晶格層光顯微術用於微流體晶片影像流式細胞技術開發之研究

Abstract

對於活體生物樣本的觀測而言，樣本所受到之光漂白與光毒性為影響成像效果的重要因素，有鑑於目前的層光螢光顯微術之激發光普遍使用環形光罩以產生貝索光束，然而，其具有旁波瓣而導致樣本於焦平面外額外之光漂白與光毒性，故本研究改用空間光調製器產生晶格光束，以相消性干涉的方式消除旁波瓣之影響，即晶格層光顯微術。本研究結合了層光螢光顯微術以及微流體系統以開發出一套影像流式細胞儀系統，期望藉由光學系統之優化以及流體之連續性來達到高解析度與高效率之樣本檢測效果。 本研究設計了兩層式的微流道晶片以符合光學系統的空間位置，製程的部分首先使用黃光微影製程進行微流道母模之製作。第一層晶片為樣本之觀測區域，使用OSTEMER 322樹脂作為本體，並使用折射率接近於水的LUMOX®薄膜作為微流道晶片之基板，以減少光束傳遞於不同介質中的影響；第二層晶片則是採用了流道轉向的設計，使得樣本流體的出入口延伸於鏡頭的外部，避免於檢測樣本時導致晶片被鏡頭頂到。 本研究成功地開發出一套具有高解析度、高檢測效率、低光漂白及低光毒性之光學與微流體整合系統，於微流道中所量測到之層光厚度僅1~2 μm，微流體系統的部分則比較了單條型與分支型之微流道設計於光學系統下之成像效果，亦針對樣本流速的部分嘗試進行優化，最後成功得到多張樣本之切片影像，清晰地觀測到中國倉鼠卵巢細胞之粒線體結構，達到細胞內之可視化效果，並根據切片影像進行反摺積運算以及生物樣本之三維影像還原。

For the live cell imaging, photobleaching and phototoxicity to the samples are the major reasons which influence the image quality. In view of the current excitation of light sheet fluorescence microscopy, an annular mask is commonly used to generate a Bessel beam. However, Bessel beam is always accompanied by the annoying side lobes to cause the additional photobleaching and phototoxicity. Therefore, we use the spatial light modulator to generate the lattice beam which can eliminate the side lobes by destructive interference which is so-called lattice light sheet microscopy. In our research, the imaging flow cytometry is developed by the combination of the light sheet fluorescence microscopy and the microfluidic system in hopes of the high resolution and high efficiency. In our research, a two-layer microfluidic chip is designed for the compatibility of the geometry of the optical system in space. The master mold is fabricated by the photolithography. The first layer of the chip is designed for the sample observation, and it is made of the OSTEMER 322 crystal clear and the LUMOX® film. LUMOX® film is a material which has the refractive index near 1.33 to be compatible with the water dipping objectives in our system for avoiding the influence as light transmitting in different mediums. The second layer of the chip is designed to keep the chip from compressing by the objectives. In our research, a microfluidic system in combination with optical system with high resolution, high efficiency, low photobleaching and low phototoxicity is successfully developed. The thickness of the light sheet in the microchannel is measured about 1~2 μm and the two types of the microchannel design are compared by the image quality under the system. Attempts are made for the optimization of the sample velocity and we successfully capture many slice images of the samples. The structure of the mitochondrion in the CHO cell is clearly presented in the slice images, which demonstrates the intracellular visualization. Finally, deconvolution and 3D reconstruction are made from the slice images.