本論文利用全反射的方式來激發金屬奈米結構中的表面電漿子，經由區域電場的強化，來增強金屬奈米結構上螢光分子的訊號。同時我們也利用奈米壓印的製程製做出不同的金屬奈米結構來作為增強螢光訊號的基板，如：金奈米圓柱陣列與金奈米球陣列，改變其尺寸與間距的大小，去探討這些結構的光學特性，並且對於螢光訊號增強的影響。
傳統的全反射螢光顯微鏡，是將光源以超過臨界角的角度入射產生一衰逝波，以激發並擷取在衰逝波範圍內(約數百奈米)的螢光分子訊號，因此可達到奈米等級的解析度，其螢光影像亦具有非常低背景雜訊的特點。但當擷取位於細胞膜上生物分子間互相作用的動態影像時，活體細胞影像上的螢光訊號仍需被增強。為了得到更強的螢光訊號以降低偵測極限，本文利用金屬奈米粒子的局域性表面電漿子增強螢光分子附近的區域電磁場並進而強化被偵測螢光訊號強度的技術，發展出一套表面電漿子增強全反射螢光顯微術的系統。

In this thesis, we use the method of total internal reflection to excite surface plasmons on metal nanostructures, strengthening the regional electric field of metal nanoparticles to significantly enhance the fluorescent molecule signal. We also use nano-imprint process to fabricate various metal nanostructures as the large-area substrate to extensively enhance the fluorescent signal. Gold nanodisk and gold nanoparticle arrays are used for changing the sizes and interparticle spaces to study the optical enhancement properties of these structures, and the enhancement level of detected fluorescence signals.
The traditional total internal reflection fluorescence microscopy (TIRFM) applies more than the critical angle of light incidence to generate evanescent wave (about several hundred nanometers) on the dielectric film where the fluorescent molecules are significantly stimulated. By this scheme, the TIRFM can achieve the resolution of nanometer level, which offers the image with very low fluorescence background noises and advancing the signal to noise ratio. However, in the real-time live cell imaging, the fluorescence signals from the cell membrane containing the dynamic information of specific molecular interactions are still needed to be enhanced. In order to get stronger fluorescence signals and to reduce the microscopic detection limit, we combine TIRFM and fabricated periodic metal nanostructure arrays to respectively measure fluorescent signals on the nano-imprint lithography-generated large-area polydimethylsiloxane (PDMS) substrates via localized surface plasmon (SP) excitations by means of the evanescent wave generation.

[1] A. Schonle and S. W. Hell, “heating by absorption in the focus of an objective lens”, Opt. Lett. 23, 325-327 (1998)
[2] K. L. Kelly, E. Coronado, L. L. Zhao, and C. Schatz, “The Optical Properties of Metal Nanoparticles: The Influence of Size, Shape, and Dielectric Environment”, J. Phys. Chem. B 107, 668-677 (2003)
[3] T. Hirschfeld, “Optical microscopic observation of single small molecules”, Appl. Opt. 15, 2965 (1976)
[4] C. Wilkerson, P. Goodwin, W. Ambrose, J. Martin, and R. Keller, “Detection and lifetime measurement of single molecules in flowing sample streams by lase-induced fluorescence”, Appl. Phys. Lett. 62, 2030 (1993)
[5] H. Lu and X. Xie, “Single-molecule spectral fluctuations at room temperature”, Nature, 385, 143 (1997)
[6] J. Trautman, J. Macklin, L. Brus, and E. Betzig, “Near-field spectroscopic of single molecules at room temperature”, Nature, 369 , 6475 (1994)
[7] K. Aslan, J. R. Lakowicz, and C. D. Geddes, “Plasmon light scattering in biology and medicine: new sensing approaches, visions perspectives”, Curr. Opin. Chem. Biol. 9, 538 (2005)
[8] K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, “Metal-enhanced fluorescence: an emerging tool in biotechnology” Curr. Opin. Chem. Biol. 16, 55 (2005)
