近來已有許多研究者開發出各種顯微造影系統，能觀察小動物的腦皮層血管，此外，這些系統也著重於從結構性影像與功能性影像的研究。而本研究中所著眼的目標在於驗證是否有機會使用非侵入性檢測的方法應用於以小鼠為模式生物的穿顱影像。以雙光子顯微術為例，這套系統具備良好的解析度，足以觀察小屬腦皮層中的微細血管，然而在使用特定的波長下其造影深度相當有限，無法充足因應小鼠腦皮層一定造影深度的需求、而更關鍵的問題是我們必須對活體施打對比劑，屬於侵入式檢測方法；又如雷射散斑對比造影系統，它通常被應用於檢測血流速度，但此種造影系統無法獲得深度上的資訊。考量了各種實驗條件，我們選用光學解析度的光聲顯微術作為研究穿顱小鼠腦的技術，目前我們已經開發出雷射掃描式光學解析度光聲顯微術可被應用於觀察小鼠耳朵血管的結構性資訊，我們進一步想了解是否能觀察腦皮層中的血管，但並非每一種光學解析度的光聲顯微系統都具備穿顱造影的功能，因此在本篇研究中我們想驗證是否雷射掃描式光學解析度光聲顯微術的系統架構下有機會做到穿顱式造影。在如此的客製化系統下，使用以532奈米之特定波長脈衝式雷射可在無需鑽開小鼠頭骨的前提下達到有效的穿顱造影；當系統獲得小鼠腦皮層微細血管的三維度資訊，我們也額外設計電刺激的實驗並提供這套系統功能性資訊的分析方法。本篇研究確認在雷射掃描式光學解析度光聲顯微術的系統下，基於虛擬點偵測的概念，可以提高訊躁比、並且擁有較高的解析度與造影速度的狀態下進行小鼠腦之穿顱式造影。

Recently, a variety of microscopy has not only been proposed to observe cerebral microvasculature for small animals, but to develop structural and functional imaging. Most of all, it is very important to develop a microscopic system to achieve transcranial imaging of microvasculature in a model organism for the demand of non-invasive diagnosis. For some microscopic systems, like two-photon microscopy, it could be applied to imaging of microvasculature in the mouse cerebral cortex, while it has limitations of penetration depth and using contrast agents in an invasive way. Laser speckle contrast imaging is usually used to measure the blood flow but we could not get the information of depth in the cerebral cortex for small animals. We consider that optical-resolution photoacoustic microscopy (OR-PAM) would provide high lateral resolution and is crucial to achieve the imaging of cerebral microvasculature in a model organism of mice. In the present, we have developed the laser-scanning optical-resolution microscopy (LSOR-PAM) system to image microvessels in mouse ear with high signal-to-noise ratio, high resolution and high imaging speed but compromising to field of view. We then wonder about the fact that whether we can accomplish the goal of imaging of cerebral microvessels without drilling and opening the mouse skull using the customized OR-PAM system. We verify that we can achieve transcranial imaging of microvasculature if we use the pulsed laser with a specific wavelength of 532 nm which is suitable for penetrating the mouse skull while retaining high imaging speed under the LSOR-PAM system based on the concept of virtual-point detection. Furthermore, we design an experiment that introduced electrical stimulation to a mouse with one pair of electrodes inserting under the skin of its forelimb, and provide the methods of analyzing the functional information of cerebral microvessels when we get the three dimensional data after scanning with LSOR-PAM.

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