臺灣博碩士論文加值系統

本研究目的主要使用金奈米桿與銀奈米粒子，利用其獨特的光學
特性，做為細胞影像觀察之媒介，並加上PI與DAPI二種染劑，與金奈
米粒桿、銀奈米粒子比較在雷射/超快雷射掃描式共軛焦顯微鏡觀察
之表現。使用電化學法合成金奈米桿，氧化還原法合成銀奈米粒子，
之後以紫外光/可見紅/近紅外光光譜儀及穿透式電子顯微鏡確認。實
驗中使用特徵吸收峰在600nm的金奈米桿，其長短軸比約2.13。而球
形銀奈米的特徵吸收峰在400nm，將金奈米桿、銀奈米粒子與人類乳
導管癌細胞(MDA-MB-435S)共培養，使用不同波長之雷射(包括
458nm、488nm、514nm、561nm、633nm)做為激發光源，藉由使用二
個不同波段之濾波器，分別接收奈米粒子的散射光與染劑的螢光，在
雷射共軛焦顯微鏡下觀察。奈米粒子的散射光與染劑的螢光可以同時
被接收且沒有訊號互相干擾的情形，並且影像可以利用軟體同時呈現，
奈米粒子由於其表面電漿共振效應(SPR)，可散射不同雷射光，因此
在各個波長下都有表現。另外對細胞做光學掃描切片，觀察奈米粒子
在細胞內的位置，而使用穿透式電子顯微鏡，觀察到奈米粒子在細胞
質中的囊泡內，細胞利用內噬作用將奈米粒子帶入細胞質的囊泡中。
在囊泡中的奈米粒子團聚使得SPR頻寬更寬，故適當選擇雷射與濾波
器範圍，可分離奈米粒子的散射光與染劑的螢光。進一步利用超快雷
射(800 nm)激發金屬奈米粒子與PI染劑，可取得雙光子細胞影像。總
之，金屬奈米粒子可以與螢光染劑結合使用，做為生物標記。
關鍵字:表面電漿共振效應、雷射掃描共軛焦顯微鏡、金屬奈米粒子、
奈米桿、細胞影像、生物標記、螢光染劑。

The purpose of this research is to use metal nanoparticles (gold nanorods and
silver nanoparticles) as biomarkers for the cellular imaging of laser scanning confocal
microscopy (LSCM). The gold nanorods were synthesized by using electrochemical
method, whereas the silver nanoparticles were synthesized by oxidation-reduction
method. The absorption spectra and the morphologies of these metal nanoparticles
were identified by the Uv/Vis-Near IR spectrophotometer and TEM, respectively. For
our experiment, the aspect ratio of the gold nanorods is about 2.13, and the peak of
absorption spectrum is at 600 nm. For the silver nanoparticles, the peak of the
absorption spectrum is at 400nm. The cell of experiment is the human breast cancer
cell line (MDA-MB-435S). These cells were incubated with a medium containing
gold nanorods or silver nanoparticles, and then their nuclei were stained by Prodium
Iodide (PI) or 4,6-diamidino-2-phenylindole (DAPI). Different lasers (458, 488, 514,
561, and 633 nm) were used individually to illuminate these cells, and the ranges of
two bandpass filters were adjusted for the detection to separate the scattered light of
metal nanoparticles and the fluorescence of dyes. The advantage of this method is that
the scattered light from the metal nanoparticles and the fluorescence from PI or DAPI
can be induced simultaneously but be detected separately without crosstalk.
Subsequently, a compound cellular image of LSCM can be obtained by combining the
scattered light of metal nanoparticles and the fluorescence of dye molecules. The
scattering of light is caused by the surface plasmon resonance (SPR) of these metal
nanoparticles. In addition, cellular optical-section images of different depths were
acquired to identify that these nanoparticles are embedded in the cytoplasm. The TEM
images also show that several clusters of metal nanoparticles enclosed by vesicles are
distributed in the cytoplasm, due to the endocytosis of the cells. The aggregation of
nanoparticles makes the SPR band broadened, so that the scattered lights are observed
for different wavelength lasers. Furthermore, the two-photon cellular images were
obtained by using femtosecond ultra-pulse Ti: sapphire laser of 800 nm to excite these
nanoparticles and PI. Summarily, the metal nanoparticles can be potential biomarkers
to be combined with fluorescence dyes for the cellular imaging.
