Abstract
Organic dyes play a crucial role in live-cell imaging because of their advantageous properties, such as photostability and high brightness. Here we introduce a super-photostable and bright organic dye, Phoenix Fluor 555 (PF555), which exhibits an order-of-magnitude longer photobleaching lifetime than conventional organic dyes without the requirement of any anti-photobleaching additives. PF555 is an asymmetric cyanine structure in which, on one side, the indole in the conventional Cyanine-3 is substituted with 3-oxo-quinoline. PF555 provides a powerful tool for long-term live-cell single-molecule imaging, as demonstrated by the imaging of the dynamic single-molecule interactions of the epidermal growth factor receptor with clathrin-coated structures on the plasma membrane of a live cell under physiological conditions.
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Data availability
The raw high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy data are provided as Extended Data Fig. 9 and Supplementary Figs. 1–6, 8–23, 25, 26 and 39–41. The raw data for Figs. 4d–i and 5 are provided as Supplementary Videos 2–8. The data and all unique materials generated in this study are available from the corresponding authors D.-H.K., Y.-T.C. and S.H.R. upon reasonable request and in compliance with material transfer agreements. Source data are provided with this paper.
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Acknowledgements
This work was supported by Global Research Laboratory (GRL) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (no. NRF-2016K1A1A2912722 to S.H.R.), National Research Foundation of Korea (NRF) grant funded by the Ministry of Education Science and Technology of Korea (MEST) (no. NRF-2019R1A2C2002152 to S.H.R.), Basic Science Research Institute Fund grant no. 2021R1A6A1A10042944 (Y.-T.C.) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (nos 2023R1A2C300453411 and RS-2024-00411069 to Y.-T.C.), Glocal University 30 project (Molecular Imaging Center, POSTECH) (H.M.T. and Y.-T.C.), SUTD Kickstarter Initiative (No. SKI 2021_03_10) (X.L.). T.H. is an investigator with Howard Hughes Medical Institute. We also acknowledge the computing service of the National Supercomputing Centre (NSCC) (Singapore).
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Supervision: D.-H.K., Y.-T.C. and S.H.R.; funding: Y.-T.C. and S.H.R.; conceptualization: D.-H.K.; investigation: D.-H.K., H.M.T. and S.H.L.; analysis: D.-H.K., H.M.T., S.H.L. and Y.-T.C.; MSD analysis: S. Jazani and T.H.; HR–MS and tandem MS: S. Jang and J.S.; quantum chemical calculation: S.A.A.A. and X.L.; paper: D.-H.K., H.M.T. and S.H.L.
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Y.-T.C., S.H.L., H.M.T., D.-H.K. and S.H.R. are inventors on a patent application (10-2023-0150395). The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Super-photostability of in situ photoblued AF647 in live-cell imaging.
a. The schematic illustrates the in situ photoblueing reaction of AF647 conjugated to membrane proteins on the plasma membrane (PM) of live COS7 cells. The reaction is triggered by the illumination of a 642 nm laser, resulting in a blue-shifted absorption and emission spectrum. b. Fluorescence images of AF647 localized on the PM of live COS7 cells. Images are captured in both the far-red channel (wavelength range: 654–870 nm) and the red channel (wavelength range: 572–624 nm). The images showcase the blue shift of AF647 spectrum before and after the in situ photoblueing reaction. This experiment was performed on n = 24 different cells across 5 independent culture batches. c. Single-molecule images of photoblued AF647 acquired over a 30-minute duration. Imaging was conducted in the red channel (wavelength range: 572–624 nm) using a frame rate of 20 Hz. This single-molecule imaging was performed on n = 24 different cells across 5 independent culture batches.
Extended Data Fig. 2 Distribution of the number of photobleaching steps of PF555 single molecules intensity profiles.
The percentage of photobleaching steps for both samples were normalized from 827 intensity profiles for PF555 and 761 intensity profiles for AF555 across n = 7 independent measurements. Data are presented as mean values +/− SEM.
Extended Data Fig. 3 Cytotoxicity assessment of PF555 and CA(O4)-PF555 using the WST assay.
