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.
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The main data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper. The raw datasets generated during the imaging studies are too large to be publicly shared, yet they are available for research purposes from the corresponding authors on reasonable request. The light-sheet microscopy dataset and GitHub code for registration and quantification with the Allen brain atlas are available at github.com/alecrimi/arcAb_mouse and figshare.com/articles/arcAB_Mouse/11473707.
https://github.com/mesoSPIM/mesoSPIM-control (Image acquisition software for mesoSPIM light-sheet microscope); https://github.com/alecrimi/arcAb_mouse (regional quantification of amyloid plaque load for lightsheet microscope data). The custom MATLAB code used to collect and process the imaging data is available for research purposes from the corresponding author on reasonable request.
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D.R. acknowledges funding from the European Research Council Consolidator Grant (ERC-2015-CoG-682379), the US National Institutes of Health (UF1-NS107680) and the Swiss National Science Foundation (310030_192757). K.P.R.N. acknowledges funding from the Swedish Research Council (2016–00748). A.A. acknowledges funding from a Nomis Distinguished Scientist Award. J.K. received funding from the Swiss National Science Foundation (320030_179277), in the framework of ERA-NET NEURON (32NE30_173678/1), the Synapsis foundation and the Vontobel foundation. R.N. received funding from the Synapsis foundation career development award (2017 CDA-03), UZH Innovation (MEDEF20-021), Helmut Horten Stiftung, Vontobel Stiftung and Jubiläumsstiftung von Swiss Life. Z.C. acknowledges funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 746430 – MSIOAM. We thank L. Mu (Institute of Pharmaceutical Sciences, ETH Zurich); J. Hehl, J. Kusch-Wieser and G. Bodizs (Scientific Center for Optical and Electron Microscopy (ScopeM) of ETH Zurich); L. Kulic, D. Schuppli and P. Ravikumar (Institute for Regenerative Medicine, University of Zurich) for technical support; M. Rudin (Institute for Biomedical Engineering, University of Zurich and ETH Zurich) for providing the AOI987 contrast agent; and M. Reiss and M. Rouault (Institute for Biomedical Engineering, University of Zurich and ETH Zurich) for assistance with the animal experiments.
The authors declare no competing interests.
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Extended Data Fig. 1 Characterization of the LMI fluorescence microscope for transcranial whole-cortex imaging at single plaque resolution.
a, LMI image of fluorescent beads (1-5 μm-diameter) on a microscope slide; Repetition n = 3. b, Comparison of magnified views of wide-field (WF) and LMI images in the red boxed regions together with their signal profiles along the corresponding green lines. The Gaussian fitted full-width-at-half-maximum (FWHM) of the bead in region #1 is 18.2 μm and 8.3 μm for the WF and LMI images, respectively. The corresponding values for region #2 are 16.4 μm (WF) and 7.1 μm (LMI); For region #3: 18.9 μm (WF) and 7.4 μm (LMI); For region #4: 27.0 μm (WF) and 11.8 μm (LMI). An averaged FWHM of 20 different beads across the field-of-view (FOV) was calculated as 7.7 ± 1.4 μm (mean ± SD). c–e, The horizontal and vertical intensity profiles of one of the optical foci in the excitation illumination pattern measured with a beam profiler (SP628, Ophir Optronics). The signal intensity (represented as mean ± SD) was normalized and fitted by Gaussian function. The spot sizes along the horizontal and vertical directions were measured at 14 μm and 11 μm, respectively; Repetition n = 3. f, Curve of the spot size along the light path (z-axis). The depth-of-focus for excitation was larger than 1 mm within a spot size of 20 μm. LMI - large-field multifocal illumination. g–i, Dynamic contrast-enhanced LMI fluorescence microscopy reveals different probe dynamics in cerebral and calvarian blood vessels. To showcase the performance of the LMI fluorescence microscopy method, time-lapse imaging of the bolus passage after intravenous tail-vein injections of Cy5.5 solution was performed. (g) Cranial and intracranial vessel network shown in maximum intensity projection (MIP) over multiple image frames; (h) Analysis of perfusion dynamics (time to peak post dye injection) allows to discriminate between cerebral (fast time to peak) and calvarian (slower time to peak) vessels. As expected, the LMI approach exhibits excellent contrast and spatial resolution through the intact skull while also accurately resolving the perfusion process in the deeply embedded cerebral microvasculature. Major cerebral vessels such as anterior cerebral artery (ACA) and middle cerebral artery (MCA) were pronounced at an early stage followed by venous vessels, such as inferior cerebral vein (ICV) and superior sagittal sinus (SSS). (i) LMI image acquired at 30 min post Cy5.5 fluorescent dye injection, showing the probe retention in the suture areas. LMI - large-field multifocal illumination.