Keywords: Surface plasmon resonance, Laser scanning confocal microscopy, metal
nanoparticles, Nanorods, Cellular imaging, Biomarker, Fluorescence dye

指導教授推薦書
口試委員會審定書
授權書 ................................................... i
誌謝 ..................................................... ii
中文摘要 ................................................ iii
英文摘要 ................................................ iv
目錄 ..................................................... v
圖目錄 ................................................. viii
表目錄 .................................................. xv
第一章 緒論 ............................................ 1
1-1 前言 ............................................ 1
1-2 研究動機與目的 .................................. 3
第二章 研究背景 .......................................... 4
2-1 奈米粒子簡介.....................................4
2-1-1 金奈米粒子...................................7
2-1-2 銀奈米粒子..................................12
2-2 金屬奈米粒子的光學特性..........................13
2-3 共軛焦顯微鏡....................................16
vi
2-3-1 雷射共軛焦顯微鏡原理........................16
2-3-2 雙光子(多光子)共軛焦顯微鏡..................18
第三章 實驗方法......................................... 21
3-1 實驗藥品........................................21
3-2 儀器設備........................................22
3-3 實驗整體流程....................................23
3-4 金奈米桿的製備..................................24
3-5 銀奈米粒子的製備................................25
3-5-1 銀奈米桿的製備.............................26
3-6 紫外光/可見光-近紅外光光譜儀(UV/VIS-Near IR)光譜
分析...........................................26
3-7 穿透式電子顯微鏡(TEM)分析...................... 27
3-8 奈米粒子做為細胞影像媒介之實驗..................27
3-8-1 細胞培養.................................. 27
3-8-2 細胞毒性分析.............................. 28
3-8-3 細胞與奈米粒子共培養.......................29
3-8-4 細胞核染色.................................30
3-9 雷射/ 超快雷射共軛焦顯微鏡分析. . . . . . . . . . . .32
3-10 穿透式電子顯微鏡之細胞樣品製備.................33
vii
第四章 實驗結果與討論....................................34
4-1 金奈米桿的合成...............................34
4-2 銀奈米粒子的合成...............................35
4-3 細胞毒性分析.............................. 36
4-4 奈米粒子做為細胞影像媒介之實驗..............37
4-4-1 金奈米桿做為細胞影像媒介之實驗..........38
4-4-2 銀奈米粒子做為細胞影像媒介之實驗........44
4-4-3 螢光染劑與奈米粒子表現之比較...........47
4-4-4 超快雷射共軛焦顯微鏡之結果.............50
第五章 結論..........................................53
參考文獻...................... ....................95
附錄. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .101
圖目錄
圖2-1 雷射消熔法之實驗裝置概圖 ......................... 8
圖2-2 金屬氣相合成法示意圖............................. 9
圖2-3 化學還原法之分類概圖............................ 10
圖2-4 奈米尺寸金屬顆粒之電化學法製備概圖 .............. 11
圖2-5 奈米粒子長短軸比與吸收峰間關係 .................. 14
圖2-6 金奈米粒桿長度與顏色變化圖 ...................... 15
圖2-7 共軛焦顯微鏡成像原理............................ 17
圖2-8 射掃描影像方式 ................................. 18
圖2-9 單光子與雙光子激發示意圖 ........................ 19
圖2-10 單光子與雙光子激發方式差異 ...................... 20
圖3-1 實驗整體流程圖 ................................. 23
圖3-2 金奈米桿實驗流程圖.............................. 25
圖3-3 細胞毒性分析方法示意圖 .......................... 29
圖3-4 PI 染劑的激發與放射波長範圍 ..................... 31
圖3-5 DAPI 染劑的激發與放射波長範圍 .................. 31
圖4-1 金奈米桿的Uv/Vis-Near IR 光譜圖，縱向表面電漿共振波
長在780nm ...................................... 58
圖4-2 金奈米桿(縱向表面電漿共振在780nm)之穿透式電子顯微鏡
影像 A.放大倍率150k B.放大倍率200k，長短軸比:3.37±
0.74 ............................................ 59
圖4-3 金奈米粒子的Uv-Vis Near IR 光譜圖，縱向表面電漿共振波
長在600nm ...................................... 60
圖4-4 金奈米桿(縱向表面電漿共振在600nm)之穿透式電子顯微鏡
iv
影像 A.放大倍率150k B.放大倍率200k C.放大倍率250，
長短軸比:2.13±0.32k .............................. 61
圖4-5 銀奈米粒子的UV/VIS-Near IR 光譜圖，表面電漿共振的特
徵吸收峰在400nm ................................ 62
圖4-6 銀奈米粒子之穿透式電子顯微鏡影像 A.放大倍率50k B.
