小透明生化材料可視化成像研究與其在顯微術上的應用

Abstract

本研究中希望將高透光率的透明生化材料加以可視化的成像，以利觀察。所欲進行的成像觀察，可 分為強度型及相位型等兩部份。在強度型成像觀察中，使用單色光或多色光，其入射角由0牽逐漸加大至 布魯斯特角、臨界角、表面電漿效應的共振角及grazing incident angle，探討反射光或透過光強度對入射 角及折射率(折射率與波長有關)變化最大的條件，在該條件之下，待測材料將可形成對比度佳的影像。 在相位型成像觀察中，使用線性偏光、線性外差偏光及旋光外差偏光，入射至待測材料，並適當引入偏 光元件，作為移相器或提高幹涉信號對比度用，探討在各種入射角之下，反射光的單一偏光份量的相位 移或正交偏光份量間的相位差為最大的條件，引進相移干涉術及外差干涉術測出待測材料二度空間的相 位移分佈或相位差分佈；接著代入由Fresnel』s equations 所導出的式子中，即可求得待測材料的二度空 間折射率分佈；然後再使用折射率色度圖即可對待測材料成像。不管強度型相位型成像系統中，最後將 與顯微成像技術相結合，期能觀察微小生化透明材料，並試組出如布魯斯特顯微鏡、全反射顯微鏡、表 面電漿共振顯微鏡及正入射顯微鏡等各種顯微鏡。與傳統成像法比較，本研究方法具有無外加染料及螢 光材料等污染的乾淨成像的優點。

Because of the high transmittances, transparent biochemical materials are very difficult to be visibly imaged without some additional dyes or fluorescent materials. To overcome these drawbacks, we plan to develop some methods for visibly imaging the transparent biochemical materials by measuring their intensity variations or phase variations. In the former category, the monochromatic light or multiple-wavelength light is incident on the tested material at different angles from 0 degree to almost 90 degree. The relation curves of intensity versus the incident angle and the refractive index, which depends on the wavelength, will be derived and depicted. The maximum slopes will be found, especially at the Brewster』s angle, the critical angle, and the resonant angle of the surface plasma resonance (SPR). Under the optimal conditions, the tested transparent material can be visibly imaged with better contrast. In the latter category, we will use any of linearly polarized light, heterodyne light, or circularly polarized heterodyne light as the incident light, and some polarization components as phase-shifters or to enhance the contrast of the interference signals.We will investigate under what conditions that the phase-shift of one single polarization component or the phase difference between two orthogonal polarization components will have extreme variations. Under these conditions, we will apply the phase-shifting interferometry or the heterodyne interferometry to measure two-dimensional phase-shifts or phase differences. Substituting these measured data into the special equations derived from Fresnel』s equations, the two-dimensional distribution of refractive index of the tested material will be obtained. Then using the relation diagram between the refractive index and chromaticity, the measured two-dimensional data can be transformed into the visible image. Finally, the microscopic techniques will introduce into the above-developed optical configurations to visibly image the tiny biochemical materials. In addition, we will try to design and assemble some alternative and novel microscopes for practice to observe the tiny biochemical material, such as Brewster』s microscopes, total internal reflection microscopes, SPR microscopes, and normal incidence microscopes, etc. Comparing traditional methods, our methods have no deteriorations from additional dyes and fluorescent materials.