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7-2. Theoretical Simulation of the effective optical field of AuOA sheet
In Chapter 4, the intensity of electric field excited on the surface of AuOA sheet is simulated using the finite difference time domain (FDTD) method. The optical field calculation was performed to the perpendicular direction from the center position of the adjacent particles.
The LSPR field intensity, which was 50 times stronger at the maximum point, decayed drastically along the distance from the interface and became less than the original light intensity at 13 nm position. This result indicates that the fluorescence enhancement by LSPR could occur only less than 13 nm region in the calculation.
The energy transfer from the dye to the AuOA sheet was considered as a surface energy transfer (SET). The intensity of ‘effective’ electric field excited on AuOA sheet was calculated by multiplying the intensity of electric field and efficiency of SET. Regardless of the SET distance, the intensity of effective electric field had a maximum at the inside of the AuOA sheet and the enhancement factor was estimated to be about 10. According to the FDTD simulation, the effective LSPR field intensity on the AuOA sheet exceeds the intensity of the evanescent light at 13 nm position from the interface. In other words, high contrast imaging of nanointerface is possible by using the enhanced fluorescence from the nanointerface (~10 nm region) and low background light on the AuOA sheet. AuOA sheet enable nanointerfacial imaging with higher signal/noise ratio and observation with higher axial resolution compared with the TIFR microscope.
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7-3. Observation of cell-attached interface with AuOA sheet
Chapter 5 presents the static images of fluorescent beads and immobilized RBL-2H3 cells using AuOA sheet as an imaging substrate under a TIRF microscope. The images obtained on AuOA sheet were directly compared with the regular TIRF images by using a glass substrate half-covered by AuOA sheet. The detailed structure of focal adhesion at the cell-attached interface was clearly visualized on the AuOA sheet, while these were unclear at the regular TIRF image (on glass substrate) screened by the sheet-like actin layer on top.
Although the illumination depth must be shorter on the AuOA sheet than on glass (~1/10 in calculation), the emission intensity was eventually comparable with the aid of LSPR-enhanced fluorescence. The experiments of fluorescence beads revealed that fluorescence intensity of the beads was maximized on AuOA sheet covered with 20 nm of SiO2 layer. This must be phenomenon related to the SET distance discussed in the previous chapter. Here again the illumination depth on the AuOA sheet was much shorter than the TIRF microscope, however, the emission intensity was comparable with that on glass due to the LSPR-enhanced fluorescence. Although the FWHM values of the bead images were the same for both on AuOA sheet and on glass, the foot section of the profile exhibited small differences between them. The width at 10% of the maximum intensity was 12 pixels (780 nm) on glass and 8-9 pixels (520-585 nm) on the AuOA sheet, which suggest the slight improvement of even lateral resolution on AuOA sheet.
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7-4. Cell dynamics imaging with AuOA sheet
Chapter 6 presents the results of live-cell imaging using AuOA sheet as an imaging substrate. We propose new methodology to observe dynamics of the dye-labeled protein at the cell-attached interface in high-speed and high axial resolution due to the LSPR-mediated light enhancement and confinement effects.
Using a confocal laser microscope, venus-paxillin-labeled 3T3 live-cells were observed for time-lapse video images. The cells attached on AuOA exhibited the spreading and elongaed form in the same way as that on glass. The difference between them was photobleaching time. The fluorescence of the cells on glass was photobleached rapidly and the imaging was not possible in 6 hours. On the other hand, the fluorescence of the cells on AuOA sheet was more sustainable and the photobleaching to the same level took about 9 hours. The result indicates an effect of suppressing photobleaching on AuOA sheet.
High speed time-lapse imaging under TIRF microscope was acquired with exposure time of 500 ms and the interval of 500 ms to monitor the fast cell dynamics. We could not observe the large scale of movement of focal adhesions in such short-term observation, while we could catch the movement of the small immature focal adhesions. By using the displacement of bright spots corresponding to a few pixels, the migration rate of paxillin in the cell was estimated to be ca. 1 µm/min. This rate is faster than the 3T3 cell migration rate of 0.19 µm/min but slower than the diffusion rate of proteins in the cell membrane, 140 µm/min. This result suggest that observed paxillin are aggregated and created a domain inside of the cell or observed phenomenon is not a simple diffusion of proteins rather including the vertical movement enhanced by the light confinement effect by LSPR.