放大倍率100k。銀奈米粒子平均粒徑為23.52±5.12nm … 63
圖4-7 不同特徵吸收峰之銀奈米桿的Uv/Vis-Near IR 光譜圖，種
晶量60λ吸收峰為591nm，種晶量200λ吸收峰為541nm，
種晶量300λ吸收峰為515nm，種晶量400λ吸收峰為509nm
................................................ 64
圖4-8 不同種晶量合成出不同長短軸比之銀奈米桿顏色示意圖
.............................................. 65
圖4-9 細胞毒性測試之四組條件，細胞株為L929 纖維母細胞，A.
以新鮮培養基培養，B. 以陽性對照組培養，含0.64%phenol，
C. 以陰性對照組培養，萃取培養盤之培養液D. 以金奈米
桿萃取液培養 ................................... 66
圖4-10 細胞毒性測試之四組條件，細胞株為L929 纖維母細胞，A.
以新鮮培養基培養，B. 以陽性對照組培養，含0.64%phenol，
C. 以陰性對照組培養，萃取培養盤之培養液D. 以銀奈米
粒子萃取液培養 ................................. 67
圖4-11 MDA-MB-435S之細胞形態比較與生長情形 A.未攝入金奈
米桿B.攝入金奈米桿培養3 天 .................... 68
圖4-12 MDA-MB-435S 之細胞形態比較與生長情形 A.未攝入銀奈
米粒子B.攝入銀奈米粒子培養3 天 ................. 69
v
圖4-13 金奈米桿吞噬實驗對照組，435s 細胞染PI 染劑以458nm 雷
射激發之共軛焦顯微鏡影像，A. Filter:432-496 之雷射光影
像，B. 穿透光之細胞形態影像，C. Filter:593-636，D. A-C
之重疊影像，放大倍率為63 倍 ..................... 70
圖4-14 金奈米桿吞噬實驗對照組，435s 細胞染PI 染劑後分別在A.
458nm B.488nm C.514nm D.561nm E.633nm 雷射下
的影像，放大倍率為63 倍 ......................... 71
圖4-15 特徵吸收峰在600nm，O.D. 1.1 的金奈米桿與435s 細胞共
培養，以458nm 雷射激發之斷層掃描影像，由細胞底部掃
描至細胞頂部，放大倍率為63 倍 ................... 72
圖4-16 特徵吸收峰在600nm，O.D. 1.1 的金奈米桿與435s 細胞共
培養，以488nm 雷射激發之斷層掃描影像，由細胞底部掃
描至細胞頂部，放大倍率為63 倍 ................... 73
圖4-17 特徵吸收峰在600nm，O.D. 1.1 的金奈米桿與435s 細胞共
培養，以514nm 雷射激發之斷層掃描影像，由細胞底部掃
描至細胞頂部，放大倍率為63 倍 ................... 74
圖4-18 特徵吸收峰在600nm，O.D. 1.1 的金奈米桿與435s 細胞共
培養，以561nm 雷射激發之斷層掃描影像，由細胞底部掃
描至細胞頂部，放大倍率為63 倍 ................... 75
圖4-19 特徵吸收峰在600nm，O.D. 1.1 的金奈米桿與435s 細胞共
培養，以633nm 雷射激發之斷層掃描影像，由細胞底部掃
描至細胞頂部，放大倍率為63 倍 ................... 76
圖4-20 金奈米桿之表面電漿共振光譜與PI 染劑的激發、放射光譜，
開二個Filter，Filter-I 為接收金奈米桿之散射光，Filter-II 接
vi
收PI 染劑之放射螢光 ............................. 77
圖4-21 特徵吸收峰在600nm，O.D. 1.1 的金奈米桿與435s 細胞共
培養， 使用A. 458nm B.488nm C.514nm D.561nm
E.633nm 雷射激發之細胞底部影像，放大倍率為63 倍...78
圖4-22 特徵吸收峰在600nm，O.D. 1.1 的金奈米桿與435s 細胞共
培養， 使用A. 458nm B.488nm C.514nm D.561nm
E.633nm 雷射激發之3-D 投影影像，放大倍率為63 倍...79
圖4-23 O.D. 0.5 之金奈米桿與MDA-MB-435S 細胞培養24 小時之
穿透式電子顯微鏡影像 A.435S 細胞全貌，放大6000 倍 B.
