| Correlative light and electron microscopy(CLEM)has become a powerful tool in life sciences,particularly Cryo-CLEM,which is considered to be an imaging technology close to the natural state of biological samples.The cryofixation methods can avoid the structural changes associated with the chemical fixation methods,so that the samples can retains the atomic structure in a near-native state.Fluorescence cryo-microscopy(Cryo-FM)can identify and localize specific biomolecules or structures,but the resolution is not up to the level of analyzing their ultrastructure in the nanometer scale.Cryo-electron microscopy(Cryo-EM)has ultra-high resolution,which can be used to analyze biomacromolecule or structures with near-atomic resolution.However,it lacks the ability to identify and locate specific biomacromolecule or structures accurately.Cryo-CLEM combines these three technologies to achieve complementary advantages,enabling people to simultaneously achieve high-precision localization and high-resolution structural information of biomolecules or structures in situ.However,as a newly developed technique,Cryo-CLEM still has many technical deficiencies that have not been properly solved.In this article,we try to improve Cryo-CLEM from two aspects:the construction of the new Cryo-FM and the development of the new sample support film.The first part of this thesis mainly focuses on the establishment of an ultra-stable super-resolution fluorescence cryo-microscopy,and verifies the cryogenic super-resolution imaging capability of our system by imaging a cryo-section and an intact cell at 93 K using photoactivated localization microscopy(PALM).Compared to other existing system,our system has the following advantages.1)Ultra-high thermal stability delivers temperature fluctuations of the objective lens and sample below 0.06 K for 10 hours.2)Excellent mechanical stability results in a sample(beads)drift less than 200 nm in 3D for5 hours at 93 K.3)Custom-designed workstation and optimized protocol ensure safe and efficient transfer of vitrified samples under 135 K.4)Super-resolution imaging capability(average single molecule localization accuracy of the cryo-section and intact cell was 13.0nm and 13.6 nm,respectively).The second part of this thesis discusses the development of sample support film for Cryo-CLEM.We have developed a new type of the Formvar-ITO support film by using ITO to improve the conductivity of the Formvar film.And the super-resolution Cryo-CLEM imaging of the cryo-section suggests that the new support film can satisfy the requirements of Cryo-CLEM.In addition,in the study of the structure of the p62-positive mitochondrion-associated protein aggregates(MAPAs),we found that the MAPAs is a special multilayer membrane structure by using the constructed imaging system and the developed sample support film,and the structure obtained provides evidence for further study of its function.Compared to the previous sample support films,the new support film has the following advantages.1)Laser absorption is far less than 10 nm carbon film,in the bands of 405 nm,488 nm,561 nm and 647 nm,the optical absorption of 10 nm ITO film is about 44.4%,16.8%,14.9%and 16.1%of 10 nm carbon film;2)It can withstand about2.0 kW/cm~2 of 488 nm laser irradiation when supporting the cryo-section,which can meet the requirements of Cryo-PALM.3)The performance of the Formvar-ITO support film is better than that of the Formvar-Carbon support film under Cryo-EM,which is suitable for Cryo-EM.The two parts,which are independent of each other,and have internal close connection.The excellent temperature and mechanical stability of the ultra-stable super-resolution fluorescence cryo-microscopy,as well as the good transparency and conductivity of the new sample support film,together provide the necessary technical support for Cryo-PALM imaging of the vitrified biological samples.At the same time,the optimization and improvement of the existing Cryo-FM and sample support film provide a new solution for further perfecting Cryo-CLEM. |
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