High-resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to substantial scattering and autofluorescence in tissue at visible (350–700 nm) and near-infrared (700–1,000 nm) wavelengths. Here, we enable real-time, non-invasive multicolour imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000–2,000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established relationships between structure and photophysical properties for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable near-infrared/SWIR excitation and single-channel detection, facilitating video-rate multicolour SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1,064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-colour imaging with high temporal and spatial resolutions.
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Snapshot multicolor fluorescence imaging using double multiplexing of excitation and emission on a single detector
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Image datasets, including all raw and processed imaging data generated in this work, are available at BioImage Archive (accession number: S-BIAD27). All other datasets that support the findings of this study are contained within the manuscript and its Supplementary Information.
Custom computer programs used for the work are available at GitHub (https://gitlab.com/brunslab/ccda).
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This work was supported by grants to E.D.C. (NSF GRFP DGE-1144087, Christopher S. Foote Fellowship), O.T.B. (Emmy-Noether-Programme of DFG BR 5355/2-1, Helmholtz Pioneer Campus Institute for Biomedical Engineering), E.M.S. (UCLA, Sloan Research Award FG-2018-10855, NIH 1R01EB027172-01) and Helmholtz Zentrum München, and by shared instrumentation grants from the NSF (CHE-1048804) and NIH (1S10OD016387-01). We thank T. Schwarz-Romond, T. S. Bischof, D. Bengel (Helmholtz Zentrum München), K. N. Houk, J. R. Caram (UCLA) and L. D. Lavis (Janelia) for discussions and support and the Garcia-Garibay Group (UCLA) for instrumentation.
Material presented in this work is included in patent and patent applications US20200140404, EP3634397 and WO2018226720 with authors E.M.S and E.D.C. and PCT/EP2020065753 with authors O.T.B., E.D.C., J.G.P.L., M.W., S.R., M.S. and E.M.S.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The 7-amino-4-methyl flavylium heterocycles were accessed through flavone intermediates 13. Three routes were used to obtain flavones 13, Mentzer pyrone synthesis, Buchwald-Hartwig coupling, and acylation. Conditions and yields for each derivative can be found in the listed Supplementary Tables.
Extended Data Fig. 2 Resolution effects upon excitation multiplexing and single-channel detection in the SWIR.
a, Images of a mouse phantom acquired after injection of JuloFlav7 (3) (94 nmol) and immediate euthanasia. Images were acquired with 785 nm excitation (93 mWcm−2), 980 nm (30 mWcm−2), and 1,064 nm (11 mWcm−2) and collection 1,150–1,700 nm (48 ms, 20 fps). Displayed images are averaged over 200 frames. b, To observe resolution, 12 cross-sections were drawn over different vessels (labelled in a) and the baseline subtracted and normalized cross-sections are overlaid. ex. = excitation; LP = longpass.
Extended Data Fig. 3 Resolution effects observed upon emission multiplexing with a single excitation wavelength in the SWIR.
a, Images of a mouse phantom acquired after injection of JuloFlav7 (3) (94 nmol) and immediate euthanasia, after one freeze-thaw cycle. Images were acquired with 980 nm excitation. Laser powers and exposure time are as follows (1) 1,000 nm LP = 8.1 mWcm−2, 30 ms; 1,300 nm LP = 24 mWcm−2, 500 ms; 1,500 nm LP = 177 mWcm−2, 500 ms. Displayed images are averaged over 200 frames. b, To observe resolution, 5 cross-sections were drawn over different vessels (labelled in a) and the baseline subtracted and normalized cross-sections are overlaid. LP = longpass.
a, Whole mouse imaging at selected time-points after i.v. injection of JuloFlav7 (3) micelles (44 nmol) in PBS buffer with excitation at 1,064 nm (103 mWcm−1) and 1,150–1,700 nm collection (8 ms exposure time, 100 fps). b, Imaging of the mouse hind-limb at selected time points after i.v. injection of JuloFlav7 (3) micelles (55 nmol) with excitation at 1,064 nm (95 mWcm−1) with 1,100–17,00 nm collection (9 ms exposure time, 100 fps). Displayed images are averaged over 5 frames. Scale bars represent 1 cm. Data are representative of two replicate experiments.
Abbreviations, Supplementary Tables 1–5, Figs. 1–16, List of Supplementary Videos 1–6, figure experimental procedures, extended data and supplementary figure experimental procedures, Notes 1–5, synthetic procedures and compound characterization.
Single-channel in vivo imaging with excitation 1,064 nm at 100 fps. Image sequence was frame averaged by a factor of three to reduce file size and is displayed in real time.
Video-rate multiplexed imaging in vivo of JuloFlav7 injection. Image sequence was frame averaged by a factor of three to reduce file size and is displayed at three times the speed.
Video-rate multiplexed imaging in vivo of ICG injection. Image sequence was frame averaged by a factor of three to reduce file size and is displayed at three times the speed.
Imaging of an awake mouse in three colours. Image sequence was not frame averaged and is displayed in real time.
Orthogonal circulatory and lymphatic imaging. Image sequence was frame averaged by a factor of two to reduce file size and is displayed at three times the speed.
Two-colour demonstration of image-guided necropsy. Image sequence was frame averaged by a factor of eight to reduce file size and is displayed at five times the speed.
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Cosco, E.D., Spearman, A.L., Ramakrishnan, S. et al. Shortwave infrared polymethine fluorophores matched to excitation lasers enable non-invasive, multicolour in vivo imaging in real time. Nat. Chem. 12, 1123–1130 (2020). https://doi.org/10.1038/s41557-020-00554-5
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