臺灣博碩士論文加值系統

本論文藉由使用超解析率明視野光學顯微術來觀測直徑小於光學解析率的絲狀偽足，因為絲狀偽足的光學對比度相當低，所以我們利用具有奈米縱向解析率的非干涉式廣視野光學測繪術(Non-Interferometric Widefield Optical Profilometry, NIWOP)將樣品表面型態的變化轉換成影像訊號強度，因此由NIWOP所產生的高對比訊號是來自於樣品表面的高低變化，而非來自絲狀偽足本身的對比或是螢光物質。將NIWOP產生的高對比訊號經由影像重建運算系統產生supre-resolution NIWOP，將橫向解析率提昇至130 nm，大約為光學解析率的 ，這將使得絲狀偽足的影像更加清晰。
目前最常用來觀測絲狀偽足的技術為利用螢光標定肌動蛋白，而使得絲狀偽足得以清楚呈現。但是某些基因轉質感染(transfetion)的方式將會對細胞的活性產生影響。此外，操作螢光顯微鏡所使用的強激發光，將會使得細胞產生光漂白(photobleach)及光毒性，導致細胞的活性改變，造成細胞死亡數目增多。這套技術中使用的細胞樣品都不需要經過螢光標定或是任何前置處理，可以避免螢光顯微術在細胞活性上造成的影響。
目前我們每分鐘可取到20張高解析率的影像，為了確認此取樣速率是否足以用來觀測絲狀偽足的動態變化。我們利用“上皮細胞生長因子會影響絲狀偽足的動態變化”這個已知的現象來驗證supre-resolution NIWOP對於絲狀偽足動態變化的觀測。我們成功的量測到當CL1-0添加EGF之後，絲狀偽足的數量增加了2倍。而其伸長及收縮速率加快約20%到30%，顯示EGF可能增進肺癌細胞的移動能力。
接下來，我們使用supre-resolution NIWOP觀測一個新因子對於CL1-0絲狀偽足在短時間之內是否有影響。當添加此因子時，CL1-0絲狀偽足的數量及每10秒的數量變化會逐漸增加，顯示此新因子會影響CL1-0的絲狀偽足，並且有可能會促進癌細胞的轉移。當反應過後，絲狀偽足的數量仍比加入藥物前還多了50％左右，因為此現象也出現於對照組中，推測是因實驗時所使用的波段為600~700 nm的可見光，波段範圍接近近紅外光，因此可能稍微促進CL1-0絲狀偽足的生長。此外，我們還使用supre-resolution NIWOP觀測轉染gene A的CL1-0，藉由觀測到其絲狀偽足的大量表現，而推斷此蛋白質會促進癌細胞的轉移能力。
一般相信，絲狀偽足具有趨化性，有能力感受到外界物質的濃度差異。但是絲狀偽足是否能作為細胞感受到外界化學物質濃度變化的特徵，則尚未有人提出。因此，我們將本技術應用到微流道細胞培養晶片中的活細胞觀測。微流道細胞培養晶片所產生的藥物濃度梯度，會使CL1-0細胞體兩側分別有不同的藥物濃度。當我們加入上皮細胞生長因子後，CL1-0絲狀偽足位於濃度較高的一側，其數量較另一側為多，顯示絲狀偽足對於上皮細胞生長因子具有趨化性(chemotactic)。以微流道細胞培養晶片做細胞特性的調控與觀測，具有樣品數量少、使用藥劑量低及操控條件方便等優勢。結合我們的super-resolution NIWOP，微流道細胞培養晶片的觀測將能獲得更多的訊息。
Supre-resolution NIWOP不需要先將細胞作基因轉質感染的處理，拍攝時用來照明的光強度也比螢光顯微術所用的光強度還要低，所以不會對細胞的動態變化及存活性造成影響，因此，我們可以得到細胞在最接近自然狀況下的反應。Supre-resolution NIWOP的高對比度及高解析率的影像會使得絲狀偽足更加清晰，此外，系統目前的取樣速率為20 frame/min，已經足夠用來觀測絲狀偽足的動態變化。我們可以用此系統觀測不同的作用因子、基因或是藥物濃度梯度對於絲狀偽足的影響，進而推斷是否會促進癌細胞的轉移。

