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Quantitative assessment of near-infrared fluorescent proteins

Nature Methods volume 20, pages 1605–1616 (2023)Cite this article

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Abstract

Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins (NIR FPs), which are rapidly becoming popular probes for a variety of imaging applications. However, the diversity of NIR FPs poses a challenge for end-users in choosing the optimal one for a given application. Here we conducted a systematic and quantitative assessment of intracellular brightness, photostability, oligomeric state, chemical stability and cytotoxicity of 22 NIR FPs in cultured mammalian cells and primary mouse neurons and identified a set of top-performing FPs including emiRFP670, miRFP680, miRFP713 and miRFP720, which can cover a majority of imaging applications. The top-performing proteins were further validated for in vivo imaging of neurons in Caenorhabditis elegans, zebrafish, and mice as well as in mice liver. We also assessed the applicability of the selected NIR FPs for multicolor imaging of fusions, expansion microscopy and two-photon imaging.

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Fig. 1: Quantitative characterization of NIR FPs in cultured mammalian cells.
Fig. 2: Quantitative characterization of the selected NIR FPs expressed in L2/3 cortical neurons in mouse brain tissue.
Fig. 3: Characterization of the selected NIR FPs in neurons in live larval zebrafish at 4 dpf.
Fig. 4: Characterization of the selected NIR FPs in neurons in live C. elegans.
Fig. 5: Multicolor imaging with NIR FPs.

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Data availability

The mass spectrometry proteomics data generated in this study have been deposited in the iProX database with the dataset identifier IPX0005767000 (https://www.iprox.cn//page/SCV017.html?query=IPX0005767000). The total size of the files acquired for this study was about 2 TB, which exceeds the limit of the FigShare repository, therefore only the most essential raw datasets, that is, the raw images with metadata supporting the results in Figs. 1–5, Extended Data Figs. 1–7 and Supplementary Figs. 2, 3, 5, 7, 9, 11 are available at FigShare (https://figshare.com/authors/Hanbin_Zhang/14524646 and https://doi.org/10.6084/m9.figshare.21975770). The rest of the files are available from the corresponding author upon request. Source data files are provided with this paper. All plasmids used in this study are available from Addgene and WeKwikGene (https://wekwikgene.wllsb.edu.cn/). Source data are provided with this paper.

Code availability

The custom MatLab code for analysis photostability data is available at Zenodo (https://doi.org/10.5281/zenodo.7992722).

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Acknowledgements

The authors thank J. Pan, J. Hu, X. Bai and the Mass Spectrometry and Metabolomics Core Facility of Westlake University for protein sample mass spectrometry analysis. The authors also thank Z. Dong from Westlake University for help with the interpretation of the mass spectrometry results; the Laboratory Animal Resources Center, the Flow Cytometry Core Facility and the Microscopy Core Facility in Westlake University. Henrietta Lacks, and the HeLa cell line that was established from her tumor cells without her knowledge or consent in 1951, have made significant contributions to scientific progress and advances in human health. We are grateful to Henrietta Lacks, now deceased, and to her surviving family members for their contributions to biomedical research. This work is supported by start-up funding from the Foundation of Westlake University, National Natural Science Foundation of China grant 32050410298 and 32171093, 2020 BBRF Young Investigator Grant 28961, and MRIC Funding 103536022023 to K.D.P., the German Research Foundation (DFG, K1949/7-2) project 241961032 and VW-Vorab des Landes Niedersachsen (project HomeoHirn(ZN3673)) to R.W.K., the Foundation of Westlake University and the National Natural Science Foundation of China grant 32071151 to L.W., and the NIH BRAIN program U24NS109107 grant to M.D.

