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Near-infrared fluorescence lifetime imaging of amyloid-β aggregates and tau fibrils through the intact skull of mice


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.

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Fig. 1: Candidate heptamethine NIR fluorophores for AD imaging.
Fig. 2: Ex vivo imaging of amyloid plaques and NFTs in APP/PS1 and rTg4510 mouse brain sections.
Fig. 3: In vivo imaging of amyloid plaques and CAA with ICG, ZW800-1A and ZW800-1C in APP/PS1 mice.
Fig. 4: In vivo imaging of NFTs with ICG, ZW800-1A and ZW800-1C in rTg4510 mice.
Fig. 5: In vivo fluorescence lifetime imaging of ZW800-1C in C57BL/6J, APP/PS1 and rTg4510 mice.
Fig. 6: Two-photon microscopy and non-invasive lifetime imaging of AD pathology with ZW800-1C.

Data availability

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.

Code availability

The custom ImageJ and Matlab codes are available on request from the corresponding authors.


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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.

Author information

Authors and Affiliations



S.S.H., J.H.L., B.J.B. and H.S.C. conceived the study and designed the experiments. S.S.H., J.Y., S.A. and Y.K. performed the ex vivo imaging experiments and analysed the results. S.S.H., J.Y., J.H.L., Y.K., M.C.-R. and S.K. performed the in vivo validation experiments and analysed the results. K.B. synthesized the compounds used in the study (ZW800-1A and ZW800-1C). S.S.H. and A.T.N.K. performed the fluorescence lifetime measurements and interpreted the results. S.S.H., B.J.B. and H.S.C. wrote the manuscript. B.J.B. and H.S.C. supervised the entire project. All authors approved the final version of the manuscript.

Corresponding authors

Correspondence to Brian J. Bacskai or Hak Soo Choi.

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Nature Biomedical Engineering thanks Jan Klohs and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

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 information

Supplementary Information

Supplementary figures and caption for the supplementary video.

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Supplementary Video

In vivo through-the-skull imaging of an APP/PS1 mouse.

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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).

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