Carboxylated Photoswitchable Diarylethenes for Biolabeling and Super-Resolution RESOLFT Microscopy

Abstract

Reversibly photoswitchable 1,2-bis(2-ethyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes (EBT) having fluorescent "closed" forms were decorated with four or eight carboxylic groups and attached to antibodies. Low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and good photophysical properties were demonstrated. Alternating light pulses of UV and blue light induce numerous reversible photochemical transformations between two stables states with distinct structures. Using relatively low light intensities, EBTs were applied in biology-related super-resolution microscopy based on the reversible saturable (switchable) optical linear fluorescence transitions (RESOLFT) and demonstrated optical resolution of 75 nm.

Keywords: diarylethenes; fluorescence; optical microscopy; photochromic dyes; water solubility.

Scheme 1

Photoswitchable EBTs 1 and 2 decorated with four or eight carboxylic acid groups; see Table 1 for spectral properties of the “open” (OF, non‐fluorescent) and the “closed” (CF, fluorescent) forms.12 For compounds with R=H and R=C₆H₅ see Refs. 9a, 9b, 9c and 9d, 9e, respectively.

Figure 1

Fluorescence emission of cells immunolabeled with dye 1 (top) and 2 (bottom) showing the on/off switching cycles upon irradiation with the light pulse sequence mentioned in the text (gray). The red line indicates the maximum fluorescence signal detected within a switching cycle, while the black line shows the overall fluorescence gained per cycle. Both plots were normalized to their maximum value. The insets show one switching cycle as the mean value of 100 cycles (blue). All measurements were taken with 20 μs bins. Displayed intensities were corrected according to the dead‐time of the sensors.

Figure 2

RESOLFT images of the whole fixed Vero cells immunostained with primary antibodies against α‐Tubulin and with the diarylethene 2 attached to the secondary antibodies. All images show raw data. The RESOLFT images were recorded before confocal images. Scale bar: 1 μm. The line profiles A–D (averaged over ten adjacent lines) display the regions indicated in the RESOLFT image. The data (dots) was fitted with a Lorentzian function (solid line) for the RESOLFT (red) and the confocal (blue) image. The FWHM was determined on the fits A and B as indicated by the small black arrows.