發展三維折射率顯微術應用於細胞定量觀察與分析

Abstract

細胞之型態與細胞內的結構於可見光範圍下幾近透明，為了增加視野下對於細胞結構之分辨力，通常會採用螢光物質進行標定，但是利用螢光標定技術應用於細胞內，可能會影響細胞內生理特性。本論文的內容主要是發展三維折射率顯微術，利用演算法重建細胞三維折射率的分佈，即為重建細胞三維的結構，並應用於細胞內生物分子定性與定量的觀察。 首先，我們建構的三維折射率顯微術系統是以馬赫－曾特爾干涉為基礎建立之架構，光源採用波長為405 nm之雷射，並以分光鏡進行樣本及參考光源之分光，而再將兩光束結合，於影像偵測器處形成干涉影像。參考光源之光路上放置壓電相位移動器，用以調整與樣本光源之間的相位偏移量，並使用遞迴式相位演算法計算二維相位影像。此外，樣本光源之光路上放置一組光學掃描振鏡，用來調變光照射於樣本上之角度，藉由不同入射光之角度取得樣本之投影干涉影像，而進一步得到不同入射角度時，樣本的二維相位資訊，再藉由三維重建演算法得到樣本的三維折射率分布的資訊。利用所建立的三維折射率顯微術，首先用於量測癌細胞株與正常細胞株其細胞內折射率的分布，並分析兩者之間的差異性。由於在此系統中為了要達到抗震的效果，我們所使用的遞迴式相位演算法需要多張干涉影像才能得到一張相位影像，擷取相位影像所需要的時間限制了此系統在觀察生物上的應用。 因此，為了要縮短相位影像擷取的時間，但仍可以保有抵抗環境震動的能力，我們進一步開發共光路三維折射率顯微術系統。此共光路三維折射率顯微術系統架構，是將收集的光訊號利用光柵分光，而再利用透鏡將兩光束結合，並於影像偵測器處擷取單張高頻干涉影像，並使用希爾伯轉換演算法計算其二維相位影像。此外，我們將原本的405 nm雷射光源置換成532 nm，以減少對活細胞的傷害。我們成功的利用共光路三維折射率顯微系統重建出子宮頸癌細胞的三維折射率的分布。 另外，我們再加入一組473 nm之雷射光源，形成雙波長的共光路三維相位顯微術系統，因此我們可以獲得同一樣本在不同波長之下的折射率分布，對於具有光色散特性的分子而言，此系統可以用來定量此類分子的濃度變化。由於紅血球中富含血色素，我們利用血色素具有色散的特性，造成不同波長之下折射率的改變，我們藉由雙波長的共光路三維相位顯微術量測紅血球的折射率分布，進而得到紅血球細胞內的三維血色素濃度分布。

This dissertation mainly describes the construction, development and applications of three-dimensional (3D) refractive index (RI) microscopy. Quantification of 3D RI with sub-cellular resolution was first achieved by a tomographic phase microscope (TPM) based on a Mach-Zehnder configuration. A simple piezoelectric actuator was used to generate phase shifts between sample and reference beams of the proposed TPM system. Two-dimensional phase images under a set of illumination angles were recorded for reconstruction of 3D RI tomograms based on optical diffraction tomography (ODT). 3D RI tomograms of various cell lines were quantified for demonstrating the feasibility of the TPM system on biological applications. However, the proposed TPM system based on phase shifting interferometry required multi-shot interferograms to obtain one phase image, resulting in limited spread and extension of bio-applications. For speeding up acquisition efficiency of one RI tomogram and enhancing resistance of vibration, another common-path tomographic phase microscopy (cTPM) was developed as a novel technique for measuring 3D RI distribution. A diffraction grating was utilized to generate a reference beam that traversed a blank region of the sample in a common-path off-axis interferometry setup. In the cTPM system, 3D RI tomogram was reconstructed from single-shot phase images at multiple illumination angles implemented with ODT algorithm. The cTPM system inherently displayed high ability in resisting environmental vibration, leading to stable extraction of more accurate phase information than the TPM based on a Mach-Zehnder configuration. Besides, the cTPM system reduced the acquisition time of one RI tomogram due to the feature of single-shot phase imaging. The cTPM system was practically performed on mapping 3D RI distribution of HeLa cells. In order to show extensive potentiality in bio-applications, the cTPM system was equipped with two light sources, a 473 nm diode laser and a 532 nm diode-pumped solid-state laser. The two-wavelength cTPM system became a powerful tool for stain-free visualization of 3D hemoglobin concentrations in single red blood cells (RBCs). In addition, cellular volume and morphology of individual RBCs could be also obtained in the two-wavelength cTPM system. This technique showed promising capabilities of charactering RBCs in blood specimens from patients with various blood deficiencies.