層光螢光顯微術用於微流道影像式流式細胞技術之研究

Abstract

胚胎發育生物學仰賴活體樣本的即時影像佐證，然而傳統螢光顯微術存在光漂白與光毒性的問題，使其不利於長時間的活體觀測。本研究整合層光螢光顯微術與微流體系統的優點，開發一套低光漂白、低光毒性及高解析度的高通量影像式流式細胞儀。 本實驗以層光系統與生物樣本的限制打造兼容兩者的微流道裝置。此微流道製程有於傳統實驗室晶片，以毛細管拉針的幾何外型製作二甲基矽氧烷（Polydimethylsiloxane, PDMS）模具，再以高分子材料MY-133-V2000進行二階段灌模固化製作出裝置本體。MY-133-V2000固化後的折射率與水相符，能配合光學鏡頭的工作環境，使光束於不同介質傳遞時不因介面折射率差異影響成像品質。 依樣品注入微流道的動力來源，可分為動力推進法及重力法。動力法雖可連續觀測樣本，但科儀相機曝光時間無法跟上樣品流速會使影像帶有嚴重地動態模糊。因此，本實驗將動力來源改以生物樣本自身重力進行實驗，達成連續觀測活體樣本之目的，獲得高解析度之秀麗隱感線蟲（C. elegans）胚胎的四維影像。本影像式流式細胞儀原型，成功地保有微流體系統的優點，同時避開傳統顯微術的缺點。

Embryonic developmental biology relies on real-time specimen images as their evidence, however, conventional fluorescence microscopy exhibits photobleaching and phototoxic effects, making it a second-choice candidate for long term live observations. By combining the benefits of light sheet fluorescence microscopy and microfluidic system, we establish a minimal photobleaching, low phototoxicity, high-resolution, and high-throughput image-based flow cytometry. This experiment designs a microfluidic device that can fit within the constraints of our light sheet system and the specimen. The fabrication process of this device is unique from traditional lab-on-chips, using the geometry of pulled capillary tubes to make polydimethylsiloxane (PDMS) molds, and use polymer material MY-133-V2000 two-step curing to create the device itself. Solidified MY-133-V2000 has the same refractive index as water, which can work in harmony with our objective lenses, alleviate image distortions result from light propagating through interfaces with the different refractive indexes. Sample can be propelled by either an active source or gravity. Though active source allows one to continuously observe samples, scientific camera scan lines may out-of-sync with sample flow rates, result in severe motion blur. Therefore, this experiment uses the specimen itself as weight source, reaching our final goal - continuous live specimen observation, acquiring high-resolution C. elegans embryo volumetric in situ images. Our image-based flow cytometry prototype successfully retains the strongholds of microfluidic systems while avoiding the weakness of traditional microscope systems.