以高速三倍頻顯微術監測人體微血管內的血球

Abstract

血液常規檢查是醫院中常見的一項檢驗方式，其在臨床的疾病診斷上不僅扮演相當重要的角色且檢驗結果也常成為評估個人健康與否的重要指標。常見的檢驗項目包含：紅血球計數、白血球計數、血小板計數、血紅素含量及血球容積比等等。雖然血液常規檢查是一項臨床常用的檢查方法，但由於此方式需向病人進行抽血，其侵入性的方式常使得病人感到疼痛且亦造成病人需浪費相當長的時間等待檢驗結果。因此發展一套非侵入性且即時量測血球資訊的檢驗方式是極其重要的。如此一來，不但可減少醫療資源的浪費更可以縮短病人等待結果的時間。 現今，非線性光學顯微術如雙光子螢光顯微術與倍頻顯微術，因具有次微米三維空間解析度的能力，因此已被廣泛地運用於生物體內研究。由於倍頻顯微術本身能量守恆的特性，相較於常用的雙光子螢光顯微術，此方法擁有最小傷害性的優勢，因此更適用於臨床的應用。對於倍頻微術來說，另一項最大的優點是此技術不需要額外的對比劑幫助，就可得到生物組織的形態資訊。 在本篇論文中，我們發展一套即時的三倍頻影像系統並應用於監測人體皮膚微血管內的血球。在本研究中，激發光源採用自建的飛秒鉻貴橄欖石雷射，其輸出波長位於生物組織的穿透窗口，不僅可提供較深的穿透深度，更可減少焦點上的光傷害。在另一方面，為了觀察快速的血球運動，高速的掃瞄能力也是不可缺少的。藉由結合 16 kHz的共振掃瞄鏡，掃瞄速率可高達每秒30張影像。另外，再利用高速的影像擷取卡，將所收集的三倍頻訊號同步取樣並重組出一張512像素大小的影像。 利用此自建的高速掃瞄能力的三倍頻影像系統，對於在皮膚微血管中的血球運動可輕易地觀察到，並且進一步地應用至區分不同種類的血球，尤其是形狀較圓且無堆疊的白血球。如此一來，此技術可用來估計人體內白血球的數目，進而評估病人的白血球量是否正常。在未來的臨床實際應用上，也可用來快速地評估白血病的病人其在化學治療(化療)下白血球數量變化的情形。

Complete Blood Count (CBC) is a routine examination running in the hospital. In clinical practices, CBC plays an important role in the diagnosis of diseases. The results of inspection will be a significant indicator of evaluating individual health status. The items of checking include: erythrocytes (red blood cells, RBCs) count, leukocytes (white blood cells, WBCs) count, thrombocytes (platelets) count, Hemoglobin, Hematocrit and so on. Although CBC is a common examination in laboratory medicines, it requires a draw of blood, which is an invasive method to patients. And it cost patients’ time to wait for the results. Hence, it is highly desired to have an on-site measurement method for blood cell counts without invasive draws of blood. That can save the medical resources and the time of patients. Nowadays, laser scanning nonlinear optical microscopy, such as two-photon fluorescence microscopy (2PFM) and harmonic generation microscopy (HGM), has been widely used for in vivo biological studies with sub-micron three-dimensional (3D) spatial resolution. Owing to the nature of energy conservation of harmonic generation (HG), there is least energy deposition to tissues under observation. Compared with 2PFM, HGM is the least-invasive optical microscopic technique suitable for clinical applications. Moreover, the extra advantage of HGM is that the morphological information of tissues can be revealed without a need of extra labeling. In this thesis, we developed the video-rate third-harmonic-generation (THG) microscopy for blood cell cytometry in human skin. The excitation laser source in our research was a home-built femtosecond Cr:forsterite laser with Kerr Lens Mode-locking. With its output wavelength covering the penetration windows of most biological tissues, the Cr:forsterite laser is an indispensable excitation laser source providing deeper penetration depth and reduced on-focus photodamage. To catch up the fast motion of blood cells, the high-speed scan rate is a critical issue. Combining a 16 kHz resonant mirror and a galvanometer mirror, the 2D scan rate could achieve 30 frames per second (FPS). Taking advantage of the high-speed frame grabber, the THG signal was synchronously sampled and reconstructed to an image with 512-by-512 pixels size. With the aid of high-speed scan rate and HGM, the blood cells in human blood capillary could be clearly observed, especially for round and unstacked leukocytes. We could count the amount of white blood cells and evaluated the corresponding flux of blood volume. Thus evaluated number density agreed with the physiological range of white blood cell count. For clinical use, in the future, the technique is potential to be used to diagnose leucopenia for the patients under chemotherapy.