電場增強分子傳遞以應用於高通量多光子顯微術

Abstract

本論文研究電場加速在大組織中染色的過程。此方法使用電場來增強帶電分子（例如：核染劑、蛋白質和抗體）的擴散性，並且不會損壞帶電樣品本身。本實驗中，我們設計了兩個獨特的裝置，即雙平行電極和雙環電極，用來施加電場在腫瘤組織上。將100微米厚度的肺腺癌腫瘤組織和1毫升的Hoechst 33342染劑放入PET膜中，並將PET膜和1.6毫升的LAB培養基一起放置到24孔細胞培養板內。位於PET膜兩側的電極產生電場在腫瘤組織上。在雙光子顯微鏡掃描腫瘤組織影像後，我們分析影像藉以獲得與深度相關的平均亮度曲線。 將組織放置於兩側電極中間後，我們的結果表明，在 Hoechst 33342溶液（用ddH2O稀釋）中的帶電離子運動可以滲透到組織深處而不破壞表面，並且允許有效的染劑滲透。在施加電壓10 伏特時，有明顯的擴散發生。此外，雙環電極的設計優於雙平行電極。對於雙平行電極，由於在施加電場時電極會產生氣泡，位於腫瘤組織正下方的電極會造成PET膜移動。

In this thesis, we study new electric field method to accelerate the staining process in large tumor tissues. It enhances the apparent diffusivity of electromobile molecules, such as nuclear dyes, proteins, and antibodies, without damaging tumor tissues. In this work, we design two unique devices, for one with two-parallel electrode and the other with double-ring electrode to apply the voltage on tumor tissues with charged dyes. For experimental design, a 100 µm thick lung adenocarcinoma tumor tissue and 1 mL Hoechst 33342 containing positive ions will be loaded into a PET membrane sitting on one of 24-plate wells with LAB media. Electrodes are positioned next to the membrane so as to produce the electric field on the tumor tissue. After scanning tumor tissue images from two-photon microscopy, we could obtain a depth-dependent averaged intensity curve featuring the image. After placing the tissue at the middle of two electrodes, our results show that the movement of charged ions in the Hoechst 33342 solution (diluted with ddH2O) are able to penetrate into the depth of the tumor tissue without destroying the surface and allow efficient dye penetration at the same time. The optimal voltage for obvious diffusivity lie in 10 volts. Furthermore, we found that double-ring electrode design outperforms its parallel electrode counterpart. For two-parallel electrode, due to bubbles generated from the application of the electric field, the electrode right below the tumor tissue would result in the displacement of the PET membrane.