微小非球面鏡於微型化視頻非線性光學顯微鏡之應用

Abstract

非線性光學顯微術的發展已經有許多年了。其相對於傳統顯微鏡較為優秀的光學虛擬切片能力，來自於其光學機制-非線性訊號僅限於物鏡焦點的附近才能產生。該顯微術所使用的光源通常為近紅外光，相對於一般可見光不只對於生物體擁有較低的破壞性，更有較佳的穿透深度。典型非線性光學顯微系統的架設是直接將一般的顯微鏡與雷射光源和額外的掃瞄機制整合在一起，然而這將使的整個系統顯得笨重許多。 為使非線性光學顯微術在臨床上能更方便應用，則原本笨重的系統就必須被縮小、重新設計。在本論文中，我們研究的重心將會擺在微小化的鏡頭上。在我們的藍圖中，一個微小化的觀測鏡頭是必要的，因為一個手持大小的系統在觀測過程中可以擁有更多方便性及彈性。再者，我們希望這個鏡頭能提供寬廣的觀測視野，這樣在觀察的過程中我們便可同時揭露更多資訊；系統的掃描機制如果能夠提供夠高的畫面更新率，則在觀察過程中微小震動造成影像模糊的問題可以獲得改善，甚至可以進行動態影像觀察的應用。 非球面鏡名稱的由來在於其表面並非剛好是正球型的一部份。相對於一般簡易生產的球面鏡，其較複雜的表面是專門設計用來減少像差或是取代多重鏡組。一些非球面鏡被設計的非常小，並被應用在手機相機鏡頭、光碟機讀取頭或是雷射二極體準直鏡上。這些鏡子的尺寸都不大而且價錢也相當便宜，或許可以用來作為傳統顯微物鏡的替代品並發揮類似的功能。 在本論文中，我們將呈現我們對於高數值孔徑微小非球面鏡（0.85 NA 藍光光碟機鏡頭、0.8 NA 雷射二極體準直鏡）作為微型化非線性光學顯微系統物鏡的潛力之研究。本系統的構造非常簡單，只有5片鏡子。微小非球面鏡與不同放大倍率之筒鏡被組合起來當作系統的物鏡，微機電鏡擔任系統掃描機制的角色，一片二色的分光鏡則用來區隔入射的紅外光與反射的可見光訊號。我們研究了該系統在不同非線性光學訊號上之表現，諸如：雙光子螢光、二倍頻、三倍頻，並使用綠螢光斑馬魚來評估生物活體實驗的可行性，以及動態影像的觀察。在論文中，亦涵蓋了本設計與其他不同微小化系統設計的比較。

Nonlinear optical microscopy has been developed for many years. Due to the mechanism of nonlinear optics, the nonlinear signal can only be generated around the focus of the objective. Thus, this kind of microscopy is known for its optical virtual biopsy ability in comparison with the traditional wide field microscopy. The excitation light is infrared, which is not only less invasive but also provides better depth of penetration. Typical way to apply nonlinear optical microscopy is directly integrating a conventional microscope with a laser light source and some additional scanning mechanisms. However, it will make the whole system bulkier. In order to apply nonlinear optical microscopy in clinical applications, the system must be miniaturized and redesigned for more flexibility. In this thesis, the investigation is focused on the miniaturized imaging head. An optical imaging head with a miniaturized size, a larger field of view (FOV), and a video frame-rate is highly desired because a miniaturized system is more convenient to be manipulated during the observation and allows intravital applications. Larger field of view means we can reveal more information once simultaneously. Higher frame rate can not only deal with the image blurring problem resulted from vibrations but also allow one to reduce the imaging acquisition time, thus dynamic observation may be realized. Aspheric lenses, which are known for their complex lens surface profile designed for aberration reduction or replacement for a multi-lens system, are used in 3C products, such as cell phone cameras, optical disk drives, or laser diode collimators. With its smaller size and cheaper price, it could be an alternative to traditional objectives in miniaturized nonlinear microscopy systems. In this thesis, we present our investigation on the potential to use high numerical aperture mini aspheric lens (a blu-ray disk lens with 0.85 NA and a laser diode collimating lens with 0.8 NA) as the objective of the miniaturized nonlinear microscopy system. The structure of the system is very simple, and only five mirrors or lenses are used. The mini aspheric lens is integrated with a tube lens pair for beam size magnification. A MEMS mirror acts as a scanner, and a dichroic beam splitter separates the excitation light and the epi-collected signal. We investigate its performance for 2PF (two-photon fluorescence), SHG (second harmonic generation), and THG (third harmonic generation) microscopies. Live GFP (green fluorescence protein) zebrafish is used to estimate the feasibility of in-vivo experiment and the ability of dynamic observation. Comparison among different systems from other groups is also listed.