| 摘要: | 生物體之生理程序與功能是仰賴其內部的分子交互作用來達成，而傳統螢光顯微術用於觀測生物體內部分子、次細胞單元和細胞的行為表現，進而探索這些複雜的交互作用機制。然而，因為傳統光學成像限制，大多數用於螢光顯微成像測量的生物樣品是以在二維基材表面上培養的細胞；雖然三維支架培養技術試圖提供近似細胞微環境的表現，仍不能完全模擬生理環境的所有因素。因此、為了理解生物分子於特定生理過程的功能，細胞樣品應存在於具所有生理因子的環境，即原組織內。單分子定位顯微術結合時域聚焦多光子激發和像散成像具有提供螢光標記之生物分子的三維奈米尺度位置資訊之能力，進而可用於顯示厚樣品中蛋白質和次細胞結構的分佈和表現。但是此顯微術對於多種生物分子和深部組織成像時，螢光成像通道之數量、定位解析度和成像深度仍受限於固定波長激發、組織樣本引起之像差、和螢光訊號間的串擾等因素。因此，計畫中提出光譜解析適應性光學輔助時域聚焦多光子激發單分子定位顯微術，多種螢光標記之組織樣本的單分子造影可分別利用快速可調波長激發來實現最佳波長激發、適應性像差校正來復原點擴散函數和光譜測量來識別不同種類的螢光分子，進而增強定位解析度、造影深度和螢光成像通道之數量。同時，單分子定位顯微影像之數據也可以定量演算法來提取感興趣生物分子的相關定量資訊。此新式單分子定位顯微術具有光學切片、快速取像速度、最佳的多光子激發、三維奈米尺度定位、優越造影深度、低光漂白和光毒性、單分子偵測和光譜量測的能力，對於闡明深組織中之生物分子的分佈和交互作用機制將具有極大潛力。計畫中將以此新式系統和三維追蹤量測研究神經退化性疾病的兩個重要主題。第一項為探索類澱粉蛋白和tau蛋白毒性的次細胞區室化、類澱粉蛋白和tau蛋白的協同交互作用和疾病特異性蛋白的擴散行為，深組織多色單分子定位顯微影像分析可提供關於腦組織中毒性的寡聚合物和原纖維聚集體前軀物及病理相關蛋白的交互作用機制和潛在作用位置的證據。另一項為神經退化性疾病之腦組織和神經元細胞中具藥物裝載之奈米載體的治療機制。疾病特異性蛋白和治療性奈米載體的深組織多色單分子定位顯微影像也將有助於理解多方面的治療機制，如類澱粉蛋白寡聚化和纖維化之抑制、tau蛋白之增殖和擴散、線粒體及小膠質細胞功能和細胞活性氧和發炎反應等。因此，這些重要的結果將非常有助於啟發研究人員開發新穎精準診斷與治療神經退化性疾病的策略。 ;Living organisms carry out every function of the life processes relay on the molecule interactions inside of them. The conventional fluorescence microscopies are used to visualize the behaviors of molecules, subcellular units, and cells to explore these interactions. Because of limits of conventional optical imaging, most bio-specimens for fluorescence imaging measurement are via cells cultured on two-dimensional substrate surface. Although three-dimensional (3D) scaffold culture techniques attempt to provide an approximate cellular microenvironment, it still can’t simulate all factors of physiological environment. To understand the functions of biomolecules for the specific process, the cells specimens should be surrounded by all physiological environmental factors. Single-molecule localization microscopy (SMLM) combined with temporal focusing multiphoton excitation (TFMPE) and astigmatic imaging is possible to deliver the nanoscale-level 3D positions of fluorophore-labeled biomolecules that reveals the distribution and organization of proteins and subcellular structures in thick specimens. However, the localization resolution, imaging depth, and number of fluorescence imaging channels of TFMPE-SMLM for the multi-species biomolecules and deep-tissue imaging are still restricted due to the fixed wavelength excitation, the aberrations induced by the tissue specimens, and the crosstalk of fluorescence signals. Therefore, the spectral adaptive optics (AO)-assisted TFMPE-SMLM imaging is proposed in this proposal. The TFMPE-SMLM imaging of multifluorophore-labeled tissue specimens via the fast tunable wavelength excitation for implementing the optimum wavelength excitation, the adaptive aberration correction for restoring the point spread function, and spectral measurement for identifying the different fluorophore species can enhance the localization resolution, imaging depth, and number of fluorescence imaging channels. Furthermore, the SMLM data can be extracted the quantitative information of biomolecule of interest by the quantitative algorithms. The spectral AO-assisted TFMPE-SMLM system has great potential to elucidate molecular distribution and interaction in deep tissues via its optical sectioning, fast frame rate, optimal MPE, nanoscale-level 3D positioning, superior imaging depth, nominal photobleaching, reduced photondamage, and capabilities of single-molecule detection and spectral measurement. Therefore, the proposed system and 3D tracking measurement would be used to investigate two important subjects of neurodegenerative diseases. One is exploring subcellular compartmentalization of the beta-amyloid (Aβ) and tau toxicity, the synergistic interaction, and the spreading behaviors of the disease-specific proteins. Deep-tissue multicolor SMLM imaging analysis can provide the evidences about the interaction mechanism and potential locations of the toxic oligomers, precursors of the fibrillar aggregates, and pathology-related proteins in the brain tissue. The other is investigating therapeutic mechanism of the drug-loading nanocarriers in neuron cells and brain tissues of neurodegenerative diseases. Deep-tissue multicolor SMLM imaging of the disease-specific proteins and therapeutic nanocarriers will also help to understand the therapeutic mechanism in many aspects including the inhibition of Aβ oligomerization and fibrillization, tau propagation and spreading, mitochondrial and microglia functions, cellular reactive oxygen species and inflammatory responses. Therefore, these significant results would be highly beneficial to inspire the researchers to develop the novel precision strategies for diagnosing and treating the neurodegenerative diseases. |
