衍射光學元件用於輕便光切片顯微鏡術設計

Abstract

為了實現對活體生物樣品的卓越三維成像，需要精細的光學切片、高速圖像採集和低光損傷。紙片光螢光顯微鏡（LSFM）滿足了所有條件，因此成為在體內觀察模型生物發育過程的主要技術。樣品架的特殊要求以及紙片光螢光顯微鏡的照明和檢測臂的正交結構，造成設計輕便型系統時產生了限制。 本文提出了兩種輕便型LSFM系統的新設計方法，並進行了實驗驗證。在第一種方法中，設計了體積全像紙片光螢光顯微鏡（VH-LSFM）。為了簡化設置而不會降低其成像性能，使用了光聚合物的體積全息光柵（VHG）來代替照明臂的笨重的圓柱透鏡和物鏡。體積全息光柵具有多種優勢，例如單一繞射、寬頻帶操作、緊湊的尺寸和設計靈活性，這使得體積全像術很適合與商用顯微鏡做結合。在第二種方法中，設計了微製成紙片光片螢光顯微鏡（Metalens-LSFM）。使用超影透鏡來代替LSFM的整個照明臂。超影透鏡的微型尺寸（數百奈米）有助於大大降低整個LSFM的複雜性。這兩個輕便型LSFM系統已經過實驗表徵，並被證明具有良好的光學切片能力。 為了證明我們的系統對生物醫學的成像能力，我們對秀麗隱桿線蟲進行了體內成像。由於秀麗隱桿線蟲是研究生物學效應的重要生物，因此各種光學顯微鏡技術常以此為目的。然而，使用LSFM系統觀察整個活的秀麗隱桿線蟲的研究很少。使用我們的系統，可以即時觀察線蟲內部結構的高對比度圖像並可以達到細胞等級的分辨率。本文中討論的概念可以為生物醫學應用設計新的光片顯微鏡中找到重要的應用。

To achieve superior three-dimensional imaging of living biological samples, fine optical sectioning, high-speed image acquisition, and low photo-damage are required. Light-sheet fluorescence microscopy (LSFM) fulfills all these conditions and thus becomes a leading technique for in-vivo observations of developmental processes in model organisms. The specific requirements on the sample holder and the orthogonal structure of illumination and detection arms of LSFM imposes restrictions in designing compact systems. In this thesis, two new design methods for compact LSFM systems are proposed and experimentally demonstrated. In the first approach, a volume holographic light-sheet fluorescence microscope (VH-LSFM) is designed. To simplify the setup without degrading its imaging performance, a photopolymer-based volume holographic grating (VHG) is used to replace the bulky cylindrical lens and an objective lens of the illumination arm. VHG offers multiple advantages such as single diffraction order, broadband operation, compact size, and design flexibility which make VHG suitable for integration with the commercial microscope. In the second approach, a metalens light sheet fluorescence microscope (Metalens-LSFM) is designed. A metalens was designed to replace the entire illumination arm of the LSFM. The miniature size of the metalens (a few hundreds of nanometers) helps in drastically reducing complexity of the entire LSFM. These two compact LSFM systems have been experimentally characterized and demonstrated to have good optical sectioning capability. To demonstrate the imaging capability of our system for biomedical imaging, we perform in-vivo imaging of C. elegans, which is an important biological organism to study biological effect. Various optical microscopy techniques have been used for this purpose; however, there are only few reports on the use LSFM system to observe entire live C. elegans. With our systems, high contrast images of internal structures of C. elegans up to cellular resolution can be observed in real-time. Concepts discussed in this work may find important applications in designing new light-sheet microscopes for biomedical applications.