Supramolecular Complex of Photochromic Diarylethene and Cucurbit[7]uril: Fluorescent Photoswitching System for Biolabeling and Imaging

Abstract

Photoswitchable fluorophores─proteins and synthetic dyes─whose emission is reversibly switched on and off upon illumination, are powerful probes for bioimaging, protein tracking, and super-resolution microscopy. Compared to proteins, synthetic dyes are smaller and brighter, but their photostability and the number of achievable switching cycles in aqueous solutions are lower. Inspired by the robust photoswitching system of natural proteins, we designed a supramolecular system based on a fluorescent diarylethene (DAE) and cucurbit[7]uril (CB7) (denoted as DAE@CB7). In this assembly, the photoswitchable DAE molecule is encapsulated by CB7 according to the host-guest principle, so that DAE is protected from the environment and its fluorescence brightness and fatigue resistance in pure water improved. The fluorescence quantum yield (Φ_fl) increased from 0.40 to 0.63 upon CB7 complexation. The photoswitching of the DAE@CB7 complex, upon alternating UV and visible light irradiations, can be repeated 2560 times in aqueous solution before half-bleaching occurs (comparable to fatigue resistance of the reversibly photoswitchable proteins), while free DAE can be switched on and off only 80 times. By incorporation of reactive groups [maleimide and N-hydroxysuccinimidyl (NHS) ester], we prepared bioconjugates of DAE@CB7 with antibodies and demonstrated both specific labeling of intracellular proteins in cells and the reversible on/off switching of the probes in cellular environments under irradiations with 355 nm/485 nm light. The bright emission and robust photoswitching of DAE-Male3@CB7 and DAE-NHS@CB7 complexes (without exclusion of air oxygen and addition of any stabilizing/antifading reagents) enabled confocal and super-resolution RESOLFT (reversible saturable optical fluorescence transitions) imaging with apparent 70-90 nm optical resolution.

Figure 1

(a) Structures of photochromic diarylethenes as guest molecules (DAE, DAE-Male1, DAE-Male2, DAE-Male3, and DAE-NHS). (b) Structure of a cucurbit[7]uril host molecule (CB7). (c) DFT-optimized geometry and the photoswitching reaction of the DAE@CB7 complex. The cylinder of a DAE@CB7 complex has a length of 2.4 nm and a diameter of 1.3 nm.

Scheme 1. Synthesis of (a) Symmetric DAE and (b) Asymmetric DAEs: DAE-COOH, DAE-Male1, DAE-Male2, DAE-Male3, and DAE-NHS

For abbreviations, see the Supporting Information.

Figure 2

(a) Normalized absorption and PL spectra of DAE (black dashed lines for the initial state and black solid lines for the PSS) and DAE@CB7 complex (orange dashed lines for the initial state and orange solid lines for the PSS) in aqueous solution (10 μM). Irradiation with UV light to reach PSS states. (b) ITC profiles: integrated heat (experimental) and fitted curve of titrating 1000 μM DAE into 100 μM CB7 in water at 25 °C. (c) Job’s plot derived from PL intensities observed for complexation of DAE and CB7 ([DAE] + [CB7] = 10 μM, χ_CB7 is the molar fraction of CB7 in a mixture). (d) ¹H NMR spectra of the open-ring isomer DAE upon addition of CB7 in D₂O ([DAE] = 2 mM).

Figure 3

Reversible photoswitching of free DAE and DAE@CB7 complex in aqueous solutions: (a) zoom into the first 80 cycles; (b) full span of 3800 cycles.

Figure 4

(A–D) Confocal images of fixed Cos7 cells stained with secondary antibodies against microtubules labeled with the indicated DAE before (top) and after (bottom) CB7 addition. The markers are switched on before the recording of each pixel, with a short pulse of 355 nm light. (E) Average signal intensity of selected single filaments before (green) and after (orange) CB7 addition. (F) Normalized intensity distribution of selected single filaments (N = 50). Scale bar in A–D: 10 μm.

Figure 5

Photoswitching fatigue resistance observed on a confocal microscope for the labeled secondary antibodies on microtubules on fixed Cos7 cells. (A) Confocal image without and with activation (355 nm) after a switching experiment was performed in the indicated area (white square). Switching on and off on a sample labeled with DAE-Male3 mounted in PBS without (B) and with (C) CB7 (2 mM) and on a sample labeled with DAE-NHS without (D) and with (E) CB7 (2 mM). The insets show the differences between two successive substeps (symbols) along with a biexponential fit (lines). (F) Boxplots of the mean (amplitude averaged) characteristic switching time (20 measurements on different positions for each case). Scale bar on A: 2 μm.

Figure 6

Confocal (A) and RESOLFT (B) images of microtubules on fixed CV1 cells stained with secondary antibodies labeled with DAE-Male3 and mounted in PBS supplemented with CB7 (2 mM). (C–E) Line profiles along indicated lines show the experimental data (circles) and fitted data (lines). (F,G) Magnifications of the areas indicated in (A). The corresponding results with DAE-NHS (with CB7) at the same conditions: confocal (H) and RESOLFT (I) images and (J,K) line profiles. (M,N) Magnifications of the areas indicated in (H). Confocal data (black symbols and lines) were fitted with a Gaussian function and RESOLFT data (red symbols and lines) with a Lorentzian function. Scale bars: 2 μm (A, B, H, I); 0.5 μm (F, G, M, N).