COS7 cells were incubated with various concentrations of CA-AF555, CA-PF555, PF555, and Paclitaxel for up to 36 h under cell growth conditions. The WST assay was performed on n = 3 independent culture batches. Data are presented as mean values +/− SD.
Extended Data Fig. 4 The versatility and photostability of CA(O4)-PF555 for labeling Halo-B2AR and Halo-FIS1.
a, c. TIRF images of Halo-B2AR (panel a) and Halo-FIS1 (panel b) over 600 s in live COS7 cells labeled with CA(O4)-PF555 and CA-AF555. Halo-FIS1 expressed COS7 cells were fixed and permeabilized before labeling with CA(O4)-PF555. Scale bar = 20 μm. b, d. Photobleaching lifetime of CA(O4)-PF555 (red) and CA-AF555 (blue). The intensity profiles of both samples were measured from a total of n = 33 cells for CA(O4)-PF555, and n = 32 cells for CA-AF555, across 10 independent culture batches. The intensity profiles were averaged, and the mean values were used to fit an exponential decay curve to determine the average photobleaching lifetime. Data points are presented as mean values +/− SD.
Extended Data Fig. 5 Brightness of CA(O4)-PF555, CA-AF555, CA-JFX549, and CA-TMR under different laser power intensities.
Single-molecule fluorescence images of the corresponding dyes on the surface of a cleaned glass coverslip under different laser power intensities. All dyes are measured in PBS solution. Scale bar = 10 μm. The intensity values of each sample on the glass surface were measured from n = 12 independent measurements.
Extended Data Fig. 6 Photoactivation and spontaneous recovery of CA(O4)-PF555.
a, d. TIRF images of Halo-EGFR in live COS7 cells labeled with CA(O4)-PF555 after 30 minutes of photobleaching with snapshots taken every 10 s over 25 minutes without (c) or with (d) a 10 s 405 nm laser pulse between snapshots. Scale bar = 20 μm. b, e. Intensity profiles from acquired images were extracted and fitted with an exponential decay function to quantify the rate and amount of spontaneous recovery (e) and photoactivation (f). c, f. Photobleaching lifetime of spontaneous recovered species (c) and photoactivated species (f) calculated by exponential decay fitting. g. The rate (left panel), amount (middle panel) and photobleaching lifetime (right panel) of photoactivation (blue) and spontaneous recovery (red) were determined by fitting the exponential decay function to the intensity profile of the CA(O4)-PF555 signal. The rate, amount and photobleaching lifetime measurements were repeated on n = 10 cells across 3 independent culture batches. Data are presented as mean +/− SD. P values are the result of a two-sided t-test. Left panel: ****P = 4.4 × 10−16. Middle panel: P = 0.0791 (ns: not significant). Right panel: ****P = 6.2 × 10−14. h. Intensity profile of CA(O4)-PF555 (black line) under repeated photoactivation by 10 s 405 nm laser pulses (pink line).
Extended Data Fig. 7 Evaluation of photobleaching lifetime of PF555 under PCA/PCD conditions in live COS7 cells.
a. Representative single-molecule images of HALO-EGFR labeled with CA-AF647, CA(O4)-PF555, and CA-TMR, both with and without the addition of PCA/PCD, on the PM of living COS7 cells. The images were acquired using a 561 nm laser with an intensity of 30 W/cm2 and an EM-CCD camera with a frame rate of 20 Hz. Photobleaching lifetime values were extracted from n = 7 cells across 7 independent culture batches. Scale bar = 10 µm. b. The graph depicts the time-dependent reduction in the number of fluorescence spots detected in each 50-ms frame for CA-AF647, CA(O4)-PF555, and CA-TMR on the PM of live COS7 cells. The black lines represent the best-fit single-exponential decay functions for each dye, and the derived exponential decay time constants are indicated. c. The photobleaching lifetime, represented as the exponential decay time constant, was calculated for CA-AF647, CA(O4)-PF555, and CA-TMR under both control and PCA/PCD-supplemented conditions. Photobleaching lifetime values were extracted from n = 7 cells across 7 independent culture batches. Data are presented as mean +/− SD.