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Author notes

  1. These authors contributed equally: Hanbin Zhang, Stavrini Papadaki.

Authors and Affiliations

  1. School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China

    Hanbin Zhang, Stavrini Papadaki, Xiaoting Sun, Luxia Yao, Lianfeng Wu & Kiryl D. Piatkevich

  2. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China

    Hanbin Zhang, Stavrini Papadaki, Xiaoting Sun, Luxia Yao, Lianfeng Wu & Kiryl D. Piatkevich

  3. Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China

    Hanbin Zhang, Stavrini Papadaki, Xiaoting Sun, Luxia Yao, Lianfeng Wu & Kiryl D. Piatkevich

  4. Division of Cellular and Molecular Neurobiology, Zoological Institute, Technische Universität Braunschweig, Braunschweig, Germany

    Xinyue Wang, Michel Rehbock, Reinhard W. Köster & Kazuhiko Namikawa

  5. Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT, USA

    Mikhail Drobizhev

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Contributions

H.Z. and S.P. performed characterization in cultured mammalian cells. X.S. characterized proteins in the expansion microscopy protocols. M.D. and H.Z. characterized proteins in vitro. H.Z. and X.S. performed the mouse experiments, X.W., M.R., R.W.K. and K.N. performed the zebrafish experiments, S.P., L.Y. and L.W. performed the C. elegans experiments. H.Z., S.P. and K.D.P. analyzed the data and wrote the manuscript with input from all of the authors. K.D.P. designed and oversaw all aspects of the project.

Corresponding author

Correspondence to Kiryl D. Piatkevich.

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Competing interests

K.D.P. is the co-founder of a company that pursues commercial applications of expansion microscopy and is listed as an inventor on several patent applications concerning development of new expansion microscopy methods. All other authors have no competing interests.

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Nature Methods thanks Erik Snapp, Lin Tian, and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Quantitative assessment of intracellular brightness and photostability of NIR FPs in live HEK cells.

(a) Intracellular normalized brightness of NIR FPs imaged in three channels: (i) Cy5-LP, (ii) Cy5-BP and (iii) Cy5.5 under 55 mW/mm2 excitation power (n > 2000 cells for each NIR-FP from three independent transfections in each channel). Brightness for each FP was normalized to the EGFP signal (here and in panel e). Asterisks (*) indicate signal-to-background ratio <2.0 throughout; in panels a-c box plots highlighted with brown correspond to top 5 NIR FPs for each panel. (b) Photobleaching half-times of NIR-FPs under Cy5 (i and ii, excitation power 58 mW/mm2) and Cy5.5 (iii, excitation power 56–58 mW/mm2; n > 93 cells for each NIR-FP from four independent transfections). (c) Product of mean brightness and mean half-time fluorescence presented as bar graph (mean ± SEM) (due to small value of SEM the error bars are not visible for some bars). (d) Normalized photobleaching curves of NIR FPs under (i) Cy5 (excitation power 58 mW/mm2) and (ii) Cy5.5 (excitation power 56–58 mW/mm2) illumination for the results shown in panel b (n > 93 cells for each NIR-FP from four independent transfections in each channel). Fluorescence was normalized to the intensity value of corresponding FP at t = 0 s. NIR FPs that exhibited signal-to-background ratio lower than 2.0 in Cy5.5 channel are not shown in the graph, however the corresponding curves are available in the source datasets except for mNeptune2.5, which was not visible in Cy5.5 channel at all and thus was not measured. (e) Side-by-side normalized brightness comparison without and with BV administration imaged in (i) Cy5-LP, (ii) Cy5-BP and (iii) Cy5.5 under 55 mW/mm2 excitation power (n > 2000 cells for each NIR-FP from three independent transfections in each channel). Outliers not shown but included in all calculations and available in the source datasets. See Supplementary Table 2 and Supplementary Dataset 1 for the detailed descriptive statistics and exact p-values.

Source data

Extended Data Fig. 2 Quantitative assessment of intracellular brightness and photostability NIR FPs in live primary cultured mouse neurons.

