Non-invasive methods for the in vivo detection of hallmarks of Alzheimer’s disease can facilitate the study of the progression of the disease in mouse models and may enable its earlier diagnosis in humans. Here we show that the zwitterionic heptamethine fluorophore ZW800-1C, which has peak excitation and emission wavelengths in the near-infrared optical window, binds in vivo and at high contrast to amyloid-β deposits and to neurofibrillary tangles, and allows for the microscopic imaging of amyloid-β and tau aggregates through the intact skull of mice. In transgenic mouse models of Alzheimer’s disease, we compare the performance of ZW800-1C with that of the two spectrally similar heptamethine fluorophores ZW800-1A and indocyanine green, and show that ZW800-1C undergoes a longer fluorescence-lifetime shift when bound to amyloid-β and tau aggregates than when circulating in blood vessels. ZW800-1C may prove advantageous for tracking the proteinic aggregates in rodent models of amyloid-β and tau pathologies.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 per month
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$79.00 per year
only $6.58 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.
The custom ImageJ and Matlab codes are available on request from the corresponding authors.
Montine, T. J. et al. National Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease: a practical approach. Acta Neuropathol. 123, 1–11 (2012).
Mathis, C., Wang, Y. & Klunk, W. Imaging beta-amyloid plaques and neurofibrillary tangles in the aging human brain. Curr. Pharm. Des. 10, 1469–1492 (2005).
Villemagne, V. L., Doré, V., Burnham, S. C., Masters, C. L. & Rowe, C. C. Imaging tau and amyloid-β proteinopathies in Alzheimer disease and other conditions. Nat. Rev. Neurol. 14, 225–236 (2018).
Poduslo, J. F. et al. Molecular targeting of Alzheimer’s amyloid plaques for contrast-enhanced magnetic resonance imaging. Neurobiol. Dis. 11, 315–329 (2002).
Flaherty, D. P. et al. Polyfluorinated bis-styrylbenzene β-amyloid plaque binding ligands. J. Med. Chem. 50, 4986–4992 (2007).
Martins, A. F. et al. PiB-conjugated, metal-based imaging probes: multimodal approaches for the visualization of β-amyloid plaques. ACS Med. Chem. Lett. 4, 436–440 (2013).
Chamberlain, R. et al. Magnetic resonance imaging of amyloid plaques in transgenic mouse models of Alzheimer’s disease. Curr. Med. Imaging Rev. 7, 3–7 (2011).
Mathis, C. A., Mason, N. S., Lopresti, B. J. & Klunk, W. E. Development of positron emission tomography β-amyloid plaque imaging agents. Semin. Nucl. Med. 42, 423–432 (2012).
Zhu, L., Ploessl, K. & Kung, H. F. PET/SPECT imaging agents for neurodegenerative diseases. Chem. Soc. Rev. 43, 6683–6691 (2014).
Krishnadas, N., Villemagne, V. L., Doré, V. & Rowe, C. C. Advances in brain amyloid imaging. Semin. Nucl. Med. 51, 241–252 (2021).
Zhuang, Z. P. et al. Structure-activity relationship of imidazo[1,2-a]pyridines as ligands for detecting β-amyloid plaques in the brain. J. Med. Chem. 46, 237–243 (2003).
Qu, W., Kung, M. P., Hou, C., Benedum, T. E. & Kung, H. F. Novel styrylpyridines as probes for SPECT imaging of amyloid plaques. J. Med. Chem. 50, 2157–2165 (2007).
Valotassiou, V. et al. SPECT and PET imaging in Alzheimer’s disease. Ann. Nucl. Med. 32, 583–593 (2018).
Tong, H., Lou, K. & Wang, W. Near-infrared fluorescent probes for imaging of amyloid plaques in Alzheimer’s disease. Acta Pharm. Sin. B 5, 25–33 (2015).
Klunk, W. E. et al. Imaging Aβ plaques in living transgenic mice with multiphoton microscopy and methoxy-X04, a systemically administered Congo red derivative. J. Neuropathol. Exp. Neurol. 61, 797–805 (2002).
Heo, C. H. et al. A quadrupolar two-photon fluorescent probe for in vivo imaging of amyloid-β plaques. Chem. Sci. 7, 4600–4606 (2016).
