臺灣博碩士論文加值系統

多數癌症病人的死亡是因為癌細胞轉移造成的。雖然越來越多的研究指出淋巴管的新生是預測腫瘤轉移到淋巴結的指標，但就現在的影像技術而言，在活體中觀察淋巴系統是不容易的。而在觀測淋巴系統的影像技術中，光學影像逐漸受到重視，因為光學影像除了沒有輻射問題，還具有良好的解析度。在此論文中，我們提出多功能光學同調斷層影像技術，此技術為非侵入性，並可在活體中同時觀察組織內的結構、血管、以及淋巴管的影像。用此項技術，我們探討了自發的黑色素瘤轉移前的腫瘤微環境變化，並做量化分析，發現比起腫瘤厚度的改變，淋巴新生與血管新生是更明顯的。因此，相較於傳統的，只能提供結構資訊的光學同調斷層影像技術，我們所提出的多功能光學同調斷層影像技術更有著早期偵測黑色素瘤轉移的潛力。

Metastasis is the cause of the majority of cancer deaths. Although a growing body of evidence indi-cates that tumor-induced lymphangiogenesis is a predictive indicator of metastasis to lymph nodes and might also be a target for prevention of metastasis, visualization of lymphatic vessels within the tissue in vivo has been challenging due to the limitations of current imaging techniques. Optical imaging of lym-phatic vessel has been attracting vast attentions since it does not involve any radiation and can achieve very high resolution. Here we introduce multi-functional optical coherence tomography (MFOCT) as a label-free technology for providing microstructural and microcirculatory imaging (both blood and lym-phatic vessels simultaneously). Using unique MFOCT-based approaches, we present a previously unex-plored in vivo longitudinal observation of angiogenesis, lymphangiogenesis and microstructural changes in the ear of the mice with spontaneously induced melanoma before metastasis. Quantitative analysis of density, diameter, and tortuosity of the skin blood and lymphatic vessels shows a significant change earli-er than tumor could be visually inspected or by measuring tumor thickness, thereby demonstrating the potential of MFOCT to help the early detection of the metastatic melanoma.

Contents
致謝 i
中文摘要 ii
Abstract iii
Contents iv
List of Figures vi
List of Tables xii
Chapter 1: Introduction - 1 -
1-1 Overview - 1 -
1-2 Motivation - 6 -
1-3 Thesis Structure - 7 -
Chapter 2: Optical Coherence Tomography - 8 -
2-1 Principle of OCT - 8 -
2-1-1 Time domain OCT - 11 -
2-1-2 Fourier domain OCT - 11 -
2-2 OCT Parameters - 13 -
2-2-1 Resolution - 13 -
2-2-2 Depth - 13 -
2-2-3 Sensitivity - 14 -
2-3 Functional OCT - 14 -
Chapter 3: Experimental Method - 16 -
3-1 MFOCT System - 16 -
3-1-1 System setup - 16 -
3-1-2 Scanning protocol and image acquisition process - 17 -
3-2 Animal Model and Experimental Procedure - 18 -
3-3 Image Processing Method - 20 -
3-3-1 Mean spectrum subtraction - 20 -
3-3-2 Dispersion compensation - 22 -
2-3-3 Region of interest (ROI) segmentation - 24 -
3-3-3 Angiography - 25 -
3-3-4 Lymphangiography - 28 -
3-4 Quantitative Analysis Method - 31 -
Chapter 4: Results - 33 -
4-1 MicroPET Images - 33 -
4-2 Microstructural Change - 34 -
4-3 Lymphangiogenesis and Angiogenesis - 39 -
4-4 Amelanotic Melanoma - 50 -
Chapter 5: Discussion - 53 -
Chapter 6: Conclusions and Prospects - 55 -
Reference - 57 -

