Break the unbroken limits toward super-resolution microscopy

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=λ/2nsinθ, where d is the resolvable feature size, λ is the wavelength of light, n is the index of refraction of the medium being imaged in, and the term “nsinθ” 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=λ/(2nsinθN1/2), (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=λ/(2nsinθ(1+I/Isat)1/2. 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.KEYWORDS:
Fluorescence super-resolution microscopy; Upconversion nanoparticles; Stimulated emission depletion microscopy; multiphoton microscopy
RERENCES
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[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.
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[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.; Ågren, 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.; Ågren, H., Fast upconversion super-resolution microscopy with 10 μs per pixel dwell times. Nanoscale 2019, 11, 1563-1569.

Speaker

Qiuqiang Zhan
Centre for Optical and Electromagnetic Research, South China Academy of Advanced Optoelectronics, South China Normal University
China

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