Living organisms carry out every function of the life processes relay on themolecule interactions inside of them. The conventional fluorescencemicroscopies are used to visualize the behaviors of molecules, subcellular units,and cells to explore these interactions. Because of limits of conventional opticalimaging, most bio-specimens for fluorescence imaging measurement are via cellscultured on two-dimensional substrate surface. Although three-dimensional (3D)scaffold culture techniques attempt to provide an approximate cellularmicroenvironment, it still can’t simulate all factors of physiological environment.To understand the functions of biomolecules for the specific process, the cellsspecimens should be surrounded by all physiological environmental factors.Single-molecule localization microscopy (SMLM) combined with temporalfocusing multiphoton excitation (TFMPE) and astigmatic imaging is possible todeliver the nanoscale-level 3D positions of fluorophore-labeled biomolecules thatreveals the distribution and organization of proteins and subcellular structures inthick specimens. However, the localization resolution, imaging depth, andnumber of fluorescence imaging channels of TFMPE-SMLM for the multi-speciesbiomolecules and deep-tissue imaging are still restricted due to the fixedwavelength excitation, the aberrations induced by the tissue specimens, and thecrosstalk of fluorescence signals. Therefore, the spectral adaptive optics (AO)-assisted TFMPE-SMLM imaging is proposed in this proposal. The TFMPE-SMLMimaging of multifluorophore-labeled tissue specimens via the fast tunablewavelength excitation for implementing the optimum wavelength excitation, theadaptive aberration correction for restoring the point spread function, and spectralmeasurement for identifying the different fluorophore species can enhance thelocalization resolution, imaging depth, and number of fluorescence imagingchannels. Furthermore, the SMLM data can be extracted the quantitativeinformation of biomolecule of interest by the quantitative algorithms. The spectralAO-assisted TFMPE-SMLM system has great potential to elucidate moleculardistribution and interaction in deep tissues via its optical sectioning, fast framerate, optimal MPE, nanoscale-level 3D positioning, superior imaging depth,nominal photobleaching, reduced photondamage, and capabilities of singlemolecule detection and spectral measurement. Therefore, the proposed systemand 3D tracking measurement would be used to investigate two importantsubjects of neurodegenerative diseases. One is exploring subcellularcompartmentalization of the beta-amyloid (Aβ) and tau toxicity, the synergisticinteraction, and the spreading behaviors of the disease-specific proteins. Deeptissue multicolor SMLM imaging analysis can provide the evidences about theinteraction mechanism and potential locations of the toxic oligomers, precursorsof the fibrillar aggregates, and pathology-related proteins in the brain tissue. Theother is investigating therapeutic mechanism of the drug-loading nanocarriers inneuron cells and brain tissues of neurodegenerative diseases. Deep-tissuemulticolor SMLM imaging of the disease-specific proteins and therapeuticnanocarriers will also help to understand the therapeutic mechanism in manyaspects including the inhibition of Aβ oligomerization and fibrillization, taupropagation and spreading, mitochondrial and microglia functions, cellularreactive oxygen species and inflammatory responses. Therefore, thesesignificant results would be highly beneficial to inspire the researchers to developthe novel precision strategies for diagnosing and treating the neurodegenerativediseases.