Chemical Manipulation For Multiscale And High-Resolution Immunofluorescence Imaging

Immunolabeling is the most direct technique to characterize molecular phenotypes,and the labeling methods for large volume samples are constantly developed and innovated along with the rise of tissue clearing techniques.However,corresponding multiscale imaging methods are always conflict between speed,contrast/resolution,and specimen volume,due to the limitation of the optical principle.It is difficult to simultaneously satisfy the requirements of fast,high resolution and multiscale imaging for immunofluorescence samples by optimizing optical parameters at the physical level.Chemical manipulation is a unique idea to improve the performance of optical imaging,and many relevant methods have been developed.Within the optical diffraction limit,the chemical sectioning imaging enables the sectioning capacity in a wide-field microscope by manipulating fluorescence molecular“on-off”state,which satisfies imaging requirements of fast and high-resolution.To break through the optical diffraction limit,another chemical manipulation method,the tissue expansion,enables traditional optical imaging to achieve resolution beyond the diffraction limit by manipulating the sample size.Combined with the sequential sectioning imaging strategy,the two methods both have the potential to achieve fast and multiscale imaging.However,the existing chemical sectioning strategy is only suitable for samples labeled fluorescent proteins,not working for immunolabeled samples,and the existing swollen hydrogels used in expansion microscopy are soft and fragile,which are incompatible with sequential sectioning imaging systems.Focusing on the above problems,this study has respectively developed immunofluorescence chemical sectioning and immunofluorescence expansion tomography for fast,high resolution and multiscale immunofluorescence imaging,providing an effective means to promote the systematic study of immunofluorescence labeled elaborate subcellular structures.The main research contents are as follows:(1)Establishment of a new fluorescence switch manipulation method of fluorescent dyes for the immunofluorescence chemical sectioning.Based on the structural analysis of Alexa dyes,ferric ion and desferrioxamine were found to manipulate the fluorescence“on-off”switch of Alexa dyes.Using these dyes in immunolabeling and chemical sectioning imaging,the out-of-focus interference below surface layer was suppressed in wide-field imaging.Combined with the wide-field sequential sectioning imaging system,immunofluorescence chemical sectioning obtained the location information of axonal buttons and nerve factors in dopaminergic neural circuit of immunolabeled mouse brain tissue with volume of 4200×3200×1000μm~3,at the scanning speed of 1.36×10~8 voxel/s and the voxel resolution of0.116×0.116×1μm~3,achieving compatibility of speed,resolution,and multiscale imaging in immunofluorescence.(2)Development of a machinable expansion hydrogel for achieving immunofluorescence expansion tomography.By systematical studying effects of each monomer component on the mechanical strength and expansion factor,and it was found that the swellable hydrogel with sodium 2-acryla-mido-2-methyl-1-propanesulfonic acid(AMPS-Na)as a superabsorbent reagent had higher mechanical strength than the existing swellable hydrogels,which could meet the requirements of sequential sectioning.Simultaneously,the expansion factor of the new hydrogel was comparable to that of the existing hydrogel(～4×),which could meet the imaging requirements beyond the optical diffraction limit.The performance parameters of the new hydrogel in immunolabeled tissue were further studied,which verified that the new hydrogel satisfied the indexes of expansion microscopic imaging.Combined with high-throughput light sheet tomography,dendritic spines morphological information of an individual neuron in immunolabeled brain tissue with volume 4600×4200×1000μm~3(20.7×18.9×4.5 mm~3 after expansion)was obtained at1.66×10~9 voxel/s imaging speed and 144×144×101 nm~3 voxel resolution,providing a methods for axially extended super-resolution multiscale imaging.