In this thesis, we explore the dynamics of cellular filopodia with diameters around 200 nm by using super-resolution bright-field optical microscopy. Filopodia are positively related to cell motility because of the low optical contrast of filopodia, we use an optical technique with nanometer depth sensitivity, non-interferometric widefield optical profilometry (NIWOP), to obtain the high contrast signal for super-resolution image restoration. The image contrast of NIWOP comes from the topographic variations of sample, rather than fluorescence labeling.
In super-resolution NIWOP, the lateral resolution is improved to 130 nm. Compared with the original optical resolution of 250 nm, the lateral resolution is reduced by 50%. At present, our system has an image-acquisition rate of 20 frames/min. In order to confirm that the lateral resolution and the image-acquisition rate is suitable for observing the dynamics of cancer-cell filopodia, we compare the numbers and dynamics of filopodia before and after the treatment of epidermal growth factor (EGF), which is known to affect the dynamics of filopodia.
In our work, The cell line is human lung adenocarcinoma cell lines CL1-0, which is less invasive, and therefore with fewer filopodia. We found that the treatment of EGF raises the number of filopodia by nearly a factor of 2. In addition, we observe the growth and activities of single filopodia of a CL1-0 cell. In the culturing condition, we measure that the filopodia exhibit an average elongation rate of 90 nm/sec, and an average shrinkage rate of 75 nm/sec. After the treatment of EGF, the elongation and shrinkage rates increase to 110 nm/sec and 100 nm/sec respectively. With the treatment of EGF, the increase on the number and the dynamics of filopodia implies the enhancement of cell motility.
We also use the super-resolution NIWOP to observe the effects of a new factor on the number of CL1-0 filopodia. After the new factor treatment, the average number of filopodia also increases, and the maximum occurs at 8-10 minutes afterwards. This result verifies that the new factor could affect the filopodia of CL1-0, and it may promote the metastasis of lung cancer cells. Moreover, with super-resolution NIWOP, we observed that the number of filopodia in gene A-transfected CL1-0 is more than that in mock-transfected CL1-0 cells. We speculate that gene A could also enhance cancer cell migration and invasion.
It is proposed that filopodia could sense the chemical materials around a cell. However nobody demonstrates that filopodia can be used to characterize the chemical gradient around a cell. Therefore, we apply super-resolution NIWOP to a transparent microfluidic cell culturing chip to demonstrate the chemo-gradient sensitivity of filopodia on a single cell. Owing to the specially designed fluidic field in the chip, we may control the concentration of chemical materials around a single cell. Placing a CL1-0 cell at high gradient of EGF, the number of filopodia on the high-concentration side is evidently more than that on the low-concentration side. This experiment proves that the quantity of filopodia depends the chemo-gradient of EGF, even on the same cell.
The non-fluorescence observation technique developed in this thesis is very attractive for living cell analyses. Without the high-intensity illumination required by fluorescence imaging, cells stay in their natural states before and after the observation. The results are thus more directly related to their behavior in native environments. On a CL1-0 lung cancer cell, the high contrast and resolution of the super-resolution NIWOP images make the characterization of filopodia much easier and more accurate. Furthermore, the imaging-acquisition rate is as fast as 20 frame/min, which is suitable for dynamic observations. With super-resolution NIWOP, we can easily observe the effect on filopodia by the treatment of various factors, genes or chemical gradient of drug and then conjecture that if they can enhance or suppress cancer-cell migration and invasion.

第一章 細胞運動特性的研究方法

第二章 絲狀偽足與細胞運動的相關性
2.1 絲狀偽足(filopodium)
2.2 絲狀偽足與細胞運動的相關性
2.3 常用於研究絲狀偽足的方法

第三章 如何在明視野光學顯微術中得到超越繞射極限的解析率
3.1 超解析率螢光顯微術
3.1.1 影像重建運算系統
3.1.2 超解析率螢光顯微術的應用
3.2 螢光顯微術的缺點
3.3 超解析率非干涉式廣視野光學測繪術(super resolution NIWOP)
3.3.1 廣視野光學切片影像
3.3.2 差動共焦顯微術

第四章 儀器架設與操作
4.1 NIWOP系統架設
4.2 橫向、縱向解析率、動態範圍及切片能力
4.3 影像重建運算系統的應用
第五章 細胞樣品備置
5.1 細胞株與細胞培養
5.2 拍攝NIWOP影像的樣品備製
5.3 分別轉染與篩選穩定表現pAcGFP-actin和pEGFP-C1的細胞株
5.3.1 將pAcGFP-actin和pEGFP-C1分別轉染至細胞中
5.3.2 分別篩選穩定表現pAcGFP-actin和pEGFP-C1的細胞株(selection of stable clone)

第六章 實驗結果
6.1 Super-resolution NIWOP對於橫向解析率的提昇
6.2 利用上皮細胞生長因子驗證super-resolution NIWOP適於觀測絲狀偽足動力學的變化
6.2.1 上皮細胞生長因子(epidermal growth factor, EGF)
6.2.2 EGF對於絲狀偽足動態上的影響
6.3 一個新的因子對於絲狀偽足動態的暫態影響
6.4 高度表現gene A的CL1-0對於絲狀偽足動態的影響

