Localisation Super-resolution Imaging Using Germanium Quantum Dots
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Novel fluorescent quantum dots of small size, tunable light emission wavelength and high compatibility with biological systems are of great significance to light microscopy super-resolution imaging. In this thesis, colloidal germanium quantum dots of 3.8nm size have been investigated as a novel fluorescent probe for cell imaging. Two single molecule localisation super-resolution methods were explored: one utilised QDs blinking statistics and the other one was based on intrinsic QDs size dispersion. We found that the blinking super-resolution strategy which combined the usage of blinking QDs and spinning disk confocal imaging has led to less than seven minutes collection time for 2000 image frames. High precision temporal separation of single molecules has been achieved on Ge QDs and CdSe QDs labelled fixed Hela cell. The spectroscopic super-resolution strategy that combined the usage of size dependent light emission QDs and spectra imaging, resulted in a 1.6 seconds data acquisition time. Spectroscopic separation and high precision single molecule localisation has been demonstrated using Ge QDs and CdSe QDs labelled fixed Hela cell samples. We compared various localisation algorithms when applied to the two superresolution methods we studied. We found that they did not work well with our data. Consequently, we developed two MATLAB-based localisation algorithms. The first algorithm used the independent component analysis (ICA) model to analyse the blinking stochastic imaging data, whilst the other used the Gaussian mixed model (GMM) to analyse the spectroscopic separation imaging data. We also conducted comparative toxicity tests of these novel Ge QDs with a typical off the-shelf system. The cell toxicity of Ge QDs was found to be less than that of CdSe/ZnS QDs. For instance, 25 nM Ge QDs in 1 mL Hela cell solution did not cause observable cells apoptosis in 24 hours. It caused 15% cells apoptosis after 3 days, rather than 35% for CdSe QDs at the same concentration. In addition, long term live cell imaging with QDs revealed that Ge QDs had not significantly changed cellular morphology within a 90 hour period.
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