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63BREAK THE UNBROKEN LIMITS TOWARD SUPER-RESOLUTION MICROSCOPY
QIUQIANG ZHAN
Centre for Optical and Electromagnetic Research, South China Academy of Advanced Optoelectronics, South China
Normal University, Guangzhou 510006, P. R. China
*E-mail: zhanqiuqiang@m. scnu.edu.cn
Abstract
The resolution of an optical imaging system, a microscope, is always theoretically limited due to the physics of diffraction [1]. Memorial to Ernst Karl Abbe, who approximated the diffraction limit of a microscope as, d=A/2nsin0, where d is the resolvable feature size, I is the wavelength of light, n is the index of refraction of the medium being imaged in, and the term "nsin0" representing the numerical aperture[1]. Theoretically, the full width at half maximum (FWHM) of the point spread function (PSF) for the N-photon microscopic imaging could be described by the formula d=A/(2nsin6W12), (N>2), which, in principle, helps to improve the resolution. It is challenging to resolve the contradiction of high-order nonlinearity and required short excitation wavelength. Lanthanide-doped photon upconversion nanoparticles (UCNPs) are capable of converting low-intensity near-infrared light to UV and visible emission through the synergistic effects of light excitation and mutual interactions between doped ions[2]. To overcome these problems, we propose visible-to-visible four-photon ultrahigh resolution microscopic imaging by using a common cost-effective 730-nm laser diode to excite the prepared Nd3+-sensitized upconversion nanoparticles with the obtained lateral resolution as high as 161-nm[3]. The stimulated emission depletion (STED) microscopy that has broken the diffraction limit of optical microscopic imaging has become crucial methods for molecularly-resolved imaging in the life sciences and beyond [4,5], with the resolution governed by d=A/(2nsin6(1+I/Isat)V2 In 2015, as shown in (Fig.1), we firstly demonstrated the potential of UCNPs for multi-photon super-resolution microscopy [1]. In 2017, our group has developed a novel low-power CW laser enabled STED mechanism using optimized lanthanide upconversion nanoparticles [7]. We have experimentally achieved highly efficient, absolutely non-bleaching cytoskeleton STED imaging at subcellular scale. These findings have great potential in super-resolution microscopy [8]. Can we break the theoretical limit of Isat, like breaking the diffraction limit? Yes, very recently we have successfully broken the limit of Isat by two orders using new depletion mechanism for further pull down the laser power for super-resolution.
Figure 1:(a) Proposed mechanism of luminescence generation of795-nm laser excited NaYF4:Yb
UCNPswith/without 1140-nm irradiation. (b) Luminescence intensity of NaYF4:Yb3+/Er3+ UCNPs under 795-nm CW excitation with/without 1140-nm irradiation. Inset figure: the amplified luminescence spectra from 350 nm to 500 nm[6].
Figure 2: Immunofluorescence labeling of cellular cytoskeleton protein desmin with antibody conjugated UCNPs and super-resolution imaging. (a) The multiphoton imaging under 975 nm excitation of some cytoskeleton structures and desmin proteins in HeLa cancer cells incubated with anti-desmin primary antibody and immunostained with UCNPs (~11.8 nm in diameter) bioconjugated with goat Anti-rabbit IgG secondary antibody. (b) The same region with (a) imaged in the super-resolution mode (975 nm excitation and the 810 nm STED laser beam). Scale bars: 2 pm. (c-n) Magnified areas selected from a, b (marked by white dotted squares) and line profile analyses; Images in c, f, i and l are taken from the white dotted squares in a; Images in d, g, j and m are taken from the white dotted squares in b. (e, h, k, n) Line profiles analyses of several areas indicated by arrow heads in c and d, fand g, i and j, and l and m, respectively[7].
Rerences
[1] Abbe, E., Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv für Mikroskopische Anatomie 1873,9, 413-468.
[2] Xu, C. T.; Zhan, Q.; Liu, H.; Somesfalean, G.; Qian, J.; He, S.; Andersson-Engels, S., Upconverting nanoparticles for pre-clinical diffuse optical imaging, microscopy and sensing: Current trends and future challenges. Laser & Photonics Reviews 2013,7, 663-697.
[3] Wang, B.; Zhan, Q.; Zhao, Y.; Wu, R.; Liu, J.; He, S., Visible-to-visible four-photon ultrahigh resolution microscopic imaging with 730-nm diode laser excited nanocrystals. Opt Express 2016,24, A302-A311.
[4] Hell, S. W.; Wichmann, J., Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 1994,19, 780-782.
[5] Willig, K. I.; Rizzoli, S. O.; Westphal, V.; Jahn, R.; Hell, S. W., STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 2006,440, 935-939.
[6] Wu, R.; Zhan, Q.; Liu, H.; Wen, X.; Wang, B.; He, S., Optical depletion mechanism of upconverting luminescence and its potential for multi-photon STED-like microscopy. Opt Express 2015,23, 32401-32412.
[7] Zhan, Q.; Liu, H.; Wang, B.; Wu, Q.; Pu, R.; Zhou, C.; Huang, B.; Peng, X.; Ägren, H.; He, S., Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles. Nature Communications 2017,8, 1058.
[8] Peng, X.; Huang, B.; Pu, R.; Liu, H.; Zhang, T.; Widengren, J.; Zhan, Q.; Ägren, H., Fast upconversion superresolution microscopy with 10 ^s per pixel dwell times. Nanoscale 2019,11, 1563-1569.
