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Fluorescence-amplified nanocrystals in the second near-infrared window for in vivo real-time dynamic multiplexed imaging

Nature Nanotechnology volume 18, pages 1195–1204 (2023)Cite this article

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Abstract

Optical imaging in the second near-infrared window (NIR-II, 1,000–1,700 nm) holds great promise for non-invasive in vivo detection. However, real-time dynamic multiplexed imaging remains challenging due to the lack of available fluorescence probes and multiplexing techniques in the ideal NIR-IIb (1,500–1,700 nm) ‘deep-tissue-transparent’ sub-window. Here we report on thulium-based cubic-phase downshifting nanoparticles (α-TmNPs) with 1,632 nm fluorescence amplification. This strategy was also validated for the fluorescence enhancement of nanoparticles doped with NIR-II Er3+ (α-ErNPs) or Ho3+ (α-HoNPs). In parallel, we developed a simultaneous dual-channel imaging system with high spatiotemporal synchronization and accuracy. The NIR-IIb α-TmNPs and α-ErNPs facilitated the non-invasive real-time dynamic multiplexed imaging of cerebrovascular vasomotion activity and the single-cell-level neutrophil behaviour in mouse subcutaneous tissue and ischaemic stroke model.

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Fig. 1: Emerging NIR-IIb fluorescence amplification of α-TmNPs.
Fig. 2: Mechanism investigation for NIR-II fluorescence amplification.
Fig. 3: Real-time dynamic NIR-II multiplexed imaging with spatiotemporal synchronization.
Fig. 4: Real-time dynamic multiplexed imaging of cerebral vessel haemodynamics.
Fig. 5: Real-time dynamic multiplexed imaging of neutrophil responses during acute inflammation.

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Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

The code that has been used for this work is available from the corresponding authors upon request.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (NSFC; 22088101, 21725502, 51961145403, 21904023, 11974097, 22161160320, 32171377), New Cornerstone Science Foundation through the XPLORER PRIZE and the Research Program of Science and Technology Commission of Shanghai Municipality (20JC1411700, 20490710600, 20S31903700, 21QA1406700, 22JC1400400).

Author information

Author notes

  1. These authors contributed equally: Yiwei Yang, Ying Chen.

Authors and Affiliations

  1. Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers and iChem, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Fudan University, Shanghai, China

    Yiwei Yang, Ying Chen, Peng Pei, Yong Fan, Shangfeng Wang, Hongxin Zhang, Dongyuan Zhao & Fan Zhang

  2. Human Phenome Institute, Zhangjiang Fudan International Innovation Center, Fudan University, Shanghai, China

    Bin-Zhi Qian

  3. Centre for Reproductive Health, College of Medicine and Veterinary Medicine, Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK

    Bin-Zhi Qian

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Contributions

F.Z. and Y.F. conceived and designed the experiments. Y.Y. synthesized the nanoparticles. Y.Y. and P.P. conducted the NIR-II dual-channel synchronous imaging in vitro. Y.C. and Y.Y. conducted the imaging of the synchronous dynamic tracking of cerebrovascular vasomotion and neutrophil behaviours in mouse subcutaneous tissue and ischaemic stroke model. S.W., H.Z. and Y.Y. built the NIR-IIb spatiotemporal synchronized dual-channel NIR-IIb imaging system. Y.Y., F.Z. and Y.F. wrote the paper. F.Z., Y.F., Y.Y., Y.C., S.W., B.-Z.Q. and H.Z analysed the results, figures and supplementary information. F.Z., Y.F., Y.Y., H.Z. and D.Z. discussed the mechanism of Tm3+-based cubic-phase core–shell-structured nanoparticles. D.Z. and F.Z. also contributed to the discussions about the dual-channel NIR-IIb imaging system. All the authors contributed to discussing and editing the paper.

Corresponding authors

Correspondence to Yong Fan or Fan Zhang.

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Nature Nanotechnology thanks Zhihong Liu, Riccardo Marin 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 Characterization of different activators based core/multi-shell-shell nanoparticles with different crystal phases.

a, TEM images and corresponding sizes of cubic-phase (α) and hexagnal-phase (β) NaYF4:Yb/A (A = Tm, Er and Ho) core and final core-shell-shell nanocrystals with same components. Scale bar, 20 nm. To eliminate the effect of nanoparticle size on fluorescence intensity comparision, α- and β-NaYF4:Yb/A@NaYbF4@NaYF4 (A = Tm, Er and Ho) were synthesized with similar core diameter followed by the identical epitaxial growth of outer shells. b, Fluorescence emission spectra of TmNPs, ErNPs and HoNPs with α- and β-phase.

