白血球在明視野顯微影像下流動型態變異之探討

Abstract

血液檢測為健康檢查中重要參考指標之一，血液中各個角色的數量與比例都可以作為臨床上評估狀況的參考，如紅血球數量可以判斷是否貧血、白血球多寡可以判斷癌症、HIV 等等。傳統血液抹片檢查為相當常見的檢查項目，但相當耗時耗力，而為了達到自動高速計數的目標，科學家們開始發展了血球計數器、血液分析儀，目前最為精確的儀器為流式細胞分選儀，與傳統的血液抹片檢查相比，流式細胞分選儀可以達到相當高速的血球計數且分選特定系細胞目的。 流式細胞分選儀中有四大系統: 液相系統、光學系統、電子系統、分選系統。利用前散射光(Forward Scattering)、側散射光(Side Scattering)、螢光(Fluorescence)作為限制條件以分選出不同種類細胞。然而，流式細胞分選儀常為達到更高的分選精確性，需要添加染劑，但這不僅需要雷射維護費用、染劑費用，亦有可能對於細胞造成傷害，因此本研究室積極尋找其他細胞影像上特徵以提升分選細胞的 可能性。 本研究室先前嘗試以倍頻顯微術觀察中性粒白血球(Neutrophil)、單核球(Monocyte)、淋巴球(Lymphocyte)的三倍頻強度，從影像中可以發現中性粒細胞比淋巴球、單核球三倍頻訊號更強，且細胞內有許多小核體，淋巴球尺寸最小、細胞內常有一圓核並伴隨一圓型亮點，單核球細胞尺寸最大。藉由三倍頻強度、自體紅螢光強度、大小可以作為三維度限制條件分選此三類白血球。 然而，無論是流式細胞儀或倍頻顯微術皆需要依賴雷射光源，為了減少雷射維護與染劑使用上的費用，本研究積極發展一種嶄新方式分選白血球種類，主要利用血球的大小與機械性質的不同在明視野與不須染劑的情況下達到分選中性粒白血球(Neutrophil)、單核球(Monocyte)、淋巴球(Lymphocyte)的目的。首先，本研究對於靜置狀態下的白血球分別以雷射光源的Leica TCS SP5 II 多光子雷射共軛焦顯微鏡與明視野下的螢光顯微鏡Leica DMI 3000B 搭配CCD 做型態上變形分析。再者，將中性粒白血球(Neutrophil)、單核球(Monocyte)、淋巴球(Lymphocyte)分別以微注射幫浦加壓入微流道中，觀察細胞在不同流力場中所受正向應力、剪應力時所產生的形變。研究結果中可以發現在高流速狀態下，淋巴球細胞有較大的變形，而大小也有顯著的差異，意味著明視野顯微影像的確適合應用在高流速環境下的血球觀察，在未來，本研究室希望以雷射層照顯微術以高速成像方式觀察更高流速的細胞影像，以影像上的資訊達到無須染色方式增加流式細胞分選儀的分辨率。從長遠來看，本研究對於血球細胞於流體動力學上的價值除了可以應用離體血液檢查儀器上提高血液分析品質外，在未來亦可以做為非侵入式血液檢查發展中血液流體動力學研究上的參考指標。

Blood test is one of the most important indicators of physical examination. The amount and proportion of every role in blood can be direction of medical assessment. For instant, the amount of red blood cell could be the sign of Anemia. Also, the variety of amount of white blood cell could be a guide of HIV, cancer, and other disease. In more detail, traditionally, biopsy is a relatively common inspection, but it wastes many time and artificially efforts. Thus, in order to achieve automatically cell counting goal, in the past decades, scientists have beginning to develop CBC (Complete Blood Count), and Blood analysis technology. In the moment, the most accurate and precise machine is Flow cytometer (FCM). Compare to traditional biopsy inspection, FCM can not only count blood cell with higher speed but also can sort specific cell and particles. FCM was divided into four parts: microfluidic system, optical system, electric system, and sorting system. The intensity of forward scattering, side scattering, and fluorescence serve as the index to distinguish different kinds of cells. Nevertheless, in order to achieve higher accuracy, it frequently is required to add particular biomarker, which probably damage intact cell, moreover, increase the cost for laser maintenance. Therefore, currently our Lab devote to develop cell sorting with higher quality depended on the characteristics of cell image in many ways. Previously, our Lab differentiates Neutrophils, Monocytes, Lymphocytes by the intensity of Third-Harmonic Generation. From the cell image, we can find the intensity of Third-Harmonic Generation of Neutrophils is higher than Monocytes and Lymphocytes. Besides, Neutrophils included many small nucleuses. With the intensity of Third-Harmonic Generation, auto-fluorescence and size and size, we can divide these three kinds of leukocytes into three groups. However, both Flow Cytometry and Harmonic Generation technique need to depend on laser source, so this thesis devote to develop an innovative way to distinguish different kinds of leukocytes without the maintenance of laser source and the waste of biomarker. To achieve our goal, the main approach in this thesis is that by size of leukocytes and morphodynamic properties of leukocytes under bright-field to sort different kinds of leukocytes without biomarker. First, we observe the morphology of stationary leukocytes on cover glass by Leica TCS SP5 II confocal microscope with laser source, resonant scanner and Leica DMI 3000B Fluorescence microscope with CCD under bright-field. Secondly, we pμmp Neutrophils, Monocytes and Lymphocytes into microfluidic channel and observe the deformation of cells suffer from normal stress and shear stress under different flow field. From the result, we can find the lymphocytes deform significantly under higher speed. Moreover, the difference of size of kinds of leukocytes is significantly obvious that means bright-field image is able to be applied to observe blood cells in high speed. In near future, our lab plan to use laser light sheet microscopy to observe cell image with higher speed, which can increase the sorting precision of Flow cytometry without biomarker. In the long run, the value of this thesis not only helps increase the quality and accuracy of blood sorting machine in vitro, but also can be served as haemodynamic reference for in vivo developing non-invasive blood inspection.