雙頻率偏極化共焦雷射掃描顯微鏡的成像理論建立及其降低樣本引入球面像差能力之評估

The Study of Image Formation Theory in Two-Frequency Polarized Confocal Laser Scanning Microscope and Its Ability on Reduction of Specimen-Induced Spherical Aberrations

吳政雄(Jheng-Syong Wu)

指導教授 ： 周晟 ； 李正中

國立中央大學/理學院/光電科學研究所/博士(2012年)

摘要

在傳統共焦顯微鏡中，樣本與環境間折射率的不匹配或樣本內的折射率變化皆會引入球面像差，而此球面像差會破壞共焦顯微鏡斷層影像的品質，尤其是對於生物樣本。本研究論文提出極化光子對共焦雷射掃描顯微鏡的成像理論，並且以實驗驗證此降低樣本引入球面像差的能力。極化光子對共焦雷射掃描顯微鏡使用日曼雷射為光源，其輸出一道線性極化光子對光束。由於線性極化光子對共路徑傳播與光學外差偵測的特性，極化光子對共焦雷射掃描顯微鏡具有降低樣本引入球面像差與提升軸向解析度的能力。對一個具有散射特性的樣本進行成像時，極化光子對共焦雷射掃描顯微鏡基於具有空間同調篩選、極化篩選及空間過濾篩選，亦能降低樣本的散射效應。極化光子對共焦雷射掃描顯微鏡的實驗已驗證其同時降低樣本引入球面像差與散射效應的能力。此外，實驗上我們比較與討論正交極化與平行極化光子對共焦雷射掃描顯微鏡的軸向反應曲線，對於引入弱球面像差情況時，平行極化光子對共焦雷射掃描顯微鏡具有較佳的軸向反應曲線；然而，對於引入強球面像差情況時，正交極化光子對共焦雷射掃描顯微鏡反而具有較佳的軸向反應曲線。因此，對於生物樣本，藉由選擇適合的極化態極化光子對共焦雷射掃描顯微鏡可以有較佳的軸向反應曲線。

關鍵字

偏極化 ； 光學同調 ； 雙頻率雷射 ； 光學外差干涉 ； 球面像差 ； 共焦顯微鏡

並列摘要

The spherical aberration induced by refractive-index mismatch results in the degradation on the quality of sectioning images in conventional confocal laser scanning microscope (CLSM), especially for a biological specimen. In this research, we have derived the theory of image formation in a two-frequency polarized confocal laser scanning microscope (TFCLSM) and conducted experiments to verify the ability of reducing spherical aberration in TFCLSM. A Zeeman laser is used as the light source and produces the linearly polarized two-frequency laser beam. With the features of common-path propagation of LPPP and optical heterodyne detection, TFCLSM shows the ability of reducing the specimen-induced spherical aberration and improving the axial resolution (13%~23%) simultaneously. TFCLSM also reduce the scattering effect when imaging into a scattering specimen, based on the spatial coherence gating, polarization gating and spatial filtering gating. In experiments, the ability to reduce the specimen-induced spherical aberration and scattering effect simultaneously in TFCLSM was verified. In addition, we experimentally compare and discuss the axial responses of the orthogonal linearly polarized two-frequency confocal laser scanning microscope (O-TFCLSM) and the parallel linearly polarized two-frequency confocal laser scanning microscope (P-TFCLSM). The axial response of the P-TFCLSM showed better performance than that of the O-TFCLSM under weak spherical aberration conditions. However, the opposite was true under serious spherical aberration. These results imply that a pair of proper polarizations in TFCLSM can have a better axial response for a biological specimen.

並列關鍵字

optical heterodyne ； spherical aberration ； confocal microscope ； polarization ； coherence ； two-frequency laser

參考文獻

1. M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, Singapore, 1996).

2. T. Wilson, and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic Press, London, 1984).

4. J. B. Pawley, Handbook of Biological Confocal Microscopy (Plenum Press, New York, 1995).

5. P. Calzavara-Pinton, C. Longo, M. Venturini, R. Sala, and G. Pellacani, “Reflectance confocal microscopy for in vivo skin imaging,” Photochem. Photobiol. 84, 1421-1430 (2008).

6. M. Goetz, A. Ziebart, S. Foersch, M. Vieth, M. J. Waldner, P. Delaney, P. R. Galle, M. F. Neurath, and R. Kiesslich, “In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor,” Gastroenterology 138, 435-446 (2010).