a, Spectrofluorometric measurements show emission peak of HS-169 in the concentration range varying from 0 to 120 µM and 200 nM Aβ42 fibril (incubation time 10 min); Data are shown as mean ± SD; Repetition n = 3. b, Fluorescence intensity of HS-169 (60 µM) and Aβ42 fibrils in the concentration range varying from 0 to 200 nM. c, Linear relation between Aβ42 fibril concentration and fluorescence intensity was observed, Data are presented as mean ± SD. Repetition n = 3. d–g, Comparison of Aβ load at 2 h and 24 h post-injection of the HS-169 probe in arcAβ mouse. (d) LMI images acquired at 2 h time point post-injection of HS-169 in a 22-month arcAβ mouse (d). LMI images acquired at 24 h post-injection of HS-169 in a 22-month arcAβ mouse with the same dose (e). Contrast-to-noise ratio (CNR) comparison between LMI and widefield (WF) fluorescence images acquired at 2 h and 24 h post-injection of HS-169 in one mouse brain (f,g). The CNR was calculated via (sig - bg)/noise; where sig and bg are the mean fluorescence intensities in ROIs as indicated in d and e; noise was calculated as the standard deviation in the ROI in a background region. Both image pattern and the CNR values of the LMI images acquired at these two time points are comparable, while there is a reduction in the CNR values in the WF image at 24 h compared to 2 h post-injection. Data is presented as mean ± SD (analysis of the 6 ROIs over one mouse brain).
a–d, Using microsphere, maximum intensity projection (MIP) along the axial direction of the vMSOT image of a 30 µm microsphere positioned in the centre of the array (a). Vertical profile of the vMSOT image along the line indicated in a (b). Fitted Gaussian curve is indicated. The spatial resolution can be estimated via the mean square difference between the width of the fitted curve and the actual microsphere diameter, resulting in a value of 113 µm. A compounded vMSOT image obtained by raster scanning the microsphere across the field of view (c). The reconstructed microsphere size for positions corresponding to various distances from the centre of the spherical array geometry, as indicated in panel c (d). e–i, Transcranial imaging of mouse brain. Maximum intensity projection (MIP) along the axial direction of the vMSOT image of a 30 µm microsphere positioned in the centre of the array’s field-of-view when an excised murine skull is placed between the microsphere and the array (e). Vertical profile of the vMSOT image along the line indicated in e (f). Fitted Gaussian curve is indicated. The mean square difference between the width of the fitted curve and the microsphere diameter corresponds to a spatial resolution of 157 µm. Ratio of amplitudes of the optoacoustic signals recorded without and with the skull as a function of the angle of each detection element with respect to the central axis of the array (g). Signals were averaged for each ring of elements. MIP of the vMSOT image of the brain of a 15-months arcAβ mouse (unmixed signal corresponding to oxygenated hemoglobin) through the intact scalp and skull (h). One-dimensional vMSOT signal profile along the white line indicated in h (i). The width of the fitted Gaussian curve is 161 µm.
a, Top-view maximum intensity projection (MIP) of the volumetric vMSOT brain image recorded from a 5-months old nude mouse when the illumination was provided from a single direction through the central aperture of the array. b, The corresponding image when the brain is illuminated from four different directions using the multi-arm fiber bundle. c,d, Cross-sections of the 3D image in b for the indicated positions. The horizontal (xy) section corresponds to a depth of 3 mm from the scalp surface. The illumination wavelength was 800 nm.
a, 3D rendering of vMSOT data in arcAβ mouse brain unmixed for AOI987 distribution. b, Baseline-subtracted single wavelength vMSOT image acquired at 650 nm. c, Image acquired at 600 nm excitation wavelength reveals the major cerebral vessels. d, Overlay of a and c. e,g, Overlay of a on MRI structural data. f, Time-lapse curve of the unmixed AOI987 absorbance. h, The corresponding curve for the baseline-subtracted signal at 650 nm. AOI987 was injected i.v. at 30 s. The temporal evolution of the unmixed AOI987 absorbance and baseline-subtracted signals was analysed in four different brain regions: cortex (red), superior sagittal sinus (green), hippocampus (blue), vessel (grey) on the cortical surface indicated in e,h. norm, normalized; ab, absorbance.
a, vMSOT image of an unspecific Cy5.5 probe distribution in a 14-month old arcAβ mouse versus non-transgenic littermate (NTL). An amount of 100 µl at a concentration of 1 mg/ml was injected intravenously. Four different time points are shown before and after Cy5.5 injection. Scale bar = 1 mm. b, Averaged Cy5.5 signals in the brains of 14-month old arcAβ mice and NTL mice at different time points post-injection (n = 3 in each group). Data are presented as mean ± SD. No significant differences were observed in probe distribution between groups.
