To understand the cell, it is necessary to study its dynamics at high resolution in space and time in a way that does not adversely affect it. Recently developed superresolution (SR) microscopy breaks the diffraction limit and offers the requisite spatial resolution but usually at the cost of slow imaging speed and excessive damage. Applying reversibly switchable fluorescent proteins (RSFPs) in the saturated depletion-based SR techniques, such as nonlinear structured illumination microscopy (SD NL-SIM) or reversible saturable optical fluorescence transition (RESOLFT) microscopy, has greatly reduced the illumination intensity thus enabled live cell SR imaging. However, one major challenge in live-cell SR is the absence of optimal fluorescent probes. Limited by the inherent optical properties of existing switchable fluorescent protein Dronpa and rsEGFP, such as small number of switching cycles, low fluorescence signal, poor contrast, it is difficult to achieve the desired resolution in a live cell SR imaging.
To circumvent the problem, Professor XU Pingyong at the Institute of Biophysics(IBP), Chinese Academy of Sciences(CAS) recently developed a new type of monomer RSFP Skylan-NS (sky lantern for nonlinear structured illumination). Cooperated with Eric Betzig, a researcher and Nobel laureate at HHIM, and LI Dong a postdoc in Betzig’s postdoc who is now a professor in IBP, they applied Skylan-NS to their previously developed SR imaging technique, patterned activation nonlinear SIM (PA NL-SIM) (Science, 2015). PA NL-SIM is much more compatible with noninvasive live-cell imaging at a resolution below 100 nm than other SR modalities. This technique uses specific RSFPs, however, the properties that strongly influence their suitability for PA NL-SIM have not been enumerated, measured, and compared. In the current paper, the researches performed such a comparison, by evaluating the photophysical properties of Skylan-NS, against two other RSFPs, rsEGFP2 and Dronpa, that have been used previously in the SR imaging modalities of RESOLFT and SD NL-SIM, respectively. They further demonstrated the superiority of Skylan-NS for PA NL-SIM by comparing the imaging performance of all three RSFPs when applied to PA NL-SIM.For the first time, they achieved low-energy (100 W / cm2), high-sampling speed (sub-second level), high-resolution (~ 60 nm) and long-term (~30 point in time) super-resolution imaging in living cells. Due to its superiority in photostability, cycle numbers and signal-to-noise ratio, Skylan-NS is one of the best fluorescent proteins applicable to the live cells SR imaging.
The research work entitled Highly Photostable, Reversibly Photoswitchable Fluorescent Protein with High Contrast Ratio for Live-cell Superresolution Microscopy was published on line in the journal Proceedings of the National Academy of Sciences on August 23, 2016. The described fluorescent protein Skylan-NS enables substantial improvements in the speed, duration, and noninvasiveness of live-cell superresolution microscopy.
Since the first photoactivatable fluorescent protein PAGFP has been developed for PALM imaging, photo-controllable fluorescent proteins play key roles in various diffraction-unlimited microscopies. Developing novel photo-controllable fluorescent proteins for different SR techniques is one of the major interests of Prof. XU Pingyong's lab. Prof. XU Pingyong and Prof. Eric Betzig are the corresponding authors. Dr. ZHANG Xi, Associate Prof. ZHANG Mingshu and Prof. LI Dong are the co-first authors of this paper. This work was also supported by the National Basic Research Program, National Key Research and Development Projects, the National Natural Science Foundation and Key Projects of CAS and Beijing Natural Science Foundation.