(a, b) Intracellular normalized brightness of NIR FPs imaged after (a) Calcium Phosphate transfection and (b) viral transduction in Cy5-LP, (ii) Cy5-BP and (iii) Cy5.5 under 55 mW/mm2 excitation power (n > 40 neurons for each NIR FP from two independent cultures). Brightness for each FP was normalized to the EGFP signal (here and in panel f). In panels a-d box plots highlighted with brown correspond to top 5 NIR FPs for each panel. (c) Photobleaching half-times of NIR FPs under Cy5 (i and ii, excitation power 55 mW/mm2) and Cy5.5 (iii, excitation power 55 mW/mm2; n > 42 neurons for each NIR FP from two independent cultures). NA – not applicable due to low fluorescence signal. (d) Product of mean brightness and mean half-time fluorescence presented in bar graph (mean ± SEM) (due to small value of SEM the error bars are not visible for some bars). (e) Normalized photobleaching curves of NIR-FPs under (i) Cy5 (excitation power 55 mW/mm2) and (ii) Cy5.5 (excitation power 55 mW/mm2) illumination for the results shown in c. Fluorescence was normalized to the intensity value of corresponding FP at t = 0 s. (f) Side-by-side normalized brightness comparison before and after BV administration imaged in (i) Cy5-LP, (ii) Cy5-BP and (iii) Cy5.5 under 55 mW/mm2 power (n > 20 neurons for each NIR FP from two independent cultures). see Supplementary Dataset 1 for the detailed descriptive statistics and exact p-values.

Source data

Extended Data Fig. 3 Quantitative characterization of NIR FPs in fixed HEK cells.

(a) NIR-to-green fluorescence ratio in fixed HEK cells imaged in (i) Cy5-LP, (ii) Cy5-BP and (iii) Cy5.5 under 55 mW/mm2 excitation power (n > 1976 cells for each NIR FP from two independent transfections in each channel). Brightness for each FP was normalized to the EGFP signal. Asterisks (*) indicate signal-to-background ratio <2.0 throughout the figure based on brightness of fixed HEK cells; in panels a, c, d box plots highlighted with brown correspond to top 5 NIR FPs for each panel. (b) Normalized photobleaching curves of NIR FPs under (i) Cy5 (excitation power 55 mW/mm2) and (ii) Cy5.5 (excitation power 55 mW/mm2) illumination in fixed HEK cells (n = 40 cells for each NIR FP from two independent transfections in each channel). Fluorescence was normalized to the intensity value of corresponding FP at t = 0 s. NIR FPs that exhibited lower than 2.0 signal-to-background ratio in Cy5.5 channel are not shown in the graph; however, the corresponding curves are available in the source datasets. (c) The products of mean NIR-to-green fluorescence ratio and mean fluorescence half-time in (i) Cy5-LP and (ii) Cy5.5 are presented in the form of bar graph (mean ± SE). (d) The products of mean absolute NIR brightness to mean fluorescence half-time in (i) Cy5-LP and (ii) Cy5.5 are presented in the form of bar graph (mean ± SE). (e) Comparison of selected NIR FPs in proExM. (e) Representative images of NIR FPs in PFA-fixed and proExM treated HEK cells acquired in Cy5-LP (NIR FP) and FITC (GFP) channels (n > 61 cells for each NIR FP from one independent transfection). The dynamic brightness range was adjusted independently to facilitate visualization of transfected cells in fixed and proExM processes for each NIR FPs. Images were obtained through single-plane scanning and without projection processing. (f) Fluorescence retention of NIR FPs in HEK cells upon PFA fixation presented as bar graph (mean of NIR retention for each protein) (Cy5-LP, n > 1974 cells for each NIR FP from two independent transfections). Black dots indicate ratio of mean brightness before and after fixation for two transfections. (g) Absolute NIR fluorescence of fixed (brown box plots with notches) and proExM treated (orange box plots with notches) HEK cells (n > 61 cells for each NIR FP from one independent transfection). (h) Retention of GFP and NIR FPs’ fluorescence in fixed HEK cells after proExM treatment (n > 61 cells for each NIR FP from one independent transfection). See Supplementary Table 2 and Supplementary Dataset 1 for the detailed descriptive statistics and exact p-values.

Source data

Extended Data Fig. 4 Fluorescence imaging of NIR FPs fusions in live HeLa cells.