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7-5. Prospects for the future
In this doctoral thesis, the necessity and importance of super-resolution fluorescence observation techniques were described. The LSPR-mediated fluorescence imaging techniques using AuOA sheet, which is the topics of this doctoral thesis, is expected to be applied to biological research and disease diagnosis. However, still some unsolved problems are remaining in this new technology.
It has already been confirmed that axial resolution is improved by using AuOA sheet.
However, the exact distance of observation area has not been discussed quantitatively yet.
Although the distance dependent enhanced electric field on AuOA sheet was calculated by FDTD simulation, this is just a qualitative tendency and cannot determine their absolute value by such a simple calculation. In order to discuss the movement of small molecules in the cell attached nanointerface, especially to solve the essential problem in the field of molecular biology, it is necessary to clarify the influence of the axial distance to the brightness quantitatively,
In addition, further investigations are needed to clarify the influence on the lateral resolution for the image on AuOA sheets. In this doctoral thesis, cameras with pixel resolutions of 128 nm and 65 nm were used for observation. The image obtained with 65 nm pixel resolution camera confirmed more detailed structure than the image obtained with 128 nm pixel resolution camera, i.e., the resolution of the original image on AuOA sheet seems to be far better than the diffraction limits. We may be able to obtain even better resolution image with high axial and lateral direction by adjusting the size and shape of nanoparticles constituting the 2D sheet as the observation substrate.
Image analysis technology using AI and deep learning has attracted attention in recent years. By analyzing a large number of images for cell-dynamics, it is possible to predict cell differentiation or to diagnose a disease. Since enormous number of images are necessary
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to be used for detecting biological phenomena occurring in high speed and continuing long time, observation technique with high time resolution and analysis using AI techniques are required. LSPR-mediated fluorescence imaging using metal nanoparticle sheet, proposed in this doctoral thesis, provides high-resolution and high-speed images without the scanning or image reconstruction. Combination of high spatial-temporal resolution images acquired by this method with AI and deep learning may lead to the technological innovation for applied biological research and medical diagnosis.
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Acknowledgment
本研究を進めるにあたり、熱心なご指導と多大なご助言を賜りました指導教官の本学先導物質化 学研究所の玉田薫 教授に感謝いたします。また、日常の議論を通じて多くの知識や示唆をいただ いた本学先導物質化学研究所の有馬祐介 准教授、龍崎奏 助教、大阪府立大学工学研究科の 岡本晃一 教授に感謝いたします。
日々の実験において技術的な指導とご助言を賜り、研究環境の面からも支えてくださいました公益 財団法人九州先端科学技術研究所の王胖胖 博士、広島大学 柳瀬雄輝 博士、本学先導物質 化学研究所の木戸秋悟 教授、久保木タッサニーヤ－ 助教、名古屋大学の臼倉英治 博士、大 分工業高等専門学校の田中大輔 講師に感謝いたします。
研究の指導、慣れない海外での生活を支えてくださいました、ドイツ ハイデルベルク大学の田中 求 教授に感謝いたします。
分子システムデバイスリーディングプログラムの活動を支えてくださったリーディング支援室の皆様 に感謝いたします。
研究室生活において、秘書として研究室を支えてくださいました、上原つくみ 氏、瓜生めぐみ 氏 に感謝いたします。研究室内での学生生活、研究生活の両方の面から支えてくださいました相田裕 輝子 氏に感謝いたします。
研究室の先輩、同期、後輩として様々な面で支えてくださいました、篠原修平 氏、立石和隆 氏、石島歩 氏、川元駿 氏、斎藤昴 氏、寺田開生 氏、荒木祥平 氏、西田知句 氏、田子森 恭平 氏、冷俊夫 氏、大藏孝太 氏、竹熊晴香 氏、長尾俊範 氏、松田倫太郎 氏、三宅雄一 郎 氏、周子尭 氏、碓木凌 氏、杉原諒一 氏、磯野晃太朗 氏、金城信哉 氏、西島孝 氏、萩 尾蓮 氏に深く感謝申し上げます。
最後になりましたが、いつも温かく見守っていただき、博士課程への進学の機会を与えてくださった 家族に心より感謝いたします。