飽和激發超解析顯微鏡之理論極限之研究

於過去的十幾年,光學顯微鏡的解析度已經進步到,超越傳統所認知的阿貝繞射極限,到達了新的領域-超解析顯微鏡. 此項技術已被應用在各種不同的領域且得到相當不錯的結果,像是結構生物學和神經科學. 而不論分子還是細胞,讓其在原本的環境下做觀察都是最好的方式,因此組織影像是光學顯微鏡相當側重的一個觀察領域. 然而目前大部分的超解析顯微鏡無法在穿過組織觀察時,同時保持其解析度. 由目前的超解析技術而言,當觀察深度到達一百微米時,就會受到組織本身的散射以及像差等光學性質所影響,而失去其超解析度. 於許多不同種類的超解析顯微鏡技術當中,飽和激發超解析顯微鏡 (於以下稱作SAX)是一項相當有潛力達成深組織超解析影像的技術. 原因在於SAX是利用時間上的調製來擷取發光源的非線性信號來達到超解析解析度,而時間上的調製又比較不會受到散射等組織的光學性質所影響. 然而傳統的以螢光為發光源的SAX,受到低訊雜比的影響,所能提供的最高解析度僅能到達λ⁄6. 為了提升SAX的解析度, 一項我們過去所做的金奈米粒子為發光源的SAX實驗給出了一個突破口. 於相同條件下,那項實驗提供了λ⁄8的解析度. 二者實驗最大的不同在於金奈米粒子的散射非線性比傳統螢光的飽和非線性來的大. 跟隨那項實驗所給出的概念,於此項研究中,我們建立了一個SAX的模擬程序,並用其來測試不同的非線性響應來找出哪種非線性響應配合SAX可以得到最好的解析度.於結果上來說,我們發現若有適合的非線性響應,SAX的解析度可以達到λ⁄50. 此外,於實驗上,我們提出了一個可能適合的非線性材料來配合SAX來得到更高的解析度.

關鍵字

超解析顯微鏡；深組織影像；飽和激發顯微鏡

並列摘要

In the last decade, resolution of optical microscopy has been ameliorated to surpass the Abbe diffraction limit, which is ~λ⁄2, enabling super-resolution observations. It has already made significant impacts in various fields, such as structural biology and neuroscience. Since molecules and cells should be studied under their own native microenvironments, tissue imaging is one vital requirement for optical imaging. However, most current super-resolution techniques cannot maintain their resolution improvement beyond 100µm depth inside a bio-tissue due to intrinsic scattering and aberration. Among the various super-resolution imaging methods, saturated excitation (SAX) microscopy provides great potential for deep tissue imaging. The reason is that SAX enhances spatial resolution by extracting emission nonlinearity with temporal modulation, which is less affected through tissue. Nevertheless, current resolution of fluorescence-based SAX microscopy rarely exceeds λ⁄6, due to inadequate signal-to-noise ratio. To further enhance SAX resolution, a hint comes from our previous study that λ⁄8 resolution was obtained by replacing the nonlinear emitter from fluorophores to Au nanoparticles, which the latter exhibits larger nonlinearity than the former. Following the concept of enhancing resolution by increasing nonlinearity, in this work, we build a SAX simulator and test it with different nonlinear responses to elucidate an optimized condition that SAX resolution reaches ~ λ⁄50. In addition, a possible nanomaterial candidate with huge nonlinearity is found, showing the feasibility of ultrahigh spatial resolution of SAX.

並列關鍵字

super-resolution microscopy ； deep tissue image ； saturated excitation microscopy

參考文獻

[1] Hooke, R. Micrographia: or some physiological descriptions of minute bodies made by magnifying glasses. Jo. Martyn and Ja. Allestry for the Royal Society, (1665).

[2] Egerton, F. N. A History of the Ecological Sciences, Part 19: Leeuwenhoek's Microscopic Natural History., Bulletin of the Ecological Society of America, (2006).

[3] Abbe, E. K. Beiträge zur Theori des Mikroskops und der mikroskopischen Wahrnehmung., Archiv fur mikroskopische Anatomy, (1873).

[4] Egner, A., Jakobs, S. & Hell, S. W. Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast. Proceedings of the National Academy of Sciences 99, 3370-3375 (2002).

[5] Gustafsson, M. G. L., Agard, D. A. & Sedat, J. W. in IS&T/SPIE's Symposium on Electronic Imaging: Science and Technology. 10 (SPIE).

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飽和激發超解析顯微鏡之理論極限之研究

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