Super-Resolution Imaging Informatics for Molecule Functions Analysis in Thick Biological Specimens(3/3)

The localization-based super-resolution fluorescence microscopy using the localization algorithm,the multi-fluorescence detections, and the axial positioning techniques has been demonstrated toachieve three dimensional (3D) multi-color fluorescence imaging with a lateral and axial resolutionof 10-20 nm and 30-50 nm to extract the significant spatial distribution and biophysical factors oftarget molecules in cell biology, neuronal, and other biomedical researches. However, thelocalization-based 3D super-resolution microscopy still has a serious drawback for the applicationsof thick biological specimens. Because the fluorescence excitation of conventional super-resolutionfluorescence localization imaging is based on single photon excitation, the drawbacks of thisapproach is photoblaching of fluorophores at other layers and the less imaging depth within thescattering specimens. Therefore, 3D dual-color super-resolution multiphoton excitation fluorescencelocalization microscopy using the temporal focusing multiphoton fluorescence astigmatic imaging isproposed in this proposal. Images of two fluorophores are obtained to locate their positions byoptical sectioning excitation using a fast temporal focusing multiphoton excitation microscope andby the simultaneous collection of data in two channels. The proposed 3D dual-color super-resolutionmultiphoton excitation fluorescence localization microscopy is useful for the dynamic study ofmolecular interactions in living thick biological specimens with a nanoscale resolution, singlemolecule detection, 3D imaging, nominal photobleaching, and superior penetration depth. Theproposed 3D dual-color super-resolution multiphoton excitation fluorescence localizationmicroscopy will used to explore two significant biomedical subjects. One is the epidermal growthfactor receptor (EGFR) activation of non-small cell lung cancer (NSCLC) with formation ofreceptor oligomers/clusters and the endocytosis modulation in the tumor associatedfibroblast-derived 3D matrices or the 3D collagen matrices. 3D dual-color super-resolutionlocalization imaging can show the significant evidence about the formation of receptoroligomers/clusters and the recruitment of receptor to CCPs for its activation of cancer related EGFRmutants in tumorigenesis. The other one is the trans-synaptic propagation of neurodegenerativedisease-specific proteins, which including Aβ, tau, α-syn, and others, in the neuronal synapse ofbrain. 3D super-resolution localization image of the pathological aggregates will also provide theevidence whether interneuronal transfer of pathological aggregates is facilitated by the trans-synaptic propagation in the neurodegenerative disease. These results will help to open a newdirection to develop drug target steps in disease related aberrant EGFR activation and the effectivetherapies of neurodegenerative diseases.