List of Figures
Figure 1-1 Schematic of lymphatic circulation. [Image adapted from website: https://www.pinterest.co.uk/pin/376121006349908479/]. - 2 -
Figure 1-2 PET-CT after radical prostatectomy. CT images are in (D), PET images 60 min after the administration of 18F-choline are in (E) and merged PET-CT images are in (F). The high intensity spot in the PET scan is a small right inguinal lymph node with likely metastasis (F: transverse plane; L: saggital plane; A: coronal plane)[10] - 3 -
Figure 1-3 Coronal 3D MR lymphography image of the lower extremities obtained after subcutaneous injection of contrast material. Abnormal, dilated lymphatic vessels extend from the left calf to the inner thigh (small arrows). Some lymphatic vessels in the contralateral normal limb appear discontinuous (small arrows). Veins appear as linear structures with lower intensity (large arrows).[11] - 4 -
Figure 1-4 Near infrared imaging of healthy lymphatics in normal subjects. Lymphatic vessels in (G) hand, (H) arm, (I) foot, ankle, and leg, and (J) lower legs. Black spots are covered injection sites.[12] - 4 -
Figure 1-5 (b) OCT lymphangiography (c)cutaneous injection of Evan’s blue dye imaged by wide-field trans-illumination with a CCD camera. [16]…………..- 5 -
Figure 2-1 Schematic of a Michelson interferometer. - 9 -
Figure 2-2 The interference pattern shows up when the optical path length of the reference mirror matches one of the reflecting structures in the sample……..- 11 -
Figure 3-1 Schematic of MFOCT system. - 16 -
Figure 3-2 Output waveform for the (a)X-galvo, (b) Y-galvo and (c) data acquisition trigger - 18 -
Figure 3-3 Schematic of the experimental timeline. - 19 -
Figure 3-4 Photograph of the mouse ear. The scanned area was highlighted with white circles. - 20 -
Figure 3-5 MFOCT images processed (a) with and (b) without mean-spectrum subtraction. - 22 -
Figure 3-6 MFOCT images processed (a) with and (b) without dispersion compensation. - 24 -
Figure 3-7 Protocol for finding the upper boundary in ROI. - 24 -
Figure 3-8 MFOCT image with selected ROI (outlined in yellow). - 25 -
Figure 3-9 (A) The magnitude and absolute real part of the cross-correlation. (B) Example voxels are showing axial and lateral global fluctuations in the phase of OCT signals.[40] - 26 -
Figure 3-10 (a) Original MFOCT angiography and (b) MFOCT angiography processed by suppressing Fourier components on the vertical coordinate axes. - 28 -
Figure 3-11 The scattering signal along one A-scan within an MFOCT image of a mouse ear. - 28 -
Figure 3-12 MFOCT images processed (a) with and (b) without histogram equalization. - 29 -
Figure 3-13 MFOCT images processed (a) with and (b) without a bandpass filter. - 30 -
Figure 3-14 (a) Minimum intensity projection of MFOCT image, the selection was outlined in yellow, (b) segmented lymphatic vessel corresponding to the selection in (a) and (c) MFOCT lymphangiography. - 31 -
Figure 3-15 Flow chart of the image processing method. - 32 -
Figure 4-1 (a) Longitudinal 18 F-FDG MicroPET/CT images of 4-HT induced mouse and (b) Details of the square regions in the corresponding images in (a). The white arrow points to the cervical lymph node. - 33 -
Figure 4-2 Mean uptake values in cervical lymph nodes relative to leg versus time (N=3). Statistical significance was evaluated with Student t-test; *P < 0.05; N: number of mice. - 34 -
Figure 4-3 (a) Histologic sections and (b) corresponding MFOCT cross sectional images from 4-HT induced mouse ear at week 0, week 3, week 4 and week 6.(Scale bar = 1 mm) - 35 -
Figure 4-4 MFOCT cross-sectional image from (a) BRAFV600E control mice, and (b) 4-HT induced group. MFOCT 3D reconstruction image of (c) BRAFV600E control mice and 4-HT induced (d) mouse ear. - 35 -
Figure 4-5 (a) MFOCT en-face image and (b) corresponding photograph of the 4-HT induced mouse ear. - 36 -
Figure 4-6 (a) Photograph, and (b) the corresponding thickness map of 4-HT induced mouse ear at week 1, week 3 and week 4. White arrow on the photograph points to melanoma on the skin surface. - 37 -
