New Protein for Superresolution Microscopy

Schema of Skylan-NS and live-cell SR imaging of actin structures  (Source: CAS / IBP)

Schema of Skylan-NS and live-cell SR imaging of actin structures (Source: CAS / IBP)

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 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) while using saturated depletion-based SR tech­niques, such as nonlinear structured illu­mination microscopy (SD NL-SIM) or reversible saturable optical fluore­scence transition (RESOLFT) microscopy, greatly reduces the illu­mination intensity, thus enabling 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 the existing switchable fluores­cent proteins Dronpa and rsEGFP, such as the small number of switching cycles, low fluores­cence signal, and poor contrast, it is difficult to achieve the desired reso­lution in live-cell SR imaging. To circumvent the problem, Xu Pingyong at the Institute of Biophysics IBP of the Chinese Academy of Sciences recently developed a new type of monomer, RSFP Skylan-NS – sky lantern for nonlinear structured illu­mination.

Xu, in coope­ration with Eric Betzig and Li Dong, applied Skylan-NS to their previously developed SR imaging technique, patterned acti­vation nonlinear SIM (PA NL-SIM). 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 their study, the researchers performed such a comparison, by eva­luating the photo­physical properties of Skylan-NS against two other RSFPs, i.e., rsEGFP2 and Dronpa, which 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, high-sampling speed on the sub-second level, high-resolution of approx. 60 nm and long-term 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 live-cell SR imaging. (Source: CAS)

Reference: Xi Zhang et al.: Highly Photostable, Reversibly Photoswitchable Fluorescent Protein with High Contrast Ratio for Live-cell Superresolution Microscopy, Proc. Nat. Ac. Sci., online 25 August 2016; DOI: 10.1073/pnas.1611038113

Link: Key Lab. of RNA Biology, Inst. of Biophysics, Chinese Academy of Sciences, Beijing, China

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