Proceedings Article | 10 October 2020
KEYWORDS: Upconversion, Super resolution microscopy, Image resolution, Photonic microstructures, Diffraction, Nanoparticles, Stimulated emission depletion microscopy, Optical imaging, Microscopes, Point spread functions
The resolution of an optical imaging system, a microscope, is always theoretically limited due to the physics of diffraction. Memorial to Ernst Karl Abbe, who approximated the diffraction limit of a microscope as, d=λ/2nsinθ, λ is the light wavelength, n is the index of refraction of the medium being imaged in, and the term “nsinθ” representing the numerical aperture. The resolution of multiphoton (N-photon) fluorescence microscopy is described by the formula d=λ/(2nsinθN1/2), (N≥2), which can theoretically help to improve the resolution. However, it is challenging in practice to address the paradox that higher-order nonlinearity (N) always results in longer excitation wavelength (λ). 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 interionic energy transfer. To break the limit of multiphoton imaging resolution, we proposed visible-to-visible four-photon microscopic imaging by using a 730-nm CW laser diode to excite the Nd3+-sensitized UCNPs with a 161-nm sub-diffraction resolution obtained. 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, with the resolution governed by d=λ/(2nsinθ(1+I/Isat)1/2). However, application of ultrahigh beam intensity in STED, imposed by the ultrafast spontaneous emission nature of commonly used fluorescent organic probes, often causes phototoxicity, photobleaching, and re-excitation background. In 2015, we firstly realized the optical emission depletion of near infrared UCNPs and demonstrated its large potential for super-resolution microscopy. In 2017, we developed a novel low-power CW laser enabled superresolution using designed UCNPs. We have experimentally achieved low-power, nonphotobleaching cytoskeleton STED imaging at subcellular scale. Can we break the theoretical limit of Isat, like breaking the diffraction limit? Yes, in our very recent progress we have successfully broken the theoretical limit of Isat by two orders to sharply pull down the laser power for super-resolution. Our approach using new depletion mechanism circumvents the fundamental high-intensity constraint of STED imaging and provides background-free, contrast-enhanced imaging at a spatial resolution of 1/38th of the excitation wavelength.