局部放大15 萬倍 C.局部放大30 萬倍 ............... 80
圖4-24 銀奈米粒子吞噬實驗對照組，435S 細胞染PI 染劑後分別在
A. 458nm B.488nm C.514nm D.561nm E.633nm 雷射
下的影像，放大倍率為63 倍 ....................... 81
圖4-25 特徵吸收峰在400nm，O.D. 1 的銀奈米粒子與435S 細胞共
培養，以458nm 雷射激發之斷層掃描影像，由細胞底部掃
描至細胞頂部，放大倍率為63 倍 ................... 82
圖4-26 特徵吸收峰在400nm，O.D. 1 的銀奈米粒子與435S 細胞共
培養，以488nm 雷射激發之斷層掃描影像，由細胞底部掃
描至細胞頂部，放大倍率為63 倍 ................... 83
圖4-27 特徵吸收峰在400nm，O.D. 1 的銀奈米粒子與435S 細胞共
培養，以514nm 雷射激發之斷層掃描影像，由細胞底部掃
描至細胞頂部，放大倍率為63 倍 ................... 84
圖4-28 特徵吸收峰在400nm，O.D. 1 的銀奈米粒子與435S 細胞共
培養，以561nm 雷射激發之斷層掃描影像，由細胞底部掃
vii
描至細胞頂部，放大倍率為63 倍 ................... 85
圖4-29 特徵吸收峰在400nm，O.D. 1 的銀奈米粒子與435S 細胞共
培養，以633nm 雷射激發之斷層掃描影像，由細胞底部掃
描至細胞頂部，放大倍率為63 倍 ................... 86
圖4-30 銀奈米粒子與435S 細胞共培養，使用A. 458nm B.488nm
C.514nm D.561nm E.633nm 雷射激發之細胞底部影像，
放大倍率為63 倍 ................................. 87
圖4-31 銀奈米粒子與435S 細胞共培養，使用A. 458nm B.488nm
C.514nm D.561nm E.633nm 雷射激發之3-D 投影影像，
放大倍率為63 倍 ................................. 88
圖4-32 金奈米桿吞噬實驗對照組，435S 細胞染DAPI 染劑後，Filter-
Ι 使用A. 458nm B.488nm C.514nm D.561nm
E.633nm 雷射激發金奈米桿，Filter-Π使用405nm 雷射激發
DAPI 影像之細胞底部影像，放大倍率為100 倍 ....... 89
圖4-33 特徵吸收峰在600nm，O.D. 1 的金奈米桿與435S 細胞共培
養， 染DAPI 染劑， Filter- Ι 使用A.458nm B.488nm
C.514nm D.561nm E.633nm 雷射激發金奈米桿，Filter-
Π使用405nm 雷射激發DAPI 影像之細胞底部影像，放大倍
率為100 倍 ...................................... 90
圖4-34 銀奈米粒子吞噬實驗對照組，435s 細胞染DAPI 染劑後，
Filter-Ι使用A. 458nm B.488nm C.514nm D.561nm
E.633nm 雷射激發金奈米桿，Filter-Π使用405nm 雷射激發
DAPI 影像之細胞底部影像，放大倍率為100 倍 ....... 91
圖4-35 特徵吸收峰在400nm，O.D.1 的銀奈米粒子與435s 細胞共
viii
培養，染DAPI 染劑，Filter-Ι使用A.458nm B.488nm
C.514nm D.561nm E.633nm 雷射激發銀奈米粒子，Filter-
Π使用405nm 雷射激發DAPI 影像之細胞底部影像，倍率為
100 倍 .......................................... 92
圖4-36 特徵吸收峰在600nm，O.D. 1.1 的金奈米桿與435S 細胞共
培養，染PI 染劑，使用超快雷射激發金奈米桿，A.
Filter:600-800nm B. Filter:500-550nm，C. A.與B.之合成圖，
倍率為63 倍 ..................................... 93
圖4-37 特徵吸收峰在400nm，O.D. 1 的銀奈米粒子與435S 細胞共
培養，染PI 染劑，使用超快雷射激發銀奈米粒子，A.
Filter:400-500nm B. Filter:500-600nm，C. Filter:600-700nm，
倍率為63 倍 ..................................... 94
ix
表目錄
表一 奈米粒子中表面原子數佔原子數之比率與粒徑的關係 ..... 4
表二 雷射光源之對應波長與功率大小 ...................... 32
表三 金奈米桿之細胞毒性測試結果.........................56
表四 銀奈米粒子之細胞毒性測試結果.......................56
表五 各波長雷射下二組Filter範圍，Filter-Ι接收金奈米桿之散射光，
Filter-Π 接收螢光染劑PI 的放射螢光....................56
表六 各波長雷射下二組Filter 範圍，Filter-Ι 接收銀奈米粒子之散射
光，Filter-Π 接收螢光染劑PI 的放射螢光.................57
表七 各波長雷射下二組Filter範圍，Filter-Ι接收金奈米桿之散射光，
Filter-Π 接收螢光染劑DAPI 的放射螢光.................57
表八 各波長雷射下二組Filter 範圍，Filter-Ι 接收銀奈米粒子之散射
光，Filter-Π 接收螢光染劑DAPI 的放射螢光..............57

參考文獻
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