Extended Data Fig. 8 Quantum chemical calculation of PF555 and Cy5.
a. Calculated ionization potential from the S1 (left) and the T1 (right) states of Cy5 and PF555. b, e. Calculated energy levels of various states of Cy5 (panel b) and PF555 (panel e). The insets show the oscillator strength (f), and spin–orbit-coupling (ζ). The top panels show the optimized geometries of Cy5 and PF555 in the ground state. Potential energy surface of Cy5 (panel c) and PF555 (panel f) as a function of rotations along the polymethine bridge (as indicated by red arrows). Electron and hole distributions in the locally excited (LE) and the twisted intramolecular charge transfer (TICT) states of Cy5 (panel d) and PF555 (panel g).
Extended Data Fig. 9 LC-MS analysis of in vitro photoblued AF647.
a. HPLC chromatography comparison between before (dash) and after photoblueing of AF647 (solid). Mobile phase gradient: H2O with 0.1% TFA (solvent A) and MeOH with 0.1% TFA (solvent B); t = 0-5 min, 10%B hold, t = 5–55 min, 10%B to 50%B; flow rate: 1.2 ml/min; column: C18 column (Phenomenex, 5 μm, 100 Å, 4.6 × 250 mm); column oven: 30 °C. b. positive (left) and negative mode (right) mass spectra of Cy3. Chemical formula of Cy3: C34H44N2O14S4 (calculated m/z: 832.2). c. positive (left) and negative mode (right) mass spectra of peak 1. Chemical formula of peak 1: C35H44N2O15S4 (calculated m/z: 860.2). d. positive (left) and negative mode (right) mass spectra of peak 2. Chemical formula of peak 2: C40H52N2O17S4 (calculated m/z: 960.2). Mass analysis condition: ion source – API-ES; drying gas flow – 11.3 l/min; nebulizer pressure – 35 psi; drying gas temperature – 350 °C; capillary voltage – 3000 V.
Extended Data Fig. 10 Identification of super-photostable photoblued PF610 derived from the photoblueing of Sulfo-Cy7.
a. In vitro photobleaching lifetime analysis of PF610 and AF647. The upper panel displays bulk fluorescence images of PF610 in the far-red channel (654–870 nm). Scale bar = 20 μm. The lower panel shows the averaged fluorescence intensity profiles fitted with exponential decay curves of PF610 (red line and dots) and AF647 (blue line and dots). b. The single-molecule intensity profile of PF610. The upper panel features single-molecule images of PF610 in the far-red channel (654–870 nm). Scale bar = 1 μm. The lower panel presents the fluorescence intensity profile of single PF610 molecules over time. c. HPLC chromatography comparison before (dash) and after (solid) the Sulfo-Cy7 photoblueing reaction. Different colors in the chromatogram indicate different absorption wavelengths, ranging from 500–630 nm, of the corresponding compounds. d. Excitation (yellow line) and emission spectrum (pink line) of PF610 measured in distilled water.
Supplementary information
Supplementary Information
Supplementary Method and Figs. 1–41.
Supplementary Video 1
The raw movie for PF555-labeled EGFR in a live cell. The raw movie for single-molecule Halo-EGFR labeled with CA(O4)-PF555 acquired using TIRFM at 20 Hz on a single live COS7 cell in DMEM at 37 °C and 5% CO2. The movie was compressed using an MPEG-4 standard. Scale bar, 2 µm.
Supplementary Video 2
The raw movie and single-particle tracking of CA(O4)-PF55-labeled Halo-EGFR in a live cell for Fig. 4d. Left: The raw movie for single-molecule Halo-EGFR labeled with CA(O4)-PF555 acquired using TIRFM at 20 Hz on a single live COS7 cell in DMEM at 37 °C and 5% CO2. Right: Single-particle tracking of Halo-EGFR. The EGFR trajectory was overlaid over the time course of imaging (red lines). The movie was compressed using an MPEG-4 H.264 standard. Scale bar, 1 µm.