第七章 以微流道細胞培養晶片進行化學梯度對絲狀偽足動態的影響
7.1 微流道細胞培養晶片
7.2 晶片內穩定流場對於絲狀偽足的影響
7.3 化學濃度梯度對於絲狀偽足的影響

第八章 結論

A. J. Ridley, M. A. Schwartz, K. Burridge, R. A. Firtel, M. H. Ginsberg, G. Borisy, J. T. Parsons, and A. R. Horwitz, "Cell migration: Integrating signals from front to back," Science 302, 1704 (2003).
D. H. Geho, R. W. Bandle, T. Clair, and L. A. Liotta, "Physiological mechanisms of tumor-cell invasion and migration," Physiology 20, 194 (2005).
E.-M. Sandrine, and A. Hall, "Rho GTPases in cell biology," Nature 420, 629 (2002).
T. Takenawa, and H. Miki, "WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement," J. Cell Sci. 114, 1801 (2001).
W. B. Kiosses, S. J. Shattil, N. Pampori, and M. A. Schwartz, "Rac recruits high-affinity integrin avb3 to lamellipodia in endothelial cell migration," Nature Cell Biol. 3, 316 (2001).
J. C. Sandquist, K. I. Swenson, K. A. DeMali, K. Burridge, and A. R. Means, "Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration," J. Biol. Chem. 281, 35873 (2006).
R. A. Worthylake, and K. Burridge, "RhoA and ROCK Promote Migration by Limiting Membrane Protrusions," J. Biol. Chem. 278, 13578 (2003).
C. C. Ho, P. H. Huang, H. Y. Huang, Y. H. Chen, P. C. Yang, and S. M. Hsu, "Up-regulated caveolin-1 accentuates the metastasis capability of lung adenocarcinoma by inducing filopodia formation," Am. J. Pathol. 161, 1647 (2002).
O. Kovbasnjuk, R. Mourtazina, B. Baibakov, T. Wang, C. Elowsky, M. A. Chot, A. Kane, and M. Donowitz, "The glycosphingolipid globotriaosylceramide in the metastatic transformation of colon cancer," Proc. Natl. Acad. Sci. U.S.A 102, 19087 (2005).
A. Steffen, J. Faix, G. P. Resch, J. Linkner, J. Wehland, J. V. Small, K. Rottner, and T. E. B. Stradal, "Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes," Mol. Biol. Cell 17, 2581 (2006).
J. Faix, and K. Rottner, "The making of filopodia," Curr. Opin. Cell Biol. 18, 18 (2006).
J. Y. Shih, S. C. Yang, T. M. Hong, A. Yuan, J. J. W. Chen, C. J. Yu, Y. L. Chang, Y. C. Lee, K. Peck, C. W. Wu, and P. C. Yang, "Collapsin response mediator protein-1 and the invasion and metastasis of cancer cells," J. Natl. Cancer Inst. 93, 1392 (2001).
S. H. Pan, T. M. Hong, Y. C. Chao, Y. Y. Wu, S. C. Yang, W. L. Wang, P. S. Ieong, and P. C. Yang, "Long form collapsin response mediator protein-1(LCRMP-1) is a novel invasion enhancer: Yin-Yang regulation of cancer cell invasion by LCRMP-1 and CRMP-1," Proc. Amer. Assoc. Cancer Res. 47, 823 (2006).
M. D.Welch, and R. D. Mullins, "Cellular control of actin nucleation," Annu. Rev. Cell Dev. Biol. 18, 247 (2002).
V. D. Vacquier, "The connection of blastomeres of sea urchin embryos by filopodia.," Exp. Cell Res. 52, 571 (1968).
T. M. Svitkina, E. A. Bulanova, O. Y. Chaga, D. M. Vignjevic, S. I. Kojima, J. M. Vasiliev, and G. G. Borisy, "Mechanism of filopodia initiation by reorganization of a dendritic network," J. Cell Biol. 160, 409 (2003).
A. Biyasheva, T. Svitkina, P. Kunda, B. Baum, and G. Borisy, "Cascade pathway of filopodia formation downstream of SCAR," J. Cell Sci. 117, 837 (2003).
T. E. B. Stradal, K. Rottner, A. Disanza, S. Confalonieri, M. Innocenti, and G. Scita, "Regulation of actin dynamics by WASP and WAVE family proteins," TRENDS in Cell Biology 14, 303 (2004).
R. C. Robinson, K. Turbedsky, D. A. Kaiser, J.-B. Marchand, H. N. Higgs, S. Choe, and T. D. Pollard, "Crystal structure of Arp2/3 complex," Science 294, 1679 (2001).
A. A. Rodal, O. Sokolova, D. B. Robins, K. M. Daugherty, S. Hippenmeyer, H. Riezman, N. Grigorieff, and B. L. Goode, "Conformational changes in the Arp2/3 complex leading to actin nucleation," Nat. Struct. Mol. Biol. 12, 26 (2005).
C. Egile, I. Rouiller, X.-P Xu, N. Volkmann, R. Li, and D. Hanein, "Mechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at actin branch junctions," PLoS Biol. 3, 1902 (2005).
