A General Strategy For Developing Organic Fluorescent Probes For Live-cell Super-resolution Image

Because of abbe limits formed by light diffraction, optical imaging are thought unable to break through the diffraction limit and obtain higher spatial resolution. In recent years, the emergence of super resolution imaging technologies have been broken the diffraction limit, and thus won the2014Nobel Prize in chemistry.Single-molecule localization microscopy (SMLM) does not require sophisticated microscopes but instead critically relies on the use of photo-modulatable fluorescent probes. Organic photo-modulatable fluorophores offer several important photophysical properties, including smaller sizes, broader spectral coverage, higher fluorescence quantum yields and better photostabilities; therefore, use of organic fluorophores could potentially improve the spatial and temporal resolution of SMLM. However, the success for live-cell super-resolution image by organic fluorescent probes is very limited, seriously hindered the further development of super resolution imaging.Here we report a general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell SMLM by exploiting the remarkable cytosolic delivery ability of a cell-penetrating peptide (rR)3R2. We develop photo-modulatable organic fluorescent probes consisting of a (rR)3R2peptide coupled to a cell-impermeable organic fluorophore and a recognition unit. Our results indicate that these organic probes are not only cell permeable but can also specifically and directly label endogenous targeted proteins. Using the probes, we obtain super-resolution images of lysosomes and endogenous F-actin under physiological conditions. The main contents are summarized as follows:(1) We designed and synthesized cell-permeable organic fluorescent probes for endogenous F-actin SMLM image in living cells, including ordinary fluorescent probe (RhB-, FITC)-labeled probes and photochromic (PA-RhB)-labeled probe.(2) We use ordinary fluorescent probes to investigate distributions inside living cells of these designed probes at different concentration for series incubation time. Probes labeled with different dyes were treated differently in the course of study. According to the optimization steps above, we determined the optimal experimental conditions for F-actin by designed organic fluorescent probes.(3) We inquired RhB-, FITC-, and PA-RhB-labeled probe in the feasibility of SMLM image. PA-RhB-labeled probe has excellent optical properties in SMLM imaging:it can photoblink inside of the cells when irradiated with excitation laser without any ultraviolet light activation, and can anti-photobleach within at least30min at the maximum intensity of laser radiation without additional photoactivation. Using PA-RhB-labeled probe, we resolve the dynamics of F-actin with10s temporal resolution in live cells and discern fine F-actin structures with diameters of～80nm.(4) We proved photoactivatable dye of caged-Rh110labelled probe can be used to image lysosomes with a resolution beyond optical diffraction limit in living cells. Using caged-Rh110-labeled probe, we obtained a super-resolution image of lysosome with spatial resolution of64nm and temporal resolution of2s in live cells.