Source data

Extended Data Fig. 2 The effect of MPR process on the luminescence transitions in α-TmNPs.

a, b, Luminescence emission spectra (a) and corresponding intensities variation (b) in visible and NIR regions of α-TmNPs with increasing Tm3+ content under 980 nm laser excitation. c, Proposed MPR mediated cross-relaxation between Tm3+. d, Lifetime of α-NaYF4:Tm(x)@NaYbF4@NaYF4 at 1632 nm with varying Tm3+ content. Inset shows the lifetime changes versus Tm3+ content.

Source data

Extended Data Fig. 3 Characterization of the backward-forward energy transfer (BFET) process.

a, Simplified energy level diagram shows backward energy transfer (BET) and forward energy transfer (FET) under 808 nm excitation (absorbed by only Tm3+). Yb3+ trapped the pumped energy from energy level 3H5 of Tm3+and transfer it to lower energy level 3F4 of Tm3+. b–d, Luminescence decay curves measured at (b)1632 nm, (c) 1475 nm and (d) 800 nm from Tm3+ with increasing Yb3+ content in core. e, Luminescence emission spectra and fluorescence intensities at 980 nm emission (inset) for α-TmNPs with increasing Yb3+ content in core(e). f, Decay curves of Yb3+ (2F5/2 → 2F7/2) with increasing Yb3+ content in core. g, Normalized luminescence lifetime in (b), (c), (d), (f).

Source data

Extended Data Fig. 4 Characterization of core-shell-shell α-HoNPs and α-ErNPs with different Yb3+ content.

a, Simplified energy level diagrams of α-HoNPs and α-ErNPs show backward energy transfer (BET) and forward energy transfer (FET) under 980 nm excitation or 655 nm excitaion. b, c, Luminescence steady-state emission spectra and intensities (insets) for α-HoNPs with increasing Yb3+ content under (b) 980 nm and (c) 655 nm excitation. Insets show the luminescence intensity. d, e, Luminescence steady-state emission spectra and intensities for α-ErNPs with increasing Yb3+ content under (b) 980 nm and (c) 655 nm excitation.

Source data

Extended Data Fig. 5 Characterization of image resolution covered with biological tissues in varied NIR-II sub-regions.

a, Typical SEM image of the PS beasds (~ 10 μm) used as the substrates for cubic-phase Tm3+-, Er3+- and Ho3+-based nanoparticles encapulation. Scale bar, 20 μm. b, Fabrication procedures for polystyrene (PS) beads tagged with single component (α-TmNPs, α-ErNPs or α-HoNPs). c, Schematic of in vitro imaging experiments using the DSNPs-tagged beads imaging that covered with biological tissues. The inset shows physical picture of mouse skull with a thickness of ~1 mm. d, e Fluorescence images of the three types of PS beads tagged with α-TmNPs (Tm-Ch, 1600–1700 nm), α-ErNPs (Er-Ch, 1500–1600 nm) and α-HoNPs (Ho-Ch, 1100–1200 nm), respectively, in the absence and presence of a covered slice of (d) mouse skin or (e) skull and corresponding image resolutions. The imaging resolutions were quantified by full width at half-maximum (FWHM) of a PS bead along the white-dased lines. Exposure time: 300 ms. Scale bar, 50 μm.

Source data

Extended Data Fig. 6 Real-time dynamic NIR-IIb multiplexed imaging of Tm/Er tagged PS beads.

a, Fabrication procedures for polystyrene (PS) beads tagged with both α-TmNPs and α-ErNPs (Tm/Er tagged beads). b, NIR-IIb fluorescence images of Tm/Er-tagged PS beads in Tm-Ch or Er-Ch. The overlapping image shows well co-localization of the two channels. Scale bar, 50 μm. c, A photograph of the simultaneous dual-channel NIR-IIb fluorescence imaging system equipped with a microfluidic system. Tm/Er- tagged PS-beads were dispersed in cetane, followed by being pumped into the flow channel of the glass chip. d, Real-time multiplexed images of Tm/Er-tagged PS beads flowing in the channel of the glass chip at a fixed perfusion rate (100 mL/h). Elongated outline of the PS beads along the direction of fluid flowing was attributed to motion-blur under exposure time of ~ 300 ms. Scale bar, 50 μm. e, Statistical results of the average velocities in the Er-Ch and Tm-Ch. Quantitative results showed mean velocities of PS beads in a range of 4437.7–4657.0 μm/min in Tm-Ch and 4524.3–4675.4 μm/min in Er-Ch, with minor deviation between two imaging channels (less than 2% as shown in the right side of the figure). The data are shown as the mean ± s.d. (n = 17–18). A boxplot with minimum, maximum and median is shown. f, g, Pearson’s correlation (Pr value) analysis of (f) velocity and (g) location of the six tracked PS beads between Er- and Tm-Ch in a, indicating a high spatiotemporal synchronicity.

