掃描式貝索光束用於微流道影像流式細胞技術開發之研究

Abstract

活體細胞的3D即時影像能夠幫助我們了解細胞的結構在各種情形下產生的變化，然而傳統的螢光顯微技術，其空間解析度較差，入射光產生的大量光漂白和光毒性也限縮了傳統螢光顯微技術的發展性。層光顯微技術將入射光限制在觀察平面上，不僅大幅降低了光漂白的問題，也將背景雜訊降至最低。層光顯微技術的效能與光的厚度有關，而現今最薄的層光為透過貝索光束掃描而成。然而層光顯微技術的樣本往往固定在封閉的腔室內，觀測不同樣本時，需重新進行系統的校正而限制了層光顯微技術的效能，因此，本研究目的在於開發一套結合微流道與掃描式貝索光束的層光顯微系統，透過導入流體的概念，使層光顯微技術可以得到連續高通量高空間解析度的生物斷層影像。 本研究之光學系統係利用雷射光通過環形光罩建構貝索光束，再透過掃描振鏡使貝索光束來回掃出層光，搭配高N.A.的物鏡與高速攝影機，以獲取樣本在數十毫秒內通過層光的影像。微流道製程的部分，透過黃光微影製程定義出微流道的圖形，製作出晶圓母模，再利用PDMS進行翻模，最後再透過OSTEMER 322進行第二次的翻模，得到微流道的本體，最後黏上LUMOX薄膜，即可得到與光學系統相容之微流道晶片。 本研究成功開發出一套結合微流道與掃描式貝索光束層光系統的影像流式細胞技術，在微流道中觀察到的層光厚度半高寬為2.49微米，具有良好的軸向解析度，軸向點擴散函數之半高寬僅1.38微米，並成功捕捉到連續高解析度的生物樣本斷層影像。

3D live imaging is essential to a better understanding of cell structure under different biological process. However, the low spatial resolution, phototoxicity and photobleaching of conventional epi-fluorescence microscopy restrict its development. For the selective plane illumination microscopy(SPIM), its excitation light is confined near the focal plane, which reduces the photobleaching and minimizes the out-of-focus background noise. The performance of SPIM depends on the thickness of the light sheet. Nowadays, the thinnest light sheet is constructed by Bessel beam. Nevertheless, the samples of SPIM are usually embedded in a closed chamber. When detecting different samples, we need to adjust the system again and again. This time-consuming procedure limits SPIM experimental efficiency. Thus, our study is dedicated to developing a systems combined microfluidic channel and SPIM. By introducing the concept of flow, SPIM can get high throughput and high spatial resolution optical sectioning image. In our study, Bessel beam is constructed by letting laser beam pass an annulus mask and the light sheet is formed by the sweeping of Bessel beam. With high N.A. objective and high speed CCD, we can get images of samples passing through the light sheet in several milliseconds. In the section of the fabrication of microchannel, we print the geometry of designed microchannel pattern onto silicon wafer and the pattern is transferred onto PDMS. Then, we transferred the pattern from PDMS onto OSTEMER 322. At last, we stick LUMOX film onto OSTEMER 322. A microchip compatible to our optical system has been fabricated. We developed a system combined microfluidic channel and Bessel beam plane illumination. The thickness of light sheet detected in microchannel is 2.49 μm(FWHM) and the axial PSF is 1.38 μm(FWHM). Namely, continuous high resolution sectioning images are captured by our system.