A general strategy for developing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging

Deng Pan,Zhihong Zhang,Yina Wang,Lingsong Qin,Yu-Hui Zhang,Zhen-Li Huang,Zhe Hu,Fengwu Qiu,Shaoqun Zeng,Yilong Ma

doi:10.1038/ncomms6573

Abstract

Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. 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)3R2 peptide 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. We resolve the dynamics of F-actin with 10 s temporal resolution in live cells and discern fine F-actin structures with diameters of ~80 nm. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.

Highlights

Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes
Recent developments in single-molecule localization microscopy (SMLM), such as photo-activated localization microscopy[1,2] and stochastic optical reconstruction microscopy[3], have enabled biological structures to be defined with a spatial resolution beyond the diffraction limit, providing powerful techniques to obtain exciting new insight into the nanoscale structures and dynamics of biological samples[4,5]
The second strategy relies on the combination of a genetically encoded target protein with a separate synthetic probe consisting of a photo-modulatable organic fluorophore and a recognition unit, such as SNAP-tags[12], TMP-tags[13] or Halotags[14]

Summary

Introduction

Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. The second strategy relies on the combination of a genetically encoded target protein (or peptide) with a separate synthetic probe consisting of a photo-modulatable organic fluorophore and a recognition unit, such as SNAP-tags[12], TMP-tags[13] or Halotags[14]. We develop a new general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging by utilizing the remarkable cytosolic delivery ability of a cell-penetrating peptide (CPP), (rR)3R2 (r: D-Arg, R: L-Arg). Our results indicate that these organic probes are cell permeable but can and directly label endogenous proteins Using these probes, we obtain super-resolution images of F-actin and lysosomes in live cells. To the best of our knowledge, the probes presented here are the first cell-permeable photo-modulatable organic fluorescent probes that can directly label intracellular endogenous targeted proteins in live cells for super-resolution imaging. Considering that the organic fluorophore and the recognition unit of the probes can be replaced with other organic dyes and recognition groups, respectively, which do not need to be cell permeable anymore, greatly expanding the number of organic fluorophores and recognition groups that can be used inside live cells for SMLM

Methods

Results

Conclusion