三維折射率顯微鏡應用於定量式分析癌細胞之 細胞週期與白血球之分類

Abstract

癌症為台灣十大死因之首，並且大多來自源上皮細胞。癌化過程伴隨細胞結構的改變，影響其光學特性。許多研究透過光學檢測方式，例如散射等光學參數的測量，作為癌症檢測的工具。其中一個重要的光學參數即為折射率。折射率可視為細胞內生對比劑，能夠在不外加染色情況下分析細胞內部細微結構。透過三維折射率顯微鏡測量，能得到物體的折射率結構進行分析。而且，折射率分布在經過有限差分法，進而模擬細胞的散射特性。先前研究已提出，癌細胞的折射率較正常細胞來的高，而且癌病變組織的散射係數高於正常組織。本篇研究主要分析同一顆細胞隨時間生長下的折射率變化，在不同時期間對於散射特性的影響。藉由自製恆溫系統及細胞晶片，讓細胞在顯微鏡系統中存活一段時間。觀察比較正常細胞和癌細胞在細胞週期間生長，對於同一顆細胞，其隨時間生長下折射率的差異。結果發現，癌細胞平均折射率會有比較明顯提高的情形;然而，在正常細胞樣本中，折射率上升相較於緩慢。希望藉由癌細胞和正常細胞的差異，在不外加其他物質下，更加了解癌細胞在生長期間的生理現象，助於早期癌症的檢測上。 除了細胞週期研究外，散射特性能應用在其他細胞的分析上。本篇研究針對人體血液中的白血球進行分析，希望透過散射特性，進而區分三種白血球-單核球、嗜中性球和淋巴球。白血球實驗結果初步發現，嗜中性球折射率和乾重都較高，造成逆散射值偏高。單核球體積較大，但折射率偏低。淋巴球體積最小，折射率居中。因此，顯示出透過逆散射光譜的震盪能夠藉由體積區分單核球和淋巴球，藉由散射值的高低，能夠區隔出嗜中性球。

Cancer is the top cause of death is in Taiwan, and almost derived from epithelial tissue. Optical properties are affected during the carcinogenesis due to cell structure distinct change including nucleus enlargement and concentration. These parameters could be the strong tool to distinguish cancer and normal tissue/cells. An important parameter - refractive index, endogenous contrast source in the cell, has been successfully studied microstructure of cell without any enteral staining recently. Further, FDTD simulation provides light scattering information at different angle and wavelength of cell by refractive index distribution. Former study showed that refractive index of cancer cell is higher than normal cell, and the scattering coefficient of carcinoma is higher the normal tissue too. Here, we want to study that the difference of refractive index of single cell within cell cycle between cancer cells and normal cells. Results suggest that refractive index increases prominently for cancer cell while growing slowly for normal cell. Our finding shows that investigating refractive index of cell growing naturally without any external staining materials could give us more information of normal physiological phenomena in cancer cell. We also investigate white blood cells in human blood in order to distinguish three types of cell, monocytes, neutrophils and lymphocytes, by back-scattering spectrum analysis. Result indicates that neutrophils have the highest refractive index and dry mass. Monocytes are the largest cell with lowest refractive index while lymphocytes are smallest cell with higher refractive index than monocyte. Based on volume and diameter, we could distinguish monocyte and lymphocyte by oscillation of back-scattering spectrum. Using scattering value, neutrophils could be distinguished from the blood sample .Our finding suggest that back-scattering spectrum may help us distinguish white blood cells in vivo.