Source data

Extended Data Fig. 7 Whole body blood vessel imaging of mouse in Tm-, Er- and Ho-Ch.

a,c, NIR-II images of mouse blood vessels in (a) supine and (c) prone posistion with intravenous injection of aqueous α-TmNPs, α-ErNPs and α-HoNPs, respectively. b, d, Correponding signal-to-background ratios (SBRs) of mouse blood vessels in (b) belly and (d) prone posistion by plotting the cross-sectional intensity profiles as shown in the dased line in a and c. Scale bar, 1 cm. Exposure time: 300 ms. Whole-body blood imaging in NIR-IIb window presents SBRs of 3.0–12.5 (Tm-Ch and Er-Ch), which were ~1.0–4.3 fold higher over that in short wavelength NIR region (2.9–4.3) (Ho-Ch).

Source data

Extended Data Fig. 8 Noninvasive monitoring the specific labelling of elasitc fibres of arteries by α-Er@Af633 nanoprobes.

a, Time-line of monitoring of the α-Er@Af633 labeled elastic fibres. Free α-ErNPs without conjugation of Alexa Fluor 633 dyes were also adopted with identical experimental procedures as a control group. b, c, Real-time tracking metabolic processes of (b) α-Er@Af633 or (c) free α-ErNPs in vessel blood. Scale bar, 20 μm. Blood vessels with similar lumen diameters were monitored for comparing the metabolic behaviors of α-Er@Af633 or free α-ErNPs. d, e, Quantitative analysis of metabolic behaviors of (d) α-Er@Af633 and (e) free α-ErNPs in blood vessels by plotting the signal intensity along the whited dashed line in (b) and (c). Decreased fluorescence intensity of the vessel lumen was observed with injection of α-Er@Af 633 over time. f, Corresponding SBRs of the artery walls with injection of α-Er@Af 633 (from 3.0 to 4.4) or free α-ErNPs (from 2.8 to 1.3).

Source data

Extended Data Fig. 9 Real-time dynamic multiplexed imaging of neutrophil behaviors in acute inflammation.

a, Multiplexed dynamic tracking of a single neutrophil (blue-dashed circle) migrating towards a small neutrophil cluster (white arrow) in the inflamed site in mice ear. The vascular profile was depicted by white dotted line. Scale bar, 20 μm. b, Migratory paths of the single neutrophil (presented as x-y projections). The calculated meandering index (MI) value of ~ 0.81 (>0.8) was commonly associated with chemotaxis, indicating its potential to be activated in inflammatory microenvironment. c, Real-time imaging of neutrophil extravasation (purple-dashed circle), which is a typical characteristic of activated state. Scale bar, 50 μm.

Source data

Extended Data Fig. 10 In vivo NIR-IIb imaging of neutrophil recruitment in stroke mice through intact skull using α-Tm-aLy6G.

Imaging was performed independently on n = 4 biological replicates for each group with similar results. Neutrophils were always considered as short-lived cells with a circulation half-life of approximately 1.5 h in healthy mice, however, their circulating period can be increased by several folds if inflammation occurred. NIR-IIb signal from α-Tm-aLy6G was clearly observed in the injured left cerebral hemisphere and increased over time (also shown in Fig. 5g in the manuscript), with the brightest intensity obtained at the 24 h p.i, which indicates inflammation responsive neutrophil recruitment.

Supplementary information

Supplementary Information

Supplementary Figs. 1–44, Tables 1–5, Imaging data analyses and References.

Reporting Summary

Supplementary Video 1

Synchronous multiplexed tracking of Tm/Er-tagged beads.

Supplementary Video 2

Synchronous multiplexed dynamic tracking of artery vasoconstriction.

Supplementary Video 3

Dynamic tracking of spontaneous vasomotion of cerebral network.

Supplementary Video 4

Neutrophil dynamics in inflamed (LPS+) and normal (Ctrl) sites.

Supplementary Video 5

Multiplexed dynamic tracking of neutrophil behaviors in acute inflammation.

Supplementary Video 6

Behavioural symptom of stroke mice.

Supplementary Video 7

Synchronous multiplexed dynamic tracking of activated neutrophils.

Supplementary Data

Statistical source data for Supplementary Figs. 28, 31, 34, 42 and 43.

Source data

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Yang, Y., Chen, Y., Pei, P. et al. Fluorescence-amplified nanocrystals in the second near-infrared window for in vivo real-time dynamic multiplexed imaging. Nat. Nanotechnol. 18, 1195–1204 (2023). https://doi.org/10.1038/s41565-023-01422-2

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