Figure 4-7 Measured ear thickness at the 7 time points. Values are means ± standard deviation. Each time point was normalized to the initial value (week 0); N represents number of scanned areas. - 37 -
Figure 4-8 (a)Photograph and (b) corresponding thickness map of a 4-HT mouse ear. (Scale bar = 1mm) - 38 -
Figure 4-9 Measured ear thickness at the 4 time points. Values are means ± standard deviation. Each time point was normalized to the initial value (week 3). Statistical significance was evaluated with Student t-test; *P < 0.05; N represents number of scanned areas. - 38 -
Figure 4-10 Monitoring the angiography and lymphangiography within the 4-HT induced mouse ear. (a) photograph, (b) MFOCT lymphangiography, (c) MFOCT angiography and (d) MFOCT angiography merged with MFOCT lymphangiography. (Scale bar = 1mm) - 40 -
Figure 4-11 Monitoring the angiography and lymphangiography within the 4-HT induced mouse ear. (a) photograph, (b) MFOCT lymphangiography and (c) MFOCT angiography merged with MFOCT lymphangiography. (Scale bar = 1mm) - 41 -
Figure 4-12 Monitoring the angiography and lymphangiography within the 4-HT induced mouse ear. (a) photograph, (b) MFOCT angiography and (c) MFOCT lymphangiography. (Scale bar = 1mm) - 42 -
Figure 4-13 Monitoring the angiography and lymphangiography within the 4-HT induced mouse ear. (a) photograph, (b) MFOCT angiography and (c) MFOCT lymphangiography. (Scale bar = 1mm) - 42 -
Figure 4-14 Monitoring the angiography and lymphangiography within the 4-HT induced mouse ear. (a) photograph, (b) MFOCT angiography and (c) MFOCT lymphangiography. (Scale bar = 1mm) - 44 -
Figure 4-15 Monitoring the angiography and lymphangiography within the 4-HT induced mouse ear. (a) photograph, (b) MFOCT angiography and (c) MFOCT lymphangiography. (Scale bar = 1mm) - 45 -
Figure 4-16 Monitoring the angiography and lymphangiography within the BRAFV600E mouse ear. (a) photograph, (b) MFOCT angiography merged with MFOCT lymphangiography. (Scale bar = 1mm) - 47 -
Figure 4-17 Monitoring the angiography and lymphangiography within the 4-HT induced mouse ear. (a) photograph, (b) MFOCT angiography, (c) MFOCT lymphangiography and (d) MFOCT angiography merged with MFOCT lymphangiography. (Scale bar = 1mm) - 47 -
Figure 4-18 Quantitative measurement of (a) lymphatic and (b) blood vessel area density. Values are means ± standard deviation. Each time point was normalized to the initial value (week 0). Statistical significance was evaluated with Student t-test; *P < 0.05; **P<0.01. N represents number of scanned areas. - 48 -
Figure 4-19 Quantitative measurement of (a) lymphatic and (b) blood vessel area diameter. Values are means ± standard deviation. Each time point was normalized to the initial value (week 0). Statistical significance was evaluated with Student t-test; *P < 0.05; **P<0.01. N represents number of scanned areas. - 49 -
Figure 4-20 Quantitative measurement of (a) lymphatic and (b) blood vessel area tortuosity. Values are means ± standard deviation. Each time point was normalized to the initial value (week 0). Statistical significance was evaluated with Student t-test; *P < 0.05; **P<0.01. N represents number of scanned areas. - 49 -
Figure 4-21 Quantitative measurement of (a) lymphatic vessel density and (b) diameter. Values are means ± standard deviation. Each time point was normalized to the initial value (week 0). Statistical significance was evaluated with Student t-test; *P < 0.05; **P<0.01. N represents number of scanned areas. - 50 -
Figure 4-22 Monitoring the structural and vascular change within the 4-HT induced mouse ear. (a)Photograph, (b)MFOCT angiography merged with MFOCT lymphangiography, (c) density map of lymphatic vessel (d) diameter map of lymphatic vessel, (e) density map of blood vessel, (f) tortuosity map of blood vessel, and (g) thickness map of the 4-HT induced mouse ear. (Scale bar = 1mm)……………- 52 -
Figure 5-1 (a) Photograph, (b) MFOCT angiography and (c) MFOCT lymphangiography (d) MFOCT angiography merged with lymphangiography of the human arm………………………….- 56 -

List of Tables
Table 3-1 Specifications of the spectrometer - 17 -

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