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Targeted multicolor in vivo imaging over 1,000 nm enabled by nonamethine cyanines

Abstract

Recent progress has shown that using wavelengths between 1,000 and 2,000 nm, referred to as the shortwave-infrared or near-infrared (NIR)-II range, can enable high-resolution in vivo imaging at depths not possible with conventional optical wavelengths. However, few bioconjugatable probes of the type that have proven invaluable for multiplexed imaging in the visible and NIR range are available for imaging these wavelengths. Using rational design, we have generated persulfonated indocyanine dyes with absorbance maxima at 872 and 1,072 nm through catechol-ring and aryl-ring fusion, respectively, onto the nonamethine scaffold. Multiplexed two-color and three-color in vivo imaging using monoclonal antibody and dextran conjugates in several tumor models illustrate the benefits of concurrent labeling of the tumor and healthy surrounding tissue and lymphatics. These efforts are enabled by complementary advances in a custom-built NIR/shortwave-infrared imaging setup and software package for multicolor real-time imaging.

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Fig. 1: Synthesis and characterization of nonamethine cyanines.
Fig. 2: Probe comparison, multiplexing strategy and single-color monoclonal antibody imaging.
Fig. 3: Applications of in vivo multicolor optical imaging.

Data availability

Image datasets, including all raw and processed imaging data generated in this work, are available at BioImage Archive (accession number S-BIAD325). Source data are provided with this paper. All other datasets are included in the Supplementary Information.

Code availability

Custom code (ccda-v3.0.1beta) used to collect data in this work is available at GitHub (https://gitlab.com/brunslab/ccda/).

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Acknowledgements

This work was supported by the Intramural Research Program of the National Institutes of Health including through a NCI FLEX award to M.J.S. (BC011506). We also acknowledge funding from the Helmholtz Zentrum München, the DFG—Emmy Noether program (no. BR 5355/2-1) and from the CZI Deep Tissue Imaging (DTI-0000000248) to O.T.B. U.R.-G. gratefully acknowledges funding from the DFG grant SIREN (DFG RE 1203/38-1). This project has also been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract no. HHSN261200800001E. M.J.S. thanks J. Barchi, NCI-CCR, for nuclear magnetic resonance assistance and J. Kelley, NCI-CCR, for mass spectrometric analysis. M.S. and O.T.B. thank A. Feuchtinger, Helmholtz Munich imaging and pathology core facility, for support with the H&E staining of the cryosections. We also thank S. Glasl, P. Anzenhofer, U. Klemm, M. Warmer, S. Ramakrishnan for the excellent technical support. We also thank T. Klein, H. Robielski and B. Arus for helpful discussion.

Author information

Authors and Affiliations

Authors

Contributions

V.G.B., M.J.S. and O.T.B. designed the project. M.J.S. and O.T.B. jointly advised the project. M.J.S. and J.I. designed and carried out the computational chemistry. V.G.B. and M.P.L. performed organic synthesis and characterization. B.R. and U.R.-G. performed photophysical measurements and analyzed the data. T.S.B., J.G.P.L., M.S. and M.P.L. built the optical imaging setup. M.P.L. and N.L.P. performed phantom imaging experiments. M.N.N. and M.S. performed cell culture. M.S., C.S., C.M.R., L.R. and S.D. established animal tumor models and were responsible for animal handling and care. V.G.B., M.P.L. and M.S. performed bioconjugation chemistry and characterization. M.P.L., M.S., J.D.K., N.L.P. and J.G.P.L. designed and carried out animal imaging experiments. P.T.T. developed the imaging software. M.P.L. and M.S. processed and analyzed the imaging data. M.P.L. and M.J.S. wrote the manuscript. All authors have reviewed and approved the final version of the manuscript. Requests for materials and other correspondence should be addressed to M.J.S. and O.T.B.

Corresponding authors

Correspondence to Oliver T. Bruns or Martin J. Schnermann.

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Competing interests

V.G.B., M.J.S., J.G.P.L., M.S. and O.T.B. have filed patent applications whose value may be affected by this publication. The remaining authors declare no competing interests.

