Innovative Optical Super-Resolution Microscopy Based On Upconversion Nanometerial

Optical microscopy has facilitated the biological research owing to the features of 3-D,real-time imaging in vivo.However,the resolution of any lens-based far field microscope has been curtailed by diffraction limit.A series of novel techniques on breaking the resolution barrier of diffraction limit have been reported recently.And three founders were given Nobel Prize in Chemistry in 2014.Stimulated Emission Deption（STED）nanoscopy is the first and optically straightforward method to effectively unlock the diffraction limit for far-field nanoscopy which owns the ability of 3-D and video rate imaging.However the extreme depletion power intensity requirement of STED makes it challenging for living cell imaging.Rare-earth doped upconversion nanomaterial exhibits luminescence with multiple intermediate excited states with lifetime on microsecond to millisecond level.The unique feather of non-photobleaching and non-photoblinking benefits the material to be widely used in bioimaging and biosensing.The long lifetime is potential to overcome the high power intensity requirement in STED.This thesis explored the feather of upconversion nanomaterial and applied it to super-resolution technologies.Owing to the feathers,we achieved lower power intensity STED and high-throughput fluorescent lifetime coding and decoding localization.Presented below are the details of every chapter:Firstly,a continuous wavelength STED（CW STED）microscope based on a Ti:Sapphire Oscillator was established to achieve⁷1nm super-resolution on 20 nm fluorescent nanoparticles.Then three components of cellular skeleton and RNA were imaged in fixed cells with super-resolution.The results presented that the continuous wavelength mode could reduce the systemic complexity and cost.However,the extreme power intensity condition to achieve effective resolution was not suitable for living cell imaging.Secondly,based on the long lifetime and multi-intermediate states of NaYF₄:Yb³⁺/Tm³⁺upconversion nanomaterial,we applied the innovative intermediate-state ladder depletion mechanism.The measurement of depletion efficiency on variable dopant concentrations nanoparticles proved that for highly doped samples,the saturation intensity could be largely reduced.Comparing to the organic dye,the requirement could be reduced by two orders of magnitude on 8%and 4%Tm³⁺-doped samples.And we imaged the high dopant comcentration nanoparticles to achieve super-resolution with much lower power intensity.Thirdly,we studied the mechanism for the low power intensity depletion on highly doped upconversion nanomaterial.We found that the rate of the cross-relaxation inside the upconversion system could be largely enhanced with high dopant concentration.This would further lead to photon avalanche in the intermediate states and eventually the population inversion.The establishment of population inversion largely reduced the power intensity requirement of stimulated emission.While for low dopant concentration sample,the rate for cross-relaxation is not sufficient to achieve photon avalanche.And we further explored variable factors which could affect the whole process.At last we explored the tunable lifetime ability of rare-earth upconversion nanocrystals and established the new high-throughtput lifetime coding and decoding localization method.This could enhance the technologies as co-localization with high-throughput library of lifetime.The capacity was demonstrated to be more than one thousand.We demonstrated the separation of five different upconversion nanoparticles on the same emission band with lifetime decoding.