Supplementary Video 3
The raw movie and single-particle tracking of CA(O4)-PF555-labeled Halo-EGFR in a live cell for Fig. 4e. Left: The raw movie for single-molecule Halo-EGFR labeled with CA(O4)-PF555 acquired using TIRFM at 20 Hz on a single live COS7 cell in DMEM at 37 °C and 5% CO2. Right: Single-particle tracking of Halo-EGFR. The EGFR trajectory was overlaid over the time course of imaging (red lines). The movie was compressed using an MPEG-4 H.264 standard. Scale bar, 1 µm.
Supplementary Video 4
The raw movie and single-particle tracking of CA(O4)-PF555-labeled Halo-EGFR in a live cell for Fig. 4f. Left: The raw movie for single-molecule Halo-EGFR labeled with CA(O4)-PF555 acquired using TIRFM at 20 Hz on a single live COS7 cell in DMEM at 37 °C and 5% CO2. Right: Single-particle tracking of Halo-EGFR. The EGFR trajectory was overlaid over the time course of imaging (red lines). The movie was compressed using an MPEG-4 H.264 standard. Scale bar, 1 µm.
Supplementary Video 5
The raw movie and single-particle tracking of CA(O4)-PF555-labeled Halo-EGFR in a live cell for Fig. 4g. Left: The raw movie for single-molecule Halo-EGFR labeled with CA(O4)-PF555 acquired using TIRFM at 20 Hz on a single live COS7 cell in DMEM at 37 °C and 5% CO2. Right: Single-particle tracking of Halo-EGFR. The EGFR trajectory was overlaid over the time course of imaging (red lines). The movie was compressed using an MPEG-4 H.264 standard. Scale bar, 1 µm.
Supplementary Video 6
The raw movie and single-particle tracking of CA(O4)-PF555-labeled Halo-EGFR in a live cell for Fig. 4h. Left: The raw movie for single-molecule Halo-EGFR labeled with CA(O4)-PF555 acquired using TIRFM at 20 Hz on a single live COS7 cell in DMEM at 37 °C and 5% CO2. Right: Single-particle tracking of Halo-EGFR. The EGFR trajectory was overlaid over the time course of imaging (red lines). The movie was compressed using an MPEG-4 H.264 standard. Scale bar, 1 µm.
Supplementary Video 7
The raw movie and single-particle tracking of CA(O4)-PF555-labeled Halo-EGFR in a live cell for Fig. 4i. Left: The raw movie for single-molecule Halo-EGFR labeled with CA(O4)-PF555 acquired using TIRFM at 20 Hz on a single live COS7 cell in DMEM at 37 °C and 5% CO2. Right: Single-particle tracking of Halo-EGFR. The EGFR trajectory was overlaid over the time course of imaging (red lines). The movie was compressed using an MPEG-4 H.264 standard. Scale bar, 1 µm.
Supplementary Video 8
The raw movie and the quantitative analysis for simultaneous multicolor imaging of PF555-labeled EGFR and EGFP-CLC in a live cell. Simultaneous multicolor imaging of single-molecule EGFR and CCSs in a live COS7 cell. Left: The raw movie for multicolor overlay of single-molecule Halo-EGFR labeled with CA(O4)-PF555 (red) and EGFP-CLC (green) acquired using TIRFM at 14.3 Hz and 0.36 Hz, respectively, on a single live COS7 cell in DMEM at 37 °C and 5% CO2. Middle left: Single-particle tracking of EGFR. The EGFR trajectory was overlaid over the time course of imaging (red lines). Middle right: Boundary detection of EGFP-CLC. The boundaries of the CCSs were overlaid over the time course of imaging (red lines). Right: Overlay movie of the EGFR trajectory and the boundaries of CCSs. The movie was compressed using an MPEG-4 standard. Scale bar, 1 µm.
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Kim, DH., Triet, H.M., Lee, S.H. et al. Super-photostable organic dye for long-term live-cell single-protein imaging. Nat Methods 22, 550–558 (2025). https://doi.org/10.1038/s41592-024-02584-0
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DOI: https://doi.org/10.1038/s41592-024-02584-0
