Development Of Super-resolution Fluorescence Microscope For Expanded Tissue Samples

Stochastic optical reconstruction microscopy(STORM),as a super-resolution technology,breaks the traditional diffraction limit.It is ultra-high precision single molecule localization technology realized by using photon-switchable fluorescent labels.Using this principle,the super-resolution image obtained can reach a lateral resolution of up to 20 nm and an axial resolution of up to 50 nm,which is higher than the diffraction limit by an order of magnitude.Due to the availability of super-resolution images,STORM has become an important tool for studying cell characterization.While in terms of tissue imaging,due to the high scattering and non-uniform of tissue samples,STORM has been obstructed and restricted when applying in imaging tissue samples.In order to apply STORM in imaging tissue samples,on the one hand,the expansion technique is used to make the tissue samples uniform and transparent,and reduce the high background caused by scattering and auto fluorescence;on the other hand,we independently set up a light-sheet microscope,which has the characteristics of fast imaging speed,low photobleaching and low phototoxicity.To activate the fluorescent dyes,two-photon laser is introduced in the illumination path.Because of the nonlinearity of two-photon laser,only the fluorescent dyes in the center of the field of view can be actived,which can reduce unnecessary photobleaching of fluorescent dyes which are out of field of view.To improve resolution when imaging deeply,We add adaptive optics in the imaging path,using closed-loop correction algorithms to correct the aberrations caused by imperfect imaging system and the refractive index mismatch between the imaging buffer and the imaging objective.In this way,3D STORM super-resolution imaging in the thick tissue sample can be realized.We firstly expanded the 150?m thickness brain slice of Thyl-YFP transgenic mouse expand by 2 times,so that the sample changed to transparent and uniform,and autofluorescence can be quenched.Then,three-dimensional STORM images of neurons in the expended brain slices at a depth of 300?m were taken by using the built system,.By controlling the movement of the piezoelectric ceramic nano-stage in the axial direction,super-resolution images of sample at different depths were obtained,and they were pieced together to obtain three-dimensional super-resolution images of neurons in mouse cerebral cortex in the volume range of 100?m×100?m×30?m.