Representative images of (from top left) iRFP670-actin, miRFP670-2-actin, miRFP680-actin, miRFP713-actin, miRFP720-actin, H2B-iRFP670, H2B-miRFP670-2, H2B-miRFP680, H2B-miRFP713, H2B-miRFP720, Cx43-iRFP670, Cx43-miRFP670-2, Cx43-miRFP680, Cx43-miRFP713, Cx43-miRFP720, keratin-iRFP670, keratin-miRFP670-2, keratin-miRFP680, keratin-miRFP713, keratin-miRFP720, iRFP670-tubulin, miRFP670-2-tubulin, miRFP680-tubulin, miRFP713-tubulin, miRFP720-tubulin (n > 15 cells for each construct from two independent transfection). For each image the dynamic range was adjusted independently to facilitate visualization and images were generated through maximum projection.

Extended Data Fig. 5 Two-photon cross-section spectra of selected NIR-FPs.

Two-photon cross-section spectra of mCardinal, emiRFP670, miRFP670-2, miRFP680, mRhubarb713, miRFP713, iRFP713, and miRFP720 presented versus laser wavelength used for excitation (GM, Goeppert-Mayer units).

Source data

Extended Data Fig. 6 Quantitative characterization of the selected NIR FPs expressed in L2/3 cortical neurons in mouse brain tissue.

(a, b, c) Intracellular normalized brightness of NIR FPs imaged in acute brain slices from (a) one-month, (b) two-month, (c) three-month old mice in (i) Cy5-LP, (ii) Cy5-BP, (iii) Cy5.5 channels (excitation power 55 mW/mm2; n ≥ 100 neurons from 3 mice for each protein in each channel). Brightness for each FP was normalized to the EGFP signal (here and in panel d). (d) Intracellular normalized brightness of NIR-FPs imaged in PFA-fixed brain slices from one-month old mice in (i) Cy5-LP, (ii) Cy5-BP, (ii) Cy5.5 (excitation power 55 mW/mm2; n > 90 neurons from 2 mice for each protein in each channel). (e, f) Normalized photobleaching curves of NIR-FPs measured in (e) acute brain slices and (f) PFA-fixed brain slices in (i) Cy5-LP and (ii) Cy5.5 channels (excitation power 55 mW/mm2; n > 40 neurons from 2 mice for each protein for each channel). Fluorescence was normalized to the intensity value of corresponding FP at t = 0 s. See Supplementary Dataset 1 for the detailed descriptive statistics and exact p-values.

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Extended Data Fig. 7 Quantitative characterization of the selected NIR FPs expressed in mouse liver in vivo.

((a) Representative wide-field fluorescence images of NIR FPs expressed in mouse liver acquired in Cy5-LP, Cy5-BP, Cy5.5, and FITC channels (excitation power 55 mW/mm2 for Cy5 and Cy5.5 channels; n = 3 liver slices from 3-4 mice for each protein). (b,c,d) Intracellular normalized brightness of NIR FPs imaged in fresh liver tissue from three-month-old mice in (b) Cy5-LP, (c) Cy5-BP, (d) Cy5.5 channels (excitation power 55 mW/mm2; n > 154 cells from 3-4 mice for each protein in each channel). Brightness for each FP was normalized to the EGFP signal. See Supplementary Dataset 1 for the detailed descriptive statistics and exact p-values.

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Extended Data Table 1 List of the NIR FPs selected for quantitative assessment in cultured cells56,57,58,59
Full size table
Extended Data Table 2 Spectroscopic and biochemical properties of the selected NIR FPs measured in this study
Full size table

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2, Tables 1–11 and Figs. 1–21.

Reporting Summary

Supplementary Video 1

The dynamic protrusions in the growth cone are visualized using Lifeact-mClover3 marking F-actin, whereas EB3-emiRFP670 labels microtubule plus-ends moving towards the tip of the growth cone dominantly. Elapsed time in min : s. Scale bar, 5 µm.

Supplementary Dataset 1

Statistical analysis of the data in this paper

Supplementary Dataset 2

Oligonucleotide sequences

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Zhang, H., Papadaki, S., Sun, X. et al. Quantitative assessment of near-infrared fluorescent proteins. Nat Methods 20, 1605–1616 (2023). https://doi.org/10.1038/s41592-023-01975-z

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