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Peer review information

Nature Methods thanks Samuel Achilefu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Photostability.

Photostability of FNIR-866 and FNIR-1072 compared with IR-800CW (2 μM in 50 mM PBS, pH 7.4) using 20 mW/cm2 irradiation at 780 nm, 850 nm and 1064 nm, respectively. Experiments were run in quadruplicate and plotted as mean values with error bars derived from the standard deviation.

Source data

Extended Data Fig. 2 Chemical stability.

Chemical stability of IR-800CW, FNIR-866, FNIR-1072 (2 μM in 10% FBS/PBS) at room temperature. Experiments were run in quadruplicate and plotted as mean values with error bars derived from the standard deviation.

Source data

Extended Data Fig. 3 Phantom imaging.

Tail emission phantom comparison of 5 μM solutions of heptamethine (green bars) and nonamethine cyanines (cyan and purple bars) under various solvent and filter conditions when excited with their respective laser lines (785 nm, 890 nm, 1064 nm) at a power density of 90 mW/cm2.

Source data

Extended Data Fig. 4 Ex-vivo histological validation of tumor labeling after i.v. injection of IRDye800CW-Pan.

An athymic nude mouse bearing a MDA-MB-468 tumor (n = 1) was injected with 100 ug of IR-800CW-Pan (DOL 1.9, 1.2 nmol IR-800CW). Mouse was sacrificed 48 h after injection and the organs excised and imaged ex vivo (See Supplementary Figure 7). The tumor tissue was snap frozen in liquid nitrogen and 15 mm sections were cut. Adjacent sections were either stained with Hematoxylin/Eosin (H&E) for imaging in transmission or mounted with a DAPI containing mounting medium (ProLong™ Diamond Antifade Mountant with DAPI - ThermoFisher P36971) for imaging fluorescence. To detect fluorescence of IR-800CW and DAPI the section was imaged on a fluorescence microscope equipped with silicon based sCMOS camera and a customized excitation path coupling in a 785 nm laser for excitation. Overview images of the whole tumor section were captured by using the stitching routine of the microscope software using a 20X objective for IR-800CW-Pan and a 4X objective for DAPI. These are shown together with the H&E stained adjacent section in the top row. A magnified view including a central necrotic area and surrounding tumor cells was selected and images of this region are shown in the middle row for DAPI and IR-800CW-Pan. For visualizing the antibody signal at cellular resolution, images for DAPI and IR-800CW -Pan were also taken with a 20X objective and displayed individually and as colored overlay in the bottom row. Scale bars are displayed in the IR-800CW-Pan images. As expected the staining after in vivo labelling with the antibody is heterogeneous. The necrotic areas which can be seen in the H&E stained section are not labeled and the IR-800CW-Pan staining is more pronounced in the tumor rim and vital parts of the tumor. The images with 20X objective achieve cellular resolution and the staining of IR-800CW-Pan is localized on the cell surface. Images shown are representative from n = 3 slides.

Extended Data Fig. 5 Ex-vivo histological validation of tumor labeling after i.v. injection of FNIR-872-Pan.

An athymic nude mouse bearing a MDA-MB-468 tumor (n = 1) was injected with 114 ug of FNIR-872-Pan (DOL 1.1, 0.8 nmol FNIR-872). Mouse was sacrificed 48 h after injection and the organs excised and imaged ex vivo (See Supplementary Figure 7). The tumor tissue was snap frozen in liquid nitrogen and 15 mm sections were cut. Adjacent sections were either stained with Hematoxylin/Eosin (H&E) for imaging in transmission or mounted with a DAPI containing mounting medium (ProLong™ Diamond Antifade Mountant with DAPI - ThermoFisher P36971) for imaging fluorescence. The fluorescence of FNIR-872 and DAPI was imaged on a fluorescence microscope equipped with silicon based sCMOS camera and a customized excitation path coupling in a 785 nm laser for excitation. Overview images of the whole tumor section were captured by using the stitching routine of the microscope software using a 20X objective for FNIR-872-Pan and DAPI. These are shown together with the H&E stained adjacent section in the top row. A magnified image including a central necrotic area and surrounding tumor cells was selected and images of this region are shown in the middle row DAPI and FNIR-872-Pan. For visualizing antibody signal at cellular resolution, images for DAPI and FNIR-872-Pan were also taken with a 20X objective and displayed individually and as colored overlay in the bottom row. Scale bars are displayed in the FNIR-872-Pan images. As expected the staining after in vivo labelling with the antibody is heterogeneous. The necrotic areas which can be seen in the H&E stained section are not labeled and the FNIR-872-Pan staining is more pronounced in the tumor rim and vital parts of the tumor. The images with 20X objective achieve cellular resolution and the staining of FNIR-872-Pan is localized on the cell surface. Images shown are representative from n = 3 slides.

