Multi-photon Nonlinear Fluorescence Emission in Upconversion Nanoparticles for Super-Resolution Imaging
- Publication Type:
- Thesis
- Issue Date:
- 2020
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Due to the unique optical properties gained by converting near-infrared light to shorter wavelength emissions, upconversion nanoparticles (UCNPs) have attracted considerable interest. Their superior features, including their multi-wavelength emissions, optical uniformity, background suppression, photostability and deep penetration depth through the tissue, make them extremely suitable for biological and biomedical applications. By taking advantage of their multi-photon nonlinear emissions in UNCPs, the goal of this thesis is to develop UCNPs-based super-resolution microscopy methods to address the challenges currently facing nanoscopy, for instance complexity, stability, limited penetration through the tissue and low throughput. The methods being investigated in this thesis make concrete the specific advantages in terms of image depth, speed, overall quality, and multiplexing potentials. To unlock a new mode of deep tissue super-resolution imaging, I first developed the near-infrared emission saturation (NIRES) nanoscopy by taking advantage of near-infrared-in and near-infrared-out optical nonlinear response curve from a single upconversion nanoparticle. This approach only requires two orders of magnitude that are lower than the excitation intensity, which is generally required for conventional multi-photon dyes. This work achieves a super-resolution of sub 50 nm, less than 1/20ᵗʰ of the excitation wavelength, and can image single UCNP through a 93 μm thick liver tissue.
To improve the overall imaging quality and simplify the system setups, I further exploited the distinct nonlinear photon response curves from the two emission bands in UCNP, and explored an opportunity for a tightly focused doughnut excitation to generate distinct spectral dependent point speared functions (PSFs). With controllable PSFs from multi-channel emissions by the excitation power density, this work presents the possibility of achieving super-resolution imaging under saturated fluorescence excitation via PSF engineering. Moreover, I developed a multicolour Fourier fusion algorithm to enlarge the optical system's frequency shifting ability, and yield an enhanced imaging quality at a higher imaging speed. By realising the uniform and distinct nonlinear emission curves from different nanoparticles, this work posits a new optical encoding dimension for multiplexing imaging. Proposed here is a robust PSF engineering strategy to extract emitter properties. This work extends the multiplexing capacity of UCNPs and offers new opportunities for their applications. These methods are my contributions to the search for a stable, viable, and multifunctional optical imaging modality for the nanoscale context.
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