Improving The Performance Of Expansion Microscopy Based On Hydrogel Matrix And Immuno-HCR

In the past two decades,super-resolution microscopy imaging technology has become the hottest direction in optical microscopy,especially after the 2014 Nobel Prize in Chemistry was awarded to three scientists who made outstanding contributions to super-resolution fluorescence microscopy imaging technology.Super-resolution microscopy imaging technology,which breaks the optical diffraction limit,has attracted researchers’ attention in the fields of biology,chemistry,and materials science.Super-resolution microscopy imaging technology is widely used to solve difficult problems in their respective fields.Expansion microscopy is an emerging super-resolution fluorescence microscopy imaging technology.The method is different from traditional super-resolution fluorescence microscopy imaging technology.Traditional super-resolution fluorescence microscopy imaging technology needs specific instruments or data processing to achieve super-resolution imaging.Expansion microscopes use physical methods to expand the sample isotropically,magnify the distance between the fluorescent molecules of the sample,and use traditional fluorescence microscopes for imaging.It does not require expensive optical instruments or massive data processing.By increasing the distance between the fluorescent molecules of the sample,expansion microscope breaks the diffraction limit and realizes super-resolution imaging.Because expansion microscopy realizes super-resolution imaging from magnifying the sample,this imaging technology can be well integrated with various optical microscopes,including traditional super-resolution imaging technology.This imaging technology may become a research hotspot in the field of super-resolution imaging.Due to the diffraction of light,it has been difficult for optical microscopes to break the diffraction limit for many years,which has limited the imaging resolution of light microscopes.Improving the imaging resolution of optical microscopes is still a hot topic in microscope research.In Chapter 2,we measured the point spread function of a confocal microscope with sub-resolution fluorescent microspheres and evaluated the imaging resolution of the microscope based on full width at half maximum of point spread function.The 60 X objective lens(NA: 1.4 oil immersion)of the confocal microscope has a resolution of 270 nm in the x-axis direction,a resolution of 260 nm in the y-axis direction,and a resolution of 550 nm in the z-axis direction.The imaging of the confocal microscope was evaluated for the development of later work.Besides,we used the tubulin of the cell as a model and used the deconvolution algorithm to restore the image.By measuring the full width at half maximum of tubulin,we evaluated the resolution of raw images and deconvolved images.The result shows that the full width at half maximum of tubulin in the deconvolved image is smaller than the full width at half maximum of tubulin in the original image.This indicates that image deconvolution can effectively improve image contrast and resolution.Expansion microscopes are based on the isotropic expansion of the sample to achieve super-resolution imaging.The mainstream expansion microscopes can only achieve 4 times the expansion of the sample and the lateral resolution of ～70 nm.This novel super-resolution imaging technology is still limited researched.In Chapter 3,we changed the cross-linking density of the hydrogel network by adjusting the concentration of the cross-linking agent and examined the performance of hydrogels with different cross-linking(N,N’-methylenebisacrylamide MBAA).We got the expansion factors from 3.6 to 5.7.The performance of the expansion microscope under different expansion factors was systematically investigated,including fluorescence retention,expansion deformation,and full width at half maximum of tubulin.Based on the studies,a 0.06%-MBAA expansion microscope was proposed.0.06%-MBAA expansion microscope reveals the fine structure of tubulin,achieving a 5.7-fold expansion of the sample and a lateral resolution of ～50 nm.Finally,we used the 0.06%-MBAA expansion microscope for clathrin imaging.The clathrin coated pits that cannot be displayed in the traditional fluorescence microscope are displayed in the 0.06%-MBAA expansion microscope image.This demonstrates its powerful resolving ability.Expansion microscope realizes super-resolution imaging based on sample expansion.This process requires anchoring the target,digesting the cytoskeleton structure and cell cross-linking network,and expanding the sample,which will inevitably lead to signal loss and reduce the contrast of imaging.In Chapter 4,we took advantage of the specificity of DNA hybridization and the ease of signal amplification to enhance the contrast of imaging.We labeled the DNA sequence on the secondary antibody,used the DNA sequence to trigger the hybridization chain reaction(HCR),and developed immuno-HCR staining.This staining method significantly increases the intensity of the fluorescent signal and improves the contrast of imaging.Then,we applied the immuno-HCR staining for the expansion microscope.The imaging clearly shows the network structure of tubulin,achieves a 3.6-fold expansion of the sample,and gets an overall expansion deformation of less than 3% of the measurement distance.Immuno-HCR expansion microscope improves the resolution double times,significantly improves the imaging contrast of the expansion microscope and solves the problem of low imaging contrast of the expansion microscope.In recent years,with the development of DNA nanotechnology,a variety of DNA nanostructures have been synthesized using the specificity and predictability of DNA complementary hybridization.Moreover,many DNA dynamic nanodevices have been designed based on DNA strand displacement reactions,such as DNA walkers,DNA motors,DNA molecular machines.Researchers used these DNA nanodevices for analysis,detection,diagnosis,and therapy.However,there are few reports on DNA nanodevices on cell membranes.In Chapter 5,we designed a series of catalytic hairpin assembly(CHA)on cell membranes.Using single catalytic chains,double catalytic chains or anchored catalytic chains initiate CHA on cell membranes.We demonstrated the random movement of the double catalytic chain on the cell membrane with flow cytometry data and confocal fluorescence microscope images,and developed a DNA walker on cell membranes.Besides,we also designed a DNA motor on the cell membrane and successfully used the DNA motor to identify the target object on the cell membrane.By controlling the temperature,the stability problem of the DNA probe on the cell membrane is solved to a certain extent,which provides a strategy for the development of the DNA dynamic device on the cell membrane.