An important breakthrough in super-resolution microscopy has been made by Prof. Peng Xi’s group in College of Engineering, Peking University, in collaboration with researchers from Macquarie University, University of Technology Sydney, and Shanghai Jiaotong University. Through the strategic application of upconversion rare-earth nanoparticles (UCNPs), this work has reduced the intensity of the traditional super-resolution by 2-3 orders of magnitude. It reveals a new mechanism of stimulated emission caused by the photon avalanche effect. With only 30mW continuous laser, resolution down to 28nm has been attained, which is only 1/36 of the excitation wavelength. It is published online on February 22 on Nature. (Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy)
Limited by diffraction, the resolution of the optical microscope is constrained to above 200 nm, whereas the intracellular organelle size is only about 1 to 50 nm. Super-resolution fluorescence microscopy won the 2014 Nobel Prize for its ability to break the diffraction limit and provide finer information on subcellular organelles. Among them, the stimulated emission depletion(STED) can provide fast super-resolution imaging, thus it is highly favored by biologists.
However, one of the drawbacks of this technique is that it requires the presence of "lasers" near the focus to erase the fluorescence, causing the required laser power too large to maintain cell activity and biological properties. Meanwhile, the cost and the complexity of maintenance of high-power lasers become a barrier for this technology to be widely used. Therefore, an optimized approach which can reduce the laser power requirement and achieve super-resolution at low power, will not only enable it to better reveal the fine structure and function of living cells, but also enable this technology to be widely used in more labs.
Figure 1 28 nm super-resolution attained with only 30mW CW laser power, taking advantage of the energy level properties of high dopant UCNPs.
In this work, researchers used rare-earth upconversion nanoparticle(UCNP) with particle size of only 40nm, to achieve super-resolution by using its unique feature in energy level, and ultra-low power super-resolution has been demonstrated successfully through the intermediate energy level depletion. Traditionally, STED requires high power laser because the fluorescence has only two energy levels: ground state and excited state. Benefitted from the rich intermediate states of UCNPs, stimulated emission can be induced with very little power. A proper choice of the intermediate state can reach lever effect to effectively deplete the electrons to ground state, prohibiting their further transferring to upper energy level.
In the meantime, researchers found that such effect can only appear in the highly doped nanoparticles, whereas low-doped nanoparticles cannot be depleted effectively. Through the study of doping concentration and extinction ratio, scientists have revealed the photon avalanche effect in high dopant UCNPs, which reflects a higher nonlinearity than resonant energy transfer.
In combination of the fluorescence properties of UCNPs and the mechanism of intermediate state depletion, researchers have achieved optical resolution as high as 28nm on single UCNP particle of 40nm and 13nm. Such resolution will help to reveal the structure and function of cells in different life cycles, the virus invasion process, etc. Moreover, as the upconversion of nanoparticles using near-infrared light for excitation, this work has the potential to be used in deep tissue three-dimensional super-resolution imaging.
The co-first authors of this paper are: Dr. Yujia Liu, a joint Ph. D. student of Shanghai Jiaotong University and Macquarie University, (Supervisor: Prof. Peng Xi and Prof. Dayong Jin), Dr. Yiqing Lu in Macquarie University, and Xusan Yang, a Ph. D. candidate in College of Engineering, Peking University (Supervisor: Prof. Peng Xi). The co-corresponding authors are Prof. Peng Xi, Dr. Yiqing Lu, and Prof. Dayong Jin (University of Technology Sydney). The work has been funded by the National Natural Science Foundation of China, the National Instrumentation Project by Ministry of Science and Technology of China (China part), and the Australia ARC Fund.