A synergistic strategy to develop photostable and bright dyes with long Stokes shift for nanoscopy.

Detailed synthesis procedures, spectroscopic data of all compounds are provided in the Supplementary Information .

Materials and general methods

Unless otherwise stated, all chemical and biological reagents were purchased from commercial suppliers in analytical grade and used without further purification. All tests are performed at room temperature. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX-400 spectrometer. Mass spectra were recorded on a Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (ultrafleXtreme) or LCQ Advantage ion trap mass spectrometer from Thermo Finnigan or Agilent 1100 HPLC/MS.E.M. spectrometer. UV absorption and emission spectra were recorded on UV-1800 spectrophotometer (Shimadzu Corporation, Japan) and Hitachi F-4600 spectrofluorometer (Tokyo, Japan) respectively. Confocal microscopy was performed on a Nikon A1 plus confocal microscope. STED microscopy was performed on an Abberior Instruments STED 775/595/RESOLFT Expert Line microscope or an Abberior Instruments STED 775/660/multiphoton Infinity Line microscope. CPY-Halo and JF608-Halo are synthesized based on Hell’s work 17 , 44 and Lavis’s work 20 .

Confocal live-cell imaging

HeLa cells were incubated with the DMEM containing streptomycin (100 μg/mL), penicillin (100 U/mL), and 10% FBS at 37°C under 5 wt%/vol CO 2 for 24 h. 5 μL stock solution (1 mM) of 1–21 was added to 1 mL culture medium (final probe concentration: 5 μM), with which the live HeLa cells was incubated for 30 min before cell imaging. To evaluate ALP levels in different cell lines, live HeLa and L02 cells were cultured in 1 mL phenol-red-free DMEM containing 5 μM 11-ALP for 30 min before imaging. In the control experiment, HeLa cells were firstly cultured with Na 3 VO 4 (1 mM) for 1 h, then treated with 11-ALP (5 μL 1 mM stock solutions) and incubated for 30 min before imaging. To express H2B-Halo in living cells, plasmid LZ10 pbrebac-H2B-Halo (#91564, addgene) was transfected into HeLa cells utilizing lipo8000 (Beyotime Biotechnology) following the standard protocols 54 . Live HeLa cells were incubated with 250 nM probe for 0.5–5 h at 37 °C in a 5% CO 2 atmosphere and directly imaged using confocal microscopy without washing steps unless specifically stated. Microscopy conditions: RhB, RhB-Halo, R-3 and YL578, 11, 12, YL578-Halo, 10-Halo, 11-ALP , λ ex 561 nm; detection range 585–675 nm. R-2 and 14, 16-Halo , λ ex 488 nm; detection range 500–550 nm. 13 , λ ex 405 nm; detection range 500–550 nm. Coumarin-Halo and R 4–6, λ ex 405 nm; detection range 425–475 nm. 15 , λ ex 405 nm; detection range 570–620 nm. The photostability testing in STED microscopy U-2 OS stably expressing Vimentin-HaloTag cells 55 were seeded on glass coverslips and incubated in imaging medium that contained 50 nM YL578-Halo , 580CP-Halo, CPY-Halo, and JF608 for 6 h at 37 °C. Next, the cells were fixed with 4% PFA for 20 min and quenched for 5 min in NH 4 Cl and glycine (both 100 mM), washed in PBS, and mounted in Mowiol. The fixed samples were imaged on an Infinity Line STED microscope equipped with 775 and 660 STED lines, and 518 nm, 561 nm, 640 nm, and multiphoton excitation lines (Abberior Instruments GmbH). Imaging conditions: λ ex 561 nm, detection range 570–700 nm, STED laser 775 nm.

3D STED microscopy

Plasmid pcDNA5-FRT-Tomm20-Halo was transfected into the host cell line U-2 OS using Turbofect (ThermoFisher) following the manufacture’s protocol. U-2 OS cells transiently expressing Tomm20-HaloTag were next incubated in imaging medium that contained 50 nM YL578-Halo overnight at 37 °C. The cells were fixed with 4% PFA for 20 min and then quenched for 5 min in NH 4 Cl and glycine (100 mM), washed in PBS, and mounted in Mowiol. Imaging was performed on an Expert line STED/RESOLFT system equipped with 595 nm and 775 nm STED lines, and 355 nm, 405 nm, 485 nm, 561 nm, and 775 nm excitation lines (Abberior Instruments). The STED images of mitochondria were collected along the z -axis with a step of 35 nm. The 3D images were constructed in ImageJ with plugin z-stack depth colorcode. Imaging conditions: λ ex 561 nm, detection range 570–700 nm, STED laser 775 nm.

Live-cell STED microscopy U-2 OS stably expressing

Vimentin-HaloTag cells seeded on glass coverslips were incubated in phenol red-free imaging medium that contained 50 nM YL578-Halo or 10-Halo overnight at 37 °C and then imaged without washing with the 775 nm STED line on the Abberior Instruments Infinity Line microscope. For 16-Halo , live U-2 OS stably expressing Vimentin-HaloTag cells were treated with 500 nM probe for 90 min and washed with imaging medium. The STED imaging was collected on the Abberior Expert Line system with excitation wavelength at 485 nm and STED lines at 595 nm. Imaging conditions: YL578-Halo and 10-Halo , λ ex 561 nm, detection range 570–700 nm, STED laser 775 nm. 16-Halo , λ ex 485 nm, detection range 505–550 nm, STED laser 595 nm. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Materials and general methods

Unless otherwise stated, all chemical and biological reagents were purchased from commercial suppliers in analytical grade and used without further purification. All tests are performed at room temperature. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX-400 spectrometer. Mass spectra were recorded on a Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (ultrafleXtreme) or LCQ Advantage ion trap mass spectrometer from Thermo Finnigan or Agilent 1100 HPLC/MS.E.M. spectrometer. UV absorption and emission spectra were recorded on UV-1800 spectrophotometer (Shimadzu Corporation, Japan) and Hitachi F-4600 spectrofluorometer (Tokyo, Japan) respectively. Confocal microscopy was performed on a Nikon A1 plus confocal microscope. STED microscopy was performed on an Abberior Instruments STED 775/595/RESOLFT Expert Line microscope or an Abberior Instruments STED 775/660/multiphoton Infinity Line microscope. CPY-Halo and JF608-Halo are synthesized based on Hell’s work 17 , 44 and Lavis’s work 20 .

Supplementary information Supplementary Information Peer Review File Reporting Summary