Extended Data Fig. 6 Control for ex-vivo histological validation of tumor labeling after i.v. injection of IRDye800CW-Pan and FNIR-872-Pan.

An athymic nude mouse bearing a MDA-MB-468 tumor (n = 1) was sacrificed without injection of a contrast agent and the organs excised. The tumor tissue was snap frozen in liquid nitrogen and 15 mm sections were cut. Adjacent sections were either stained with Hematoxylin/Eosin (H&E) for imaging in transmission or mounted with a DAPI containing mounting medium (ProLong™ Diamond Antifade Mountant with DAPI - ThermoFisher P36971) for imaging fluorescence. The same settings as IR-800CW and FNIR-872 were applied and DAPI was imaged on a fluorescence microscope equipped with silicon based sCMOS camera and a customized excitation path coupling in a 785 nm laser for excitation. Overview images of the whole tumor section were captured by using the stitching routine of the microscope software. These are shown together with the H&E stained adjacent section in the top row. A magnified view including a central necrotic area and surrounding tumor cells was selected and images of this region are shown in the middle row for DAPI and the 785 nm excitation channel. For visualization with cellular resolution, images for DAPI and the 785 nm excitation channel were also taken with a 20X objective and displayed individually and as colored overlay in the bottom row with the same display settings as for FNIR-872-Pan. Scale bars are displayed in the 785 nm excitation channel images. There is no significant signal in the 785 nm excitation channel.

Extended Data Fig. 7 2-Color Imaging with FNIR-Tag-Dextran and FNIR-872-Pan.

Two color imaging using FNIR-Tag-Dex (10 kDa, 5 nmol effective dye in 100 μL PBS) and FNIR-872-Pan (100 μg, DOL 1.4, 1 nmol effective dye in 100 μL PBS) in an athymic nude mouse bearing an MDA-MB-468 xenograft tumor implanted s.c. in the right flank (n = 1). Imaging settings: Images were acquired on a NIRvana 640ST using 3×1150 long pass filters. The lasers (785 nm for FNIR-Tag-Dex and 890 nm for FNIR-872-Dex) were power matched at 100 mW/cm2 measured over the tumor for each imaging timepoint. Camera exposure times were 75 ms and 400 ms for the FNIR-Tag-Dex and FNIR-872-Pan channels, respectively. For in vivo images, the color scale is from 0-273 and 14-50 counts/ms in the 785 and 890 channel, respectively. For the ex vivo images, the color scale is from 0-440 and 7-88 counts/ms in the 785 and 890 channel, respectively.

Extended Data Fig. 8 Single Color Imaging of Hep3B Orthotopic Liver Tumors.

Side by side comparison of NIR (a) (Perkin-Elmer IVIS) and SWIR (b) detection of a representative Hep3B orthotopic bearing or naïve athymic nude mice after receiving ICG (2 mg/kg). Images were recorded at 72 h postinjection. SWIR detection enables multiplexing of a marker for healthy liver tissue (FNIR-872-Dex) to aid in delineation of the tumor margins (see Fig. 3a). Plot profiles for Hep3B and naïve mice (c, IVIS and d, SWIR), drawn as indicated by the red lines, demonstrate the enhanced resolution of imaging in the SWIR.