Extended Data Fig. 7 In vivo Aβ imaging in 8, 14 and 15-months old arcAβ mice by means of vMSOT and AOI987 probe.
Regional signal analysis revealed a higher amyloid load in the cortex and cerebellum at 15 months-of-age compared to 8 months. Data is presented as mean ± SD. Two-way ANOVA followed by post-hoc Bonferroni correction for multiple comparison was used. Comparison between 8 month (n = 4) and 15 month (n = 4) arcAβ mice showed significant increase in the cortex (p = 0.012), and in the hippocampus (p = 0.0331). No different was observed between 14 (n = 4) and 15 month (n = 4).
Extended Data Fig. 8 Ex vivo whole brain imaging using vMSOT and mesoSPIM with LOCs h-FTAA and q-FTAA.
a, 3D rendering of an anatomical vMSOT image (acquired at 600 nm) overlayed with unmixed AOI987 distribution rendered by vMSOT in NTL and arcAβ mice. b, 3D rendering of mesoSPIM data of Aβ distribution (stained with LCOs h-FTAA and q-FTAA) in NTL and arcAβ mice. c, Quantification of regional fluorescence intensity in the arcAβ mouse brain. Cortex: Ctx; Hippocampus: Hip; Midbrain: MB; Striatum: Str; Thalamus: TH. d,e, Sagittal views of the brain of 24 month-old non-transgenic littermate (NTL) (d) and arcAβ mouse (e). High Aβ plaque load was observed in the cortex, hippocampus and thalamus, while fewer plaques were observed in cerebellar and other subcortical regions. Fluorescence intensity (F.I.). Scale bar = 1 mm.
Extended Data Fig. 9 Two-photon microscopy (2PM) of ex vivo brain slices from arcAβ mice after in vivo vMSOT imaging with i.v. injection of AOI987.
a,b, Lambda spectrum mapping of a representative brain slice from an 8-month old arcAβ mouse showing the peak of signal. c,e,f, Horizontal views and zoom-ins of brain slices from 8- and 15-month-old arcAβ mice. Signals were observed in the parenchyma as well as inside a cortical vessel (zoom-in in panel f). Non-specific signal was detected in the cerebellum of 8-month-old arcAβ mouse. AOI987 signals were observed in the cerebellum of 15-month-old arcAβ mice. d, Horizontal view of brain mouse slides from Allen brain atlas are shown for anatomical reference 1. Scale bar = 0.5 mm (a), 50 µm (b,f), 1 mm (c,e), Cortex: Ctx, Cerebellum: Cb.
Extended Data Fig. 10 Illustration of the differences between 2PM and LMI imaging of CAA deposits and parenchymal plaques.
a, Illustration of the high-resolution 2PM excitation of a thin CAA vascular deposit versus parenchymal plaque. b, The corresponding illustration showing a much larger fluorescence volume excited by LMI in the large densely-filled Aβ parenchymal plaque. The incident light beams and the volumes where fluorescence responses are produced for both modalities are indicated in red and green, respectively. c,d, Representative confocal microscopic images of horizontal sections from an arcAβ mouse brain. 3D rendering of vessel-wall associated CAA and parenchymal plaque indicates the major difference in their shape and overall mass, affecting the total amount of signal detected by LMI. DAPI (blue), Alexa488-6E10 (green), HS-169 (red), CAA - cerebral amyloid angiopathy; LMI - large-field multifocal illumination. Scale bar = 20 µm.
Supplementary methods, tables and references.
High-resolution Aβ imaging.
Non-invasive in vivo volumetric multispectral optoacoustic tomography (vMSOT) of the targeted AOI987 probe distribution in an arcAβ mouse brain at 60 min after intravenous injection.
MesoSPIM using luminescent conjugated oligothiophene in the whole brain of an arcAβ mouse revealed Aβ plaques.
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Ni, R., Chen, Z., Deán-Ben, X.L. et al. Multiscale optical and optoacoustic imaging of amyloid-β deposits in mice. Nat. Biomed. Eng 6, 1031–1044 (2022). https://doi.org/10.1038/s41551-022-00906-1
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