonstrated higher retention in arcAb; compared to non-transgenic littermate mice. Immunohistochemistry showed co-localization of CRANAD-2 and Ab; deposits in arcAb; mouse brain sections, thus verifying the specificity of the probe. In conclusion, we demonstrate suitability of CRANAD-2 for fluorescence- and MSOT-based detection of Ab; deposits in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting Ab; deposits.

The abnormal deposition of beta-amyloid proteins in the brain is one of the major histopathological hallmarks of Alzheimer's disease. Currently available intravital microscopy techniques for highresolution plaque visualization commonly involve highly invasive procedures and are limited to a small field-of-view within the rodent brain. Here, we report the transcranial detection of amyloidbeta deposits at the whole brain scale with 20 m resolution in APP/PS1 and arcA mouse models of Alzheimer's disease amyloidosis using a large-field multifocal (LMI) fluorescence microscopy technique. Highly sensitive and specific detection of amyloid-beta deposits at a single plaque level in APP/PS1 and arcA mice was facilitated using luminescent conjugated oligothiophene HS-169.Immunohistochemical staining with HS-169, anti-A antibody 6E10, and conformation antibodies OC (fibrillar) of brain tissue sections further showed that HS-169 resolved compact parenchymal and vessel-associated amyloid deposits. The novel imaging platform offers new prospects for in vivo studies into Alzheimer's disease mechanisms in animal models as well as longitudinal monitoring of therapeutic responses at a single plaque level.

Background The abnormal deposition of beta‐amyloid proteins in the brain is one of the major histopathological hallmarks of Alzheimer’s disease. Currently available intravital microscopy techniques for high‐resolution plaque visualization commonly involve highly invasive procedures and are limited to small field‐of‐views within the rodent brain. Method We devised a large‐field multi‐focal illumination (LMI) fluorescence microscopy method that provides a 20 × 20 mm field‐of‐view and an axial resolution of ∼15 mm. In vivo and ex vivo transcranial LMI fluorescence microscopy and wide‐field fluorescence microscopy for amyloid deposits were performed in APP/PS1 and arcAb mouse models of Alzheimer’s disease amyloidosis using luminescent conjugated oligothiophene probe HS‐169. Immunohistochemical staining with HS‐169, anti‐Ab antibody 6E10, and conformation antibodies OC (fibrillar) of brain tissue sections Result LMI fluorescence microscopy detected amyloid‐beta deposits at a single plaque level in APP/PS1 and arcAb mice with high sensitivity and specificity. The contrast‐to‐noise ratio was approximately 24 times higher in LMI fluorescence microscopy of amyloid deposits compared to wide‐field fluorescence microscopy. Immunohistochemical staining showed HS‐169 co‐localized with 6E10 and OC stained compact parenchymal and vessel‐associated amyloid deposits. Conclusion The novel LMI in vivo amyloid imaging platform offers new prospects for studies into Alzheimer’s disease mechanisms in animal models as well as longitudinal monitoring of therapeutic responses at a single plaque level.

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