利用三維折射率顯微術與逆散射光譜區分白血球種類

Abstract

白血球在人的身體中扮演著極為重要的角色，包含作為免疫系統以對抗病原體，並有良好的防禦機制。不同的白血球也有不同的型態，去對抗不同的病原體與疾病。因此如何區分不同型態的白血球，以及不同白血球型態具有那些可比較的參數，便是一個很重要的課題。 目前臨床上定量人體內各種類白血球的主要方法是使用全血計測器(Complete Blood Count, CBC)，必須抽血且需要大型儀器。因此本研究以三維折射率顯微鏡觀察白血球的型態與折射率分佈，希望利用逆散射光譜的強度與週期性震盪來區分不同種的白血球，評估未來將此技術開發達成活體血球計數之潛力。目前先試著分辨三種主要的白血球：淋巴球Lymphocyte，嗜中性白血球Neutrophil，單核球Monocyte。三種主要白血球的從三維折射率分佈，代入FDTD數值模擬程式分析血球逆散射光譜，評估從光譜區分不同種類白血球計算得到的逆散射光譜的可行性，初步結果顯示淋巴球的震盪週期與其他兩種白血球明顯不同，且和Mie theory比對可以估計淋巴球的尺寸，而嗜中性白血球由於折射率明顯較高，其逆散射強度約為其他兩種白血球的5~10倍，因此我們預測可以利用逆散射光譜將三種白血球做區分。 在此理論基礎下，建造一個可量測細胞逆散射的光學系統架構，直接量測三種白血球的逆散射，在不同波長下的震盪頻率以及其逆散射強度，以期獲得更多可以區分白血球的實際資訊。

White blood cells (WBCs) play main roles in defending the host against harmful pathogens and disease conditions including autoimmune diseases , neurodegenerative diseases and cancer. Various subtypes of WBCs have been characterized to play different roles in the pathophysiology of diseases. Therefore, accounting the number of WBC in specific subtypes is important. We have developed common-path tomographic diffractive microscopy (cTDM) to acquire three-dimensional (3D) refractive index (RI) mappings of WBC without staining, thus getting morphological information of the cells when they are close to their natural state. Three main subtypes of WBC were analyzed, including monocyte, neutrophil and lymphocyte. We also analyzed the total phase integrated over the projected area of individual WBC, which is proportional to the dry mass. The scattering properties of WBC were obtained from 3D RI tomograms using the finite-difference time-domain (FDTD) method to demonstrate the feasibility of classifying the three subtypes of WBC based on their backscattering spectra. The FDTD simulation results indicated that backscattering characteristics of lymphocytes, monocytes and neutrophils are different from each other. Go a step further, we set a optical system to measure backscattering spectrum from WBCs directly. The results also indicated the possibility of classifying the three types of WBC based on backscattering measurements by spectral intensity and oscillation frequency because neutrophils has granular distribution in nucleus, monocytes has larger volume than the other two.