以三倍頻顯微術發展無標定式白血球影像細胞儀技術

在臨床醫學上，血液檢驗為所有疾病檢驗的第一步，其包含了化學分子檢驗與血球數量、組成比例及數量的檢驗。其中，後者的檢驗是最為直覺反應身體健康狀況的重要指標。人體內血液細胞主要包含了紅血球、白血球及血小板，對一健康人而言，三者各別的數量及比例會維持在一個範圍而不易隨著時間有太大的變動，因此了解其數量與組成的變化將有助於給予醫師一個診斷的方向。然而，這些檢驗都需要抽血後再以流式細胞儀等儀器分析，此種方式不僅會造成患者的心理負擔(如：重複抽血)，檢體在離體情況下更有機會因變質影響診斷的結果。因此，一套即時性活體內檢測工具的發展是相當重要的。　　非線性光學顯微術，因為具有次微米的三維空間解析度，已被廣泛應用在生物方面的研究。其中，倍頻技術因其能量守恆特性，不易對生物組織造成傷害，加上不需外加染劑的特點，使此光學技術更適合作為活體內檢驗的工具。本篇論文中，我們發展出一套即時性且不須染色的活體內影像流式細胞儀技術，並成功分離與辨別活體內不同型態的白血球，即是顆粒球、單核球與淋巴球。本研究使用自製的飛秒鉻貴橄欖石雷射，其工作波長位於第二個生物穿透窗口，提供較高的組織穿透性與較低的光傷害。搭配高速影像擷取卡與16 kHz共振掃瞄鏡模組，此系統提供每秒30張512 x 512像素大小的影像，得以觀察流動的血球型態。　　在這套系統下，顆粒球具有較強的三倍頻訊號，且細胞內部具有較暗的多核型態結構；單核球與淋巴球的訊號則相對較弱且強度相似，但結構上具有一接近細胞大小的細胞核，因此易與顆粒球區別。單核球與淋巴球則可由細胞大小做區別。利用三倍頻訊號強度與細胞大小配合k-means分群演算法，三種白血球可以有效的在二維圖上區別。利用此方式統計出的個別細胞數量、比例及總細胞數量結果皆與醫院常規使用的流式細胞儀結果一致，顯示出這套三倍頻活體影像流式細胞儀技術應用在活體內檢測的可行性。

關鍵字

非線性光學；倍頻顯微術；流式細胞儀；鉻貴橄欖石雷射；白血球

並列摘要

Blood tests, which analyze the composition of species in blood, are usually the first stage of clinical examination in the hospital. This includes biochemical and complete blood count analysis. The indices of the latter provide information about the health status of a person. The blood cells include erythrocytes (red blood cells, RBCs), leukocytes (white blood cells, WBCs), and thrombocytes (platelets). For a healthy person, the quantities and percentage of these cells will remain in a steady state; thus, studying the variation of cells gives doctors an aspect of diagnosis. However, these processes require blood to be drawn for further analysis (e.g. flow cytometry, histology), which not only takes time and causes pain through an invasive method, but the in vitro samples are affected by the environment. Therefore, an instrument with real-time and non-invasive analysis is highly desired for blood-cell counting. In current studies, nonlinear optical microscopy, such as harmonic generation microscopy (HGM) and two-photon microscopy (2PFM), has been widely used in biological and material studies due to its sub-micron three-dimensional (3D) spatial resolution. Obeying the energy conservation of harmonic generation, there is no energy deposition in tissue, making it more suitable for long-term observation. Additionally, the greatest advantage of HGM is that it needs no extra labeling for samples, making it more suitable for studying living biology, especially in clinics. In this thesis, we developed a real-time and label-free in vivo third-harmonic generation (THG) flow cytometer, and we further distinguished and differentiated different types of WBCs (neutrophils, monocytes, and lymphocytes). The laser source in this study was a homemade femtosecond Cr: forsterite (Cr: F) with a Kerr-lens mode-locking technique. As the wavelength falls within the second optical penetration window, this laser penetrates deeper without out-of- and on-focus photodamage. With the help of high-speed imaging acquisition software and a set of 16-kHz resonant galvanometer mirrors, the rolling cells within vessels could be revealed individually with 30 frames per second (fps) and 512 x 512 pixels in the image output. The results showed that neutrophil shows a stronger THG signal than monocyte and lymphocyte, with a dark multi-lobed nucleus within the cell. In contrast, monocyte and lymphocyte have identical THG intensity, but less than neutrophil, with a single nucleus roughly equal to the cell’s size. Taking the size of each cell into account, WBCs could be differentiated into three groups by a k-means clustering algorithm in a THG intensity-size plot. The cell count and percentage of each type of leukocyte agreed with the regular complete blood count readout, showing that the in vivo THG imaging flow cytometer will be applicable to clinical diagnosis in the future.

並列關鍵字

Nonlinear optics ； harmonic generation microscopy ； flow cytometry ； Cr:forsterite laser ； leukocytes

參考文獻

[1] A. Moldavan., “Photo-electric technique for the counting of microscopical cells,” Sci., Vol. 80, No. 2069, pp. 188-9, 1934

[2] L. A. Kamentsky, M. R. Melamed, H. Derman., “Spectrophotometer: New Instrument for Ultrarapid Cell Analysis,” Sci., Vol. 150, No. 3696, pp. 630-631, 1965

[3] Mack J Fulwyler, Particle separator, U.S. Patent, 3380584A, 1965

[4] S. Yazdanfar, M. D. Kulkarni, and J. A. Izatt., “High resolution imaging of in vivo cardiac dynamics using color Doppler optical coherence tomography,” Opt. Express, Vol. 1, No. 13, pp. 424-431, 1997

[5] Z. Chen, T. E. Milner, S. Srinivas, X. Wang, A. Malekafzali, M. J. van Gemert, J. S. Nelson., “Noninvasive imaging of in vivo blood flow velocity using optical Doppler tomography,” Opt. Lett., Vol. 22, No. 14, pp. 1119-21, 1997

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以三倍頻顯微術發展無標定式白血球影像細胞儀技術

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