Unlocking low-power stimulated emission depletion super-resolution microscopy by using upconversion nanocrystals
thesisposted on 28.03.2022, 18:53 by Yujia Liu
Optical microscopy capable of real-time 3-dimentional imaging for live cells has facilitated modern biological research, especially with the recent development of a series of novel super-resolution techniques that overcome the diffraction limit associated with conventional optical microscopes. Among them, stimulated emission depletion (STED) nanoscopy is the first and optically most straightforward method to effectively break the resolution barrier for far-field microscopes. It has been demonstrated that STED can achieve 3-D imaging even at video rate. However the very high optical intensities required for depletion remain a key challenge for STED to be applied to live biological cells and tissues. This thesis explores the potential to use luminescent probes with long decay lifetimes to lower the optical depletion intensity requirement in STED. In particular, lanthanide-doped upconversion nanomaterials involving multiple intermediate excited states with lifetimes on the microsecond-to-millisecond timescale have been investigated. Such materials have found broad applications in biosensing and imaging due to their unique feature of non-photobleaching and non-photoblinking. Here, their photon upconversion process and depletion capability have been carefully studied, in order to apply them to achieve STED super-resolution imaging at lowered intensity. The thesis is composed of the following research: Firstly, a continuous-wave STED (CW STED) microscope based on a Ti:Sapphire oscillator was established to achieve ~71 nm super-resolution on 20 nm fluorescent nanoparticles. Three components of cellular skeleton and RNA were imaged in fixed cells with super-resolution. This result shows that the CW mode operation reduces the system complexity and cost; however, it cannot relieve the extreme intensity requirement in STED to achieve super resolution for live cell imaging. Secondly, we explored and realized super-resolution imaging via an intermediate-level depletion mechanism using NaYF4:Yb3+/Tm3+ upconversion nanocrystals. Measurement of the depletion efficiency from upconversion nanocrystals with variable doping concentrations showed that the saturation intensity reduces substantially at increased Tm3+ doping concentration. In comparison to conventional organic dyes, the depletion intensity requirement for 8% Tm3+-doped samples is reduced by two orders of magnitude, allowing us to achieve moderate super-resolution on these highly-doped samples at much lower depletion intensities. Thirdly, the mechanism underpinning efficient depletion on highly doped upconversion nanocrystals was studied in detail. We found that the Tm-Tm cross-relaxation rates in the Yb-Tm photon upconversion system increase significantly at high Tm doping concentration. This leads to a photon-avalanche-like process to efficiently establish population inversion on the intermediate excited levels, enabling amplified stimulated emission to depletion/inhibit upconversion luminescence using largely reduced intensity. Meanwhile, for low doping concentration samples, the cross-relaxation rates are not sufficient to achieve photon-avalanche-enhanced stimulated emission depletion. The results embodied in this thesis demonstrate the capability to achieve low-power operation of STED using upconversion nanocrystals for super-resolution imaging for live cells. It suggest that novel luminescence nanoprobes may hold the key to next-generation optical nanoscopy. We further explored the lifetime tunability of upconversion nanocrystals for multiplexed imaging and molecular localization at high throughput, with preliminary results suggesting that upconversion nanoparticles with the same emission wavelengths can be effectively distinguished based on lifetime coding. It is expected that the new bio-friendly upconversion-STED method may find broad applications in life sciences.