Source data

Extended Data Fig. 9 Crosstalk between ICG and FNIR-872-Pan in vivo.

An athymic nude mouse bearing an MDA-MB-468 xenograft tumor implanted orthotopic in the mammary fat pad was imaged before injection (prescan) of FNIR872-Pan (140 μg, DOL 1.1, 1.1 nmol effective dye in 100 μL PBS, i.v.). The mouse was imaged again one day after injection of FNIR872-Pan (pre ICG injection) and ca 1 hour after injection of ICG intradermal (ca. 100 nmol ICG in water) at the base of the tail (post ICG injection). The signal of FNIR872-Pan was calculated as the difference of the intensity in the 890 nm channel (pre ICG injection – prescan) at the tumor. Crosstalk of FNIR872-Pan in the 785 nm channel was calculated as the difference of the intensity in the 785 nm channel (pre ICG injection – prescan) at the tumor. The signal of ICG was calculated as the difference of the intensity in the 785 nm channel (post ICG injection – pre ICG injection) at the lymphatic vessels closed to the tumor. Crosstalk of ICG in the 890 nm channel was calculated as the difference of the intensity in the 890 nm channel (post ICG injection – pre ICG injection) at the same lymphatic vessels. Crosstalk between ICG and FNIR-872-Pan is very small with a signal to crosstalk ratio of around 12 for the 785 nm ICG channel and a signal to crosstalk ratio of around 17 for the 890 nm FNIR-872-Pan channel. Imaging settings: Images were acquired with Allied Vision G-32 camera using 2X 1000 nm long pass filters and 3X 1050 nm long pass filters. Laser 785 nm (prescan dark-subtracted image taken with 50 ms exposure time and divided by a factor of 5 and dark-corrected pre-ICG injection image taken with 25 ms exposure time and divided by a factor of 2.5 to match the time-point after injection of ICG taken with 10 ms exposure time, 80 mW/cm2) and laser 890 nm (prescan dark-subtracted images were taken with 50 ms exposure time and divided by a factor of 2 to match the time-points after injection of Pan-FNIR872 taken with 25 ms exposure time, 115 mW/cm2) were sequentially triggered by CCDA software together with the 1300 nm LED (10 ms exposure time) as reflection channel. MFI of 785 nm channel is displayed in counts/10 ms, whereas MFI of 890 nm channel is displayed in counts/25 ms.

Extended Data Fig. 10 Crosstalk between ICG and FNIR-1072-Pan in vivo.

An athymic nude mouse bearing an MDA-MB-468 xenograft tumor implanted orthotopic in the mammary fat pad was imaged before injection (prescan) of FNIR-1072-Pan (103 μg, DOL 0.53, 0.4 nmol effective dye in 100 μL PBS, i.v.). The mouse was imaged again one day after injection of FNIR1072-Pan (pre ICG injection) and 2 hour after injection of ICG intradermal (ca. 100 nmol ICG in water) at the base of the tail (post ICG injection). The signal of FNIR1072-Pan was calculated as the difference of the intensity in the 968 nm channel (pre ICG injection – prescan) at the tumor. Crosstalk of FNIR1072-Pan in the 785 nm channel was calculated as the difference of the intensity in the 785 nm channel (pre ICG injection – prescan) at the tumor. The signal of ICG was calculated as the difference of the intensity in the 785 nm channel (post ICG injection – pre ICG injection) at the lymph node. Crosstalk of ICG in the 968 nm channel was calculated as the difference of the intensity in the 968 nm channel (post ICG injection – pre ICG injection) at the lymph node. Crosstalk between ICG and FNIR-1072-Pan is very small with a signal to crosstalk ratio of around 15 for the 785 nm ICG channel and a signal to crosstalk ratio of around 140 for the 968 nm FNIR-1072-Pan channel. Imaging settings: images were acquired with Allied Vision G-32 camera using 2X 1000 nm long pass filters and 3X 1050 nm long pass filters. Laser 785 nm (prescan dark corrected images taken with 100 ms exposure time and divided by a factor of 4 to match the time-points after injection of Pan-FNIR1072 taken with 25 ms exposiure time, 80 mW/cm2) and 968 nm (dark subtracted images were taken with 100ms exposure time and divided by a factor of 2 to match the time-points after injection, 350 mW/cm2) were sequentially triggered by CCDA software together with the 1300 nm LED (20 ms exposure time) as reflection channel. MFI of 785 nm channel is displayed in counts/25 ms whereas MFI of 968 nm channel is displayed in counts/50 ms.