Teoh, C. L. et al. Chemical fluorescent probe for detection of Aβ oligomers. J. Am. Chem. Soc. 137, 13503–13509 (2015).
Ntziachristos, V., Bremer, C. & Weissleder, R. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur. Radiol. 13, 195–208 (2003).
Ntziachristos, V. Fluorescence molecular imaging. Annu. Rev. Biomed. Eng. 8, 1–33 (2006).
Frangioni, J. V. In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 7, 626–634 (2003).
Licha, K. & Olbrich, C. Optical imaging in drug discovery and diagnostic applications. Adv. Drug Deliv. Rev. 57, 1087–1108 (2005).
Shcherbakova, D. M., Stepanenko, O. V., Turoverov, K. K. & Verkhusha, V. V. Near-infrared fluorescent proteins: multiplexing and optogenetics across scales. Trends Biotechnol. 36, 1230–1243 (2018).
Hong, G., Antaris, A. L. & Dai, H. Near-infrared fluorophores for biomedical imaging. Nat. Biomed. Eng. 1, 0010 (2017).
Shi, L., Sordillo, L. A., Rodríguez-Contreras, A. & Alfano, R. Transmission in near-infrared optical windows for deep brain imaging. J. Biophotonics 9, 38–43 (2016).
Kim, D. et al. Two-photon absorbing dyes with minimal autofluorescence in tissue imaging: application to in vivo imaging of amyloid-β plaques with a negligible background signal. J. Am. Chem. Soc. 137, 6781–6789 (2015).
Yang, J. et al. Oxalate-curcumin-based probe for micro- and macroimaging of reactive oxygen species in Alzheimer’s disease. Proc. Natl Acad. Sci. USA 114, 12384–12389 (2017).
Chen, C. et al. In vivo near-infrared two-photon imaging of amyloid plaques in deep brain of Alzheimer’s disease mouse model. ACS Chem. Neurosci. 9, 3128–3136 (2018).
Li, Y. et al. Fluoro-substituted cyanine for reliable in vivo labelling of amyloid-β oligomers and neuroprotection against amyloid-β induced toxicity. Chem. Sci. 8, 8279–8284 (2017).
Zhang, X. et al. Design and synthesis of curcumin analogues for in vivo fluorescence imaging and inhibiting copper-induced cross-linking of amyloid beta species in Alzheimer’s disease. J. Am. Chem. Soc. 135, 16397–16409 (2013).
Cui, M. et al. Smart near-infrared fluorescence probes with donor-acceptor structure for in vivo detection of β-amyloid deposits. J. Am. Chem. Soc. 136, 3388–3394 (2014).
Zhang, X. et al. Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer’s disease. Proc. Natl Acad. Sci. USA 112, 9734–9739 (2015).
Fu, H. et al. Amyloid-β deposits target efficient near-infrared fluorescent probes: synthesis, in vitro evaluation, and in vivo imaging. Anal. Chem. 88, 1944–1950 (2016).
Seo, Y. et al. A smart near-infrared fluorescence probe for selective detection of tau fibrils in Alzheimer’s disease. ACS Chem. Neurosci. 7, 1474–1481 (2016).
Park, K. S. et al. A difluoroboron β-diketonate probe shows ‘turn-on’ near-infrared fluorescence specific for tau fibrils. ACS Chem. Neurosci. 8, 2124–2131 (2017).
Ni, R. et al. Detection of cerebral tauopathy in P301L mice using high-resolution large-field multifocal illumination fluorescence microscopy. Biomed. Opt. Express 11, 4989–5002 (2020).
Tagai, K. et al. High-contrast in vivo imaging of Tau pathologies in Alzheimer’s and non-Alzheimer’s disease tauopathies. Neuron 109, 42–58.e8 (2021).
Park, K. S. et al. A curcumin-based molecular probe for near-infrared fluorescence imaging of tau fibrils in Alzheimer’s disease. Org. Biomol. Chem. 13, 11194–11199 (2015).
Hyun, H. et al. Central C-C bonding increases optical and chemical stability of NIR fluorophores. RSC Adv. 4, 58762–58768 (2014).
