Photoactivatable Large Stokes Shift Fluorophores for Multicolor Nanoscopy

Abstract

We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and ∼100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells, and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted tetrazine ligation and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of an imaging channel with another large Stokes shift dye have been demonstrated.

Figure 1

Photoactivatable large Stokes shift fluorescent dyes based around Si-pyronine core structure. (a) Photoactivatable PA-SiR dye generating an electrophilic 9-alkyl-Si-pyronine fluorophore with ∼20 nm Stokes shift. (b) Photoactivatable 1-vinylxanthone (PaX) dyes are stable toward nucleophiles under neutral conditions. (c) Reversible protonation of 1-vinyl-10-silaxanthone imines 1-OF–3-OF vs an irreversible photochemical ring closure. (d–f) Changes in absorption and fluorescence emission spectra of 1-OF (d), 2-OF (e), and 3-OF (f) upon UV irradiation (405 nm, d; 365 nm, e,f).

Figure 2

(a) Photoactivatable large Stokes shift fluorescent labels for fixed- (b,c) and live-cell (d–f) fluorescence imaging. (b,c) Confocal and STED images of tubulin filaments in fixed COS-7 cells labeled by indirect immunofluorescence with secondary antibodies tagged with 2-NHS (b) or 3-NHS (c). (d) Confocal image of U2OS cells stably expressing vimentin-HaloTag fusion protein labeled with 3-Halo (200 nM). (e) Confocal image of U2OS cells stably expressing vimentin-SNAP-tag fusion protein labeled with 3-BG (1 μM). (f) Confocal image of U2OS cells stably expressing vimentin-HaloTag fusion protein labeled with HTL-BCN (10 μM) followed by 3-Tz (1 μM). In the upper left corner of (b–f), the same images before photoactivation are shown. Scale bars: 2 μm.

Figure 3

Channel duplexing with photoactivatable large Stokes shift dye 3. (a–c) Confocal imaging of fixed COS-7 cells with microtubules labeled by indirect immunofluorescence with DyLight 515-LS NHS ester (ThermoFisher) and mitochondria labeled with 3-NHS before (a) and after (b) photobleaching of DyLight 515-LS and after (c) photoactivation of 3 by a 405 nm laser. (d) Combined pseudo-two-color image showing microtubules (magenta) and mitochondria (yellow) obtained by sequential imaging as shown in (a–c). (e,f) Confocal (e) and STED (f) images of mitochondria in the magnified region marked in (d). (g) Absorption and emission spectra of DyLight 515-LS (magenta) and 3 (yellow), the excitation laser line (485 nm, dashed line), and the detection window (560–700 nm, gray); STED wavelength of 775 nm. Scale bars: 10 μm (a–d), 2 μm (e–f).

Figure 4

Multicolor imaging with photoactivatable large Stokes shift labels. (a) Confocal imaging of fixed COS-7 cells labeled by indirect immunofluorescence for mitochondria (3-NHS) and vimentin (abberior STAR 512 NHS ester), and with small-molecule fluorescent probes SiR-actin (for F-actin) and DAPI (for nuclear DNA). (b) Confocal imaging of living U2OS cells stably expressing a vimentin-HaloTag construct labeled with 3-Halo and live cell-compatible probes MitoTracker Deep Red FM (specific for mitochondria), abberior LIVE 510-tubulin (microtubules), and Hoechst 33342 (DNA). Spectral unmixing of individual channels was performed according to ref (23). Scale bars: 10 μm.