多模態雷射掃瞄生物顯微術

Abstract

在這篇論文中，我們建立了一套多模態雷射掃瞄顯微系統，並使用反射式共焦顯微術、二倍頻、三倍頻及雙光子螢光顯微術來作為影像模態。共焦的技術和非線性光學的效應都可以達到顯微鏡應用所需的光學切片特性。在結合一個工作在於生物穿透較深波段的近紅外光飛秒雷射光源和一個高數值孔徑的物鏡後，這個多模態雷射掃瞄顯微術可以提供超過１公厘的穿透深度及次微米的空間解析度並在生物樣本中有著較少的光破壞及光毒性現象。利用這個多模態雷射掃瞄顯微術我們可以在活體生物取得充足的影像資訊來幫助在生物學上的研究並且和鈦藍寶石雷射為主的技術比起來可以擁有較低程度的侵入性破壞。 這樣的顯微系統可以「放大」我們在組織學和發展生物學上的視野，這些我們在後面的章節會展示。作為一個充分發展的皮膚光學切片工具，正常皮膚的反射式共焦影像和傳統組織學的圖像是非常吻合的。將反射式共焦模態和倍頻的模態結合在一起，這個顯微鏡工具可以提供比傳統的共焦雷射掃瞄顯微術更豐富的的影像資訊並有著較好的空間解析度及多模態影像，這些功能對皮膚學上的研究是非常有幫助的。 利用反射式共焦顯微術和三倍頻顯微術所取得的人皮膚影像亦提供了非常有用的證據來證明當細胞的小胞器在細胞中的體積比例增加時，光在細胞中的散射機制會是由萊利散射所主導。這說明了反射共焦信號和三倍頻信號所提供的細胞影像亮區是包含了許多小胞器如粒腺體之類的細胞質影像。我們也發現了在皮膚深的信號強度衰減是由於焦平面的擴大所導致的，這也是為什麼在皮膚深層每個影像模態的解析度都會明顯變差的原因。 在利用基因轉殖技術的協助下，使用螢光蛋白標定特定組織這個方法在發展生物學上已經成為一個強而有力的工具，因為螢光蛋白質在空間上的表現可以顯現出帶有這個基因的表現型態。這樣的基因轉殖的螢光蛋白質再搭配雙光子螢光顯微術，我們可以藉其非線性光學的效應得到擁有高三維解析度的影像。有著基因轉殖技術的輔助及利用在於生物穿透較深波段的近紅外光飛秒雷射光源，我們結合了雙光子螢光顯微術和倍頻光學顯微術來取得生物組織中的分子影像、型態學及有結構的蛋白質資訊。包含了光學切片的特性、高穿透深度、及較少量的光破壞，這個多模態的方法可以提供極佳的顯影能力，對未來要動態的研究脊椎動物胚胎的發育會是一個非常有用的工具。 多模態雷射掃瞄顯微術所提供的反射式共焦信號和非線性光學信號，讓我們可以在活體生物中利用反射共焦信號和三倍頻信號取得型態學上的資訊、利用二倍頻信號取得有結構的蛋白質資訊並利用雙光子螢光信號取得分子影像的資訊。這樣的顯微鏡系統將可以解決未來特定生物學上的問題，並在臨床醫學上能夠有非常有用的幫助。

In this thesis, a multi-modality laser scanning microscopy system is build based on a modified optical scanning microscope and a Cr:forsterite laser. Reflection confocal, second harmonic generation (SHG), third harmonic generation (THG), and 2-photon fluorescence (2PF) have been used as imaging modalities. Both confocal techniques and nonlinear natures can achieve the optical sectioning property desirable for microscopy applications. Combined with the near-infrared femtosecond source working in the biological penetration window and a high numerical aperture (NA) objective, multi-modality laser scanning microscopy can offer >1-mm penetration depth and sub-micron spatial resolution in biological samples with much-reduced photodamage and phototoxicity. Multi-modality laser scanning microscopy can thus provide sufficient information deep inside a live biological specimen for biological studies less invasively than Ti:sapphire based techniques. Such a microscopy system would “enlarge” the field of view on histology and developmental biology researches, as presented in the following chapter. As a well-developed optical biopsy tool, reflection confocal images of normal skin correlate very well with images from conventional histology. Combining reflection confocal imaging modality with SHG and THG imaging modalities, this microscopic tool can provide much more information than traditional confocal laser scanning microscopy with better spatial resolution and multiple imaging modalities that are very useful for dermatology studies. The images in human skin taken from reflection confocal and THG microscopy provided very helpful evidences that the light scattering from cells is dominated by Rayleigh scattering when the volume fraction of organelles increases, which confirms the bright reflection confocal and THG signals from cytoplasm that containing multiple organelles such as mitochondria. The signal intensity decay in deeper parts of human skin was found to be mainly caused by the focal plane broadening which was also the reason of the resolution degradation of each imaging modality. With the aid of transgenic techniques, specific tissues tagged by fluorescent protein have become a powerful tool in developmental biology studies because the spatial expression of that fluorescent protein enables to encapsulate the expression pattern of endogenous genes. Based on a 2PF microscope, images high 3-dimensional (3D) resolution can be obtained due to its nonlinear nature. We have combined 2PF microscopy with higher-harmonic optical microscopy based on the femtosceond Cr:forsterite laser with the aid of new transgenic lines tagged with HC-red fluorescent protein to obtain molecular, morphological, and structural protein information in biological tissues. With its optical sectioning property, high penetration depth, and much-reduced photodamages, this multi-modal method provides superb imaging capability for dynamic developmental studies of vertebrate embryos in the future.