Yang, C. et al. ZW800-PEG: a renal clearable zwitterionic near-infrared fluorophore for potential clinical translation. Angew. Chem. Int. Ed. 60, 13847–13852 (2021).
Landsman, M. L. J., Kwant, G., Mook, G. A. & Zijlstra, W. G. Light absorbing properties, stability, and spectral stabilization of indocyanine green. J. Appl. Physiol. 40, 575–583 (1976).
Choi, H. S. et al. Synthesis and in vivo fate of zwitterionic near-infrared fluorophores. Angew. Chem. Int. Ed. 50, 6258–6263 (2011).
Raymond, S. B., Boas, D. A., Bacskai, B. J. & Kumar, A. T. N. Lifetime-based tomographic multiplexing. J. Biomed. Opt. 15, 046011 (2010).
Venugopal, V., Chen, J., Lesage, F. & Intes, X. Full-field time-resolved fluorescence tomography of small animals. Opt. Lett. 35, 3189–3191 (2010).
Jankowsky, J. L. et al. Mutant presenilins specifically elevate the levels of the 42 residue β-amyloid peptide in vivo: evidence for augmentation of a 42-specific γ secretase. Hum. Mol. Genet. 13, 159–170 (2004).
Santacruz, K. et al. Tau suppression in a neurodegenerative mouse model improves memory function. Science 309, 476–481 (2005).
Rajamohamedsait, H. B. & Sigurdsson, E. M. Histological staining of amyloid and pre-amyloid peptides and proteins in mouse tissue. Methods Mol. Biol. 849, 411–424 (2012).
Kwan, A. C., Duff, K., Gouras, G. K. & Webb, W. W. Optical visualization of Alzheimer’s pathology via multiphoton-excited intrinsic fluorescence and second harmonic generation. Opt. Express 17, 3679–3689 (2009).
Shirani, H. et al. A palette of fluorescent thiophene-based ligands for the identification of protein aggregates. Chemistry 21, 15133–15137 (2015).
Calvo-Rodriguez, M. et al. In vivo detection of tau fibrils and amyloid β aggregates with luminescent conjugated oligothiophenes and multiphoton microscopy. Acta Neuropathol. Commun. 7, 171 (2019).
Banks, W. A. Characteristics of compounds that cross the blood-brain barrier. BMC Neurology 9, S3 (2009).
Minogue, A. M. et al. Age-associated dysregulation of microglial activation is coupled with enhanced blood-brain barrier permeability and pathology in APP/PS1 mice. Neurobiol. Aging 35, 1442–1452 (2014).
Ohtsuki, S. & Terasaki, T. Contribution of carrier-mediated transport systems to the blood-brain barrier as a supporting and protecting interface for the brain; importance for CNS drug discovery and development. Pharm. Res. 24, 1745–1758 (2007).
Sanchez-Covarrubias, L., Slosky, L., Thompson, B., Davis, T. & Ronaldson, P. Transporters at CNS barrier sites: obstacles or opportunities for drug delivery? Curr. Pharm. Des. 20, 1422–1449 (2014).
Kuchibhotla, K. V. et al. Aβ plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks. Neuron 59, 214–225 (2008).
Wang, T. et al. Three-photon imaging of mouse brain structure and function through the intact skull. Nat. Methods 15, 789–792 (2018).
Pologruto, T. A., Sabatini, B. L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).
Choi, H. S. et al. Targeted zwitterionic near-infrared fluorophores for improved optical imaging. Nat. Biotechnol. 31, 148–153 (2013).
We thank K. P. R. Nilsson for providing the HS-84 probe. This study was supported by grants from the National Institutes of Health (NIA R56AG060974 to B.J.B.; NIA K01AG072046 to S.S.H.; NCI R01CA211084 to A.T.N.K.; NIBIB R01EB022230 to H.S.C.) and the Creative Materials Discovery Program through the National Research Foundation of Korea (2019M3D1A1078938 to H.S.C.). The content expressed is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
The authors declare no competing interests.
Peer review information
Nature Biomedical Engineering thanks Jan Klohs and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Ex vivo labeling intensity of amyloid plaques and CAA by ZW800-1C.
