Application Of Expansion Microscopy For Biological Samples

Expansion microscopy(Ex M)is a super-resolution imaging technology based on the uniform expansion of biological samples with the polyelectrolyte hydrogel.It solves the problems of expensive equipment,complex operation,and limited choice of samples in other super-resolution imaging techniques.With the integration into the conventional optical microscope,Ex M has the advantage of making the sample transparent and compatible with multiple immunofluorescence staining.However,this indirect imaging method suffers from a suspectable imaging fidelity.On the one hand,it is hard to ensure the isotropic and homogeneous expansion of the polymer network.On the other hand,the linear expansion factor is calculated by a subjective image registration method,which is difficult and costs a long time.Here,we developed the photonic crystal spectrum ruler and fluorescence marker array for the measurement of linear expansion factor in Ex M.Their feasibilities in open environment and microfluidic chip have been verified in practical experiments.The specific research contents are as follows:(1)To observe C2C12 cells and tumor spheres by the Ex M,the treatment conditions were explored.According to the fluorescence retention rate,suitable dyes,anchors,and their concentrations were selected.Besides,the operation procedures including sample processing and image registration were confirmed.The resolution of 60 nm was obtained in the expansion imaging of cells,and the stability of this method was verified by the image registration method.This part determined the suitable materials and operation procedures of expansion microscope for C2C12 cells and tumor spheres,which laid the foundation for the follow-up experiment.(2)Based on the corresponding relationship between the macroscopic reflection spectrum and the microscopic lattice distance of photonic crystals,a photonic crystal expansion microscope was developed for in-situ measurement of the linear expansion factor.A photonic crystal spectrum ruler which could expand with the swellable polyelectrolyte gel network,was achieved with the self-assembled non-close packed photonic crystal hydrogel and the linear relationship between the reflection peak and local linear expansion factor.By using the photonic crystal ruler,the difference of the expansion factors between the sample area and the non-sample area was found and corrected.This work provides a quick and accurate method for the measurement of the local expansion factor,which is helpful to improve the imaging fidelity of the expansion microscopy.(3)The fluorescence marker array was prepared by two-photon polymerization laser direct writing technique,and three-dimensional super-resolution imaging of the tumor sphere was demonstrated by integrating Ex M into the microfluidic chip.The effects of printing speed,laser intensity,UV irradiation,and immersion in aqueous solution on these fluorescence markers were explored to determine the preparation parameters.By studing the deformation of markers and cells during the expansion of polyelectrolyte gel,the effect of gel shape on the expansion uniformity was eliminated.In addition,a microfluidic chip fabricated with polymethyl methacrylate was used to construct and control the expansion of the hydrogel in the chip.Combined with the fluorescence marker array,the expansion factor of the gel network was measured and the internal structure of the tumor sphere was characterized with the optimal Ex M.This part provides another measurement method for the expansion factor and proves the feasibility of integrating the expansion microscopy into the chip.