Supplementary information

Supplementary Information

Supplementary Figs. 1–9 and Supplementary Notes 1–4

Reporting Summary

Supplementary Video 1

Imaging of an awake mouse 24 h after injection of panitumumab-FNIR-872 for tumor targeting. Reflected light images of the mouse were obtained using an LED at 1,300 nm. Image sequence was acquired with a frame rate of 7 frames per second (f.p.s.).

Supplementary Video 2

Real-time multiplexed in vivo visualization in three colors of lymphatics pumping and lymph nodes in a tumor-bearing mouse 24 h after injection of panitumumab-FNIR-872 for tumor labeling and 30 min after injection of ICG intradermally (i.d.) before injection of dextran-FNIR-1072. Reflected light images of the mouse were obtained using an LED at 1,300 nm. Image sequence was acquired with a frame rate of 4 f.p.s.

Supplementary Video 3

Real-time multiplexed in vivo imaging in four colors of the injection of dextran-FNIR-1072 in a tumor-bearing mouse 24 h after injection of panitumumab-FNIR-872 for tumor labeling and 1 h after injection of ICG (i.d.) for lymphatics and lymph node visualization. Reflected light images of the mouse were obtained using an LED at 1,300 nm. Image sequence was acquired with a frame rate of 4 f.p.s.

Supplementary Video 4

Real-time multiplexed fluorescence-guided dissection of tumor in a mouse injected with panitumumab-FNIR-872 (i.v.), ICG (i.d.) and dextran-FNIR-1072 (i.v.). Reflected light images of the mouse were obtained using an LED at 1,300 nm. Image sequence was acquired with a frame rate of 4 f.p.s.

Supplementary Video 5

Real-time multiplexed fluorescence-guided dissection of lymph nodes in a mouse injected with panitumumab-FNIR-872 (i.v.), ICG (i.d.) and dextran-FNIR-1072 (i.v.). Reflected light images of the mouse were obtained using an LED at 1,300 nm. Image sequence was acquired with a frame rate of 4 f.p.s.

Supplementary Video 6

Real-time multiplexed fluorescence-guided dissection of tumor in a mouse injected with panitumumab-FNIR-1072 (i.v.), ICG (i.d.) and dextran-FNIR-872 (i.v.). Reflected light images of the mouse were obtained using an LED at 1,300 nm. Image sequence was acquired with a frame rate of 6 f.p.s.

Supplementary Video 7

Real-time multiplexed fluorescence-guided dissection of lymph nodes in a mouse injected with panitumumab-FNIR-1072 (i.v.), ICG (i.d.) and dextran-FNIR-872 (i.v.). Reflected light images of the mouse were obtained using an LED at 1,300 nm. Image sequence was acquired with a frame rate of 6 f.p.s.

Source data

Source Data Fig. 1

Singlet oxygen generation quantum yield kinetics.

Source Data Fig. 2

Tumor-to-background data.

Source Data Fig. 3

Plot profile data.

Source Data Extended Data Fig. 1

Photostability kinetic data.

Source Data Extended Data Fig. 2

Chemical stability kinetic data.

Source Data Extended Data Fig. 3

Tail emission quantification.

Source Data Extended Data Fig. 8

Hep3B plot profile data.

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Bandi, V.G., Luciano, M.P., Saccomano, M. et al. Targeted multicolor in vivo imaging over 1,000 nm enabled by nonamethine cyanines. Nat Methods 19, 353–358 (2022). https://doi.org/10.1038/s41592-022-01394-6

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  • DOI: https://doi.org/10.1038/s41592-022-01394-6

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