S. Suetsugu, H. Miki, and T. Takenawa, "Spatial and temporal regulation of actin polymerization for cytoskeleton formation through Arp2/3 complex and WASP/WAVE proteins," Cell Motil. Cytoskeleton 51, 113 (2002).
T. M. Svitkina, E. A. Bulanova, O. Y. Chaga, D. M. Vignjevic, S.-I. Kojima, J. M. Vasiliev, and G. G. Borisy, "Mechanism of filopodia initiation by reorganization of a dendritic network," J. Cell Biol. 160, 409 (2003).
W. Wood, A. Jacinto, R. Grose, S. Woolner, J. Gale, C. Wilson, and P. Martin, "Wound healing recapitulates morphogenesis in Drosophila embryos," Nat. Cell Biol. 4, 907 (2002).
Y. W. Chu, P. C. Yang, S. C. Yang, Y. C. Shyu, M. J. C. Hendrix, R. Wu, and C. W. Wu, "Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line," Am. J. Respir. Cell Mol. Biol. 17, 353 (1997).
M. M. Knight, S. R. Roberts, D. A. Lee, and D. L. Bader, "Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death," Am. J. Physiol. Cell Physiol. 284, C1083 (2003).
S. Landry, P. L. McGhee, R. J. Girardin, and W. J. Keeler, "Monitoring live cell viability: Comparative study of fluorescence, oblique incidence reflection and phase contrast microscopy imaging techniques," Opt. Express 12, 5754 (2004).
S.-W. Huang, H.-Y Mong, and C.-H. Lee, "Super-resolution bright-field optical microscopy based on nanometer topographic contrast," Microsc. Res. Tech. 65, 180 (2004).
T. H. Hsu, W. Y. Liao, P. C. Yang, C. C. Wang, J. L. Xiao, and C. H. Lee, "Dynamics of cancer cell filopodia characterized by super-resolution bright-field optical microscopy," Opt. Express 15, 76 (2007).
W. A. Carrington, R. M. Lynch, E. D. W. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, "Superresolution three-dimensional images of fluorescence in cells with minimal light exposure," Science 268, 1483 (1995).
G. M. P. v. Kempen, H. T. M. v. d. Voort, J. G. J. Bauman, and K. C. Strasters, "Comparing maximum likelihood estimation and constrained tikhonov-miller restoration," IEEE Eng. Med. Biol. Mag. 15, 76 (1996).
C. H. Lee, H. Y. Chiang, and H. Y. Mong, "Sub-diffraction-limit imaging based on the topographic contrast of differential confocal microscopy," Opt. Lett. 28, 1772 (2003).
M. Schrader, S. W. Hella, and H. T. M. v. d. Voort, "Potential of confocal microscopes to resolve in the 50-100 nm range," Appl. Phys. Lett. 69, 3644 (1996).
M. A. A. Neil, R. Juškaitis, and T. Wilson, "Method of obtaining optical sectioning by using structured light in a conventional microscope," Opt. Lett. 22, 1905 (1997).
C. H. Lee, and J. Wang, "Noninterferometric differential confocal microscopy with 2-nm depth resolution," Opt. Commun. 135, 233 (1997).
李超煌, "差動共焦顯微術及其應用," 博士論文, (國立台灣大學電機工程學研究所,1997).
C. H. Lee, H. Y. Mong, and W. C. Lin, "Noninterferometric wide-field optical profilometry with nanometer depth resolution," Opt. Lett. 27, 1773 (2002).
D. J. Stevenson, T. K. Lake, B. Agate, V. G. Chávez, K. Dholakia, and F. G. Moore, "Optically guided neuronal growth at near infrared wavelengths," Opt. Express 14, 9786 (2006).
O. Boussif, F. Lwzoualc'ht, M. Zanta, M. Mergnyt, D. Schermant, B. Demeneixt, and J.-P. Behr, "A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine," Proc. Natl. Acad. Sci. USA 92, 7297 (1995).
R. N. Jorissen, F. Walker, N. Pouliot, T. P. J. Garrett, C. W. Ward, and A. W. Burgess, "Epidermal growth factor receptor: mechanisms of activation and signalling," Exp. Cell Res. 284, 31 (2003).
D. S. Lidke, K. A. Lidke, B. Rieger, T. M. Jovin, and D. J. Arndt-Jovin, "Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors," J. Cell Biol. 170, 619 (2005).
T. D. Pollard, and G. G. Borisy, "Cellular motility driven by assembly and disassembly of actin filaments," Cell 112, 453 (2003).