Quantitative comparison of the average labeling intensity of CAA and plaques by ZW800-1C in APP/PS1 brain tissue sections. Sections were stained by incubation in 100 µM ZW800-1C solution in PBS. Significance was calculated using unpaired two-tailed t-test: ****P < 0.0001. Data are shown as mean ± s.e.m.
Extended Data Fig. 2 In vivo imaging reveals selective labeling of CAA with ICG in APP/PS1 mice.
Examples of arteries with CAA that are positively labeled with MX04 and (a) co-labeled with ICG and (b) not co-labeled with ICG. Images were acquired with confocal microscopy at 2 h post-injection.
Extended Data Fig. 3 In vivo imaging of ICG, ZW800-1A, and ZW800-1C in APP/PS1 and rTg4510 non-transgenic littermates.
Each NIR fluorophore was injected intravenously (50 nmol) and imaged 2 h post-injection with confocal microscopy. Representative images for both (a) APP/PS1 non-Tg littermates (n = 3) and (b) rTg4510 non-Tg littermates (n = 3) show low levels of periarterial labeling by all three fluorophores.
Extended Data Fig. 4 Post-mortem validation of ZW800-1C labeling in APP/PS1 and rTg4510 mice.
APP/PS1 mouse is intraperitoneally injected with MX04 and intravenously injected with ZW800-1C at 24 h and 2 h before sacrifice. Ex vivo imaging is performed on brain tissue sections, and representative images show co-stained (a) CAA and (b) amyloid plaques. (c) Tg4510 mouse is intravenously injected with HS-84 and ZW800-1C at 7 days and 2 h before sacrifice, respectively. Representative images show co-labeling of NFTs.
Extended Data Fig. 5 In vivo biodistribution study of ICG, ZW800-1A, and ZW800-1C in CD-1 mice.
25 nmol of ZW800-1C was injected in 25 g CD-1 mice 4 h prior to imaging. (a) Representative color and NIR images of ZW800-1C showing major organs in the thoracic cavity (left), abdominal cavity (middle), and after resection (right). (b) Signal-to-background ratio (SBR) of resected major organs (Or) against muscle (Mu). Abbreviations used are He, heart; Lu, lung; Li, liver; Pa, pancreas; Sp, spleen; Ki, kidney; Du, duodenum; Ga, gallbladder; BD, bile duct; Bl, bladder; In, intestine. ***P = 0.0006 and ****P < 0.0001 by two-way ANOVA with Dunnett’s multiple comparison test. (c) Comparison of the pharmacokinetic parameters of the candidate NIR fluorophores. Abbreviations used are t1/2α, distribution blood half-life; t1/2β, elimination blood half-life; Vd, the volume of distribution; AUC, area under the curve; Cl, clearance. Data are shown as mean ± s.e.m.
Extended Data Fig. 6 In vivo fluorescence lifetime imaging of ICG and ZW800-1A in C57BL/6J and APP/PS1 mice.
Histogram distribution of fluorescence lifetimes for (a) ICG and (b) ZW800-1A within blood vessels and after being bound to amyloid-β aggregates.
Extended Data Fig. 7 Two-photon imaging of ZW800-1C in APP/PS1 and rTg4510 mice.
Two-photon imaging of ZW800-1C (red) with 1,300 nm excitation. Alexa Fluor 680-70 kDa Dextran (green) was systemically injected to create a fluorescent angiogram. Representative images show (a) CAA, (b) amyloid plaques and (c) NFTs from APP/PS1 (n = 3) and rTg4510 (n = 3) mice. (d) Images of amyloid plaques taken at different depths from 7–8 months old (mo) and 22–24 mo APP/PS1 mice. (e) Comparison of the percentage volume occupied by amyloid plaques for 7–8 mo and 22–24 mo APP/PS1 mice (n = 3 mice per age group). Data are shown as mean ± s.e.m.
Supplementary figures and caption for the supplementary video.
In vivo through-the-skull imaging of an APP/PS1 mouse.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Hou, S.S., Yang, J., Lee, J.H. et al. Near-infrared fluorescence lifetime imaging of amyloid-β aggregates and tau fibrils through the intact skull of mice. Nat. Biomed. Eng 7, 270–280 (2023). https://doi.org/10.1038/s41551-023-01003-7