[9] J. Malicka, I. Gryczynski, and J. R. Lakowicz, “DNA hybridization assays using metal-enhanced fluorescence” , Biochem. Biophys. Res.Commun. 306, 213, (2003)
[10] E. Matveeva, Z. Gryczynski, J. Malicka, I. Gryczynski, and J. R. Lakowicz, “Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces”, Anal. Biochem. 334, 303 (2004)
[11] M. H. Chowdhury, K. Ray, S. K. Gray, J. Pond, and J. R. Lakowicz, “Aluminum Nanoparticles as Substrates for Metal-Enhanced Fluorescence in the Ultraviolet for the Label-Free Detection of Biomolecules”, Anal.Chem. 81, 1397 (2009)
[12] R. H. Ritchie, “plasma losses by fast electrons in the thin films,” Phys. Rev. 106, 874-881 (1957)
[13] C. J. Powell and J. B. Swan, “Effect of oxidation on the characteristics loss spectra of aluminum and magnesium,” Phys. Rev. 118, 640-643 (1960)
[14] W. A. Weimer, and M. J. Dyer, “Tunable surface plasmon resonance silver films”, Appl. Phys. Lett. , 79, 3164 (2001)
[15] H. Raether, “Surface Plasmons” , Springer , New York (1988)
[16] D. A. Schultz, “Current Opinion in Biotechnology” 14, 13 (2003)
[17] V.Owen, “Real-time optical immunosensors –a commercial reality”, Biosensors & Bioelectronics, 12, (1997)
[18] 邱國斌、蔡定平, “金屬表面電漿簡介”
[19] K. A. Willets and R. P. Van Duyne, “Localized Surface Plasmon Resonance Spectroscopy and Sensing”, Annu. Rev. Phys. Chem. 58, 267–297 (2007)
[20] G. C. Schatz, M. A. Young, and R.P. Van Duyne, “Electromagnetic mechanism of SERS. In Surface-Enhanced Raman Scattering: Physics and Applications”, Springer, p.19 (2006)
[21] A. D. McFarland and R. P. Van Duyne, “Single Silver Nanoparticles as Real-Time Optical Sensors with Zeptomole Sensitivity”, Nano Lett. 3, 8 (2003)
[22] M. A. Behlke, L. Huang, L. Bogh, S. Rose, and E. J. Devor, “Fluorescence and Fluorescence Applications”, Integrated DNA Technologies.
[23] A. Hards, “The combined AFM manipulation and fluorescence imaging of single DNA molecules” Ph.D. Thesis, Department Chemistry Ludwig Maximilian University Munich, (2004)
[24] J. A. Steyer and W. Almers, “A real-time view of life within 100 nm of the plasma membrane”, Molecular Cell Biology, 2, 268-276,(2001)
[25] N. L. Thompson and B. C. Lagerholm, “Total internal reflection fluorescence: applications in cellular biophysics”, Opin. In Biotech. 8, 58-64 (1997)
[26] Y. Harada, T. Funatsu, K. Murakami, Y. Nonoyama, A. Ishihama, and T. Yanagida, “Single-Molecule Imaging of RNA Polymerase-DNA Interactions in Real Time”, Biophysical Journal 76, 709–715 (1999)
[27] R. Y. He, G. L. Chang, H. L. Wu, C. H. Lin, K. C. Chiu, Y. D. Su, and S. J. Chen, “Enhanced live cell membrane imaging using surface plasmon-enhanced total internal reflection fluorescence microscopy” Optics Express ,14 ,9307-9316 (2006)
[28] C.H. Chen, T.H. Yu, and Y.C. Lee, “Direct patterning of metallic micro/nano-structures on flexible polymer substrates by roller-based contact printing and infrared heating” J. Micromech. Microeng. 20, 025034 (2010)
[29] Y. C. Lee, C. Y. Chiu, and S. H. Chang, “Micro-/nano-lithography based on the contact transfer of thin film and mask embedded etching” J. Micromech. Microeng., 18, 025034 (2008)
[30] F. Sun, W. Cai, and Y. Li, “Laser morphological manipulation of gold nanoparticles periodically arranged on solid supports” Appl. Phys. B, 81, 765–768 (2005)