胰島素金奈米粒子螢光耗乏特性之探討

Abstract

過去十年，能克服光波繞射極限(~λ/2)之超解析遠場螢光顯微術，因其於生物醫學研究上，可達到觀察細胞內部細微結構或蛋白質分佈至數十奈米的解析度，而成為熱門議題。在多種超解析螢光顯微術中，我們選擇研究受激放射耗乏顯微術(stimulated emission depletion microscopy, STED microscopy), 因為其容易與共軛焦雷射掃描顯微鏡(confocal laser scanning microscope)結合，並提供快速掃描與光學切片能力，在活體觀察及3D奈米成像方面皆有許多方面的應用。透過兩道雷射並利用受激放射原理對螢光分子做光操縱，以達到超解析成像的受激放射耗乏顯微術，目前已可達僅僅只有數十奈米的解析度，是活體神經突觸觀察與研究的良好工具。 執行受激放射耗乏顯微術時，我們需要一道實心的激發光去激發樣本，以及另一道強度分佈為甜甜圈形狀的耗乏光將中心之外的自發螢光耗乏掉。根據理論計算的結果，若所施加的耗乏光強度越高，光學解析度就能提高越多，然而，在此條件下，我們所用的螢光標定物便需具備絕佳的光穩定性以承受強烈雷射照射。此外，若所搭配的螢光標定物容易被激發及耗乏，也能使光學解析度有效提高。因此，檢驗材料之光穩定性及其螢光是否易被耗乏，便成為我們的目標。 目前已知有些金屬奈米粒子(metallic nanoclusters)可發螢光且比起一般有機染劑較少光漂白(photobleach). 然而，目前並無文獻探討螢光金屬奈米粒子於受激放射耗乏顯微術中之應用潛力，且考慮到此螢光標定物需能配合生醫相關研究，因此我們選擇了胰島素金奈米粒子(insulin-gold nanoclusters, IGNCs)作為我們的研究材料。 此論文中，我們檢測IGNCs，其受激放射耗乏特性、光穩定性，以及評估將IGNCs應用於受激放射耗乏顯微術中之應用潛力。 我們發現IGNCs在連續激發、耗乏過程中，其所呈現之光穩定性，以20%的光漂白(photobleach)為標準，IGNCs於105 W/cm2之激發光、109 W/cm2之耗乏光下花費約120秒，而常用於受激放射耗乏顯微術之染劑Atto565於101 W/cm2之激發光下大約花費210秒，考慮激發強度不同之下，一般掃描情況下，IGNCs呈現良好之光穩定性。同時，我們也檢測，被激發的IGNCs, 在耗乏光作用下，有螢光明顯被耗乏的效應發生，但耗乏效率僅可達57%, 而其飽和強度(saturation intensity, Is)約為2.75 GW/cm2, 比一般螢光標定物的Is (~ 106 - 107 W/cm2)高出2 - 3個數量級。深入探討其原因，是由於此種材料被激發後，電子會做系統間跨越(intersystem crossing, ISC)以及逆系統間跨越(reverse intersystem crossing, RISC)之躍遷機制，而導致螢光不易被耗乏，有較高之Is值。為了能更進一步降低耗乏時的雷射功率以及降低螢光分子的光漂白，我們使用時間閘偵測(time-gated detection)技術以及降低雷射脈衝重複率，進一步提升14%之螢光耗乏效率，並降低對螢光分子的破壞。 由於IGNCs的Is值與一般螢光標定物相比很大，螢光不易被耗乏，因此我們認為它不適合用於受激放射耗乏顯微術中，除非搭配良好的時間閘偵測技術以及將脈衝寬度、間距、波長最佳化。但我們相信，IGNCs在其它螢光操縱應用中或生物醫學研究領域，尤其是與胰島素相關的疾病研究中，應該仍能有所表現。

Super-resolution far-field fluorescence microscopy, which overcomes the diffraction limit (~ λ/2), has gotten much interest in the last decade due to its nanoscale resolving ability of observing fine structures or protein distribution in cells. Among super-resolution fluorescence techniques, we study stimulated emission depletion (STED) microscopy because it can easily be combined with confocal laser scanning microscope, then provides not only fast imaging and optical sectioning ability, but also 3D nanoscale in vivo observation. By using two laser beams and the mechanism of STED to optically control the fluorescence of fluorophores, STED microscopy can currently achieve resolution with only tens of nanometer, and become one of the best tools in revealing the morphology of dendritic spines and synapse. During the process of implementing STED microscopy, we need one laser beam (called excitation beam) to excite the sample, and the other beam (called depletion beam) with doughnut shaped intensity distribution to deplete the excited region except the center. According to the theory of STED microscopy, with great depletion intensity, comes great image resolution. However, in this condition, fluorescence markers should be good in optical stability and be capable of sustaining high power laser illumination. In addition, it would also lead to high resolution when fluorescence markers is easy to be excited and depleted. It is known that some metallic nanoclusters (NCs) have strong fluorescence and less photobleach than organic dyes. However, there is no report discussing the application potential of metallic NCs in STED microscopy. To facilitate applications to biomedical studies, we chose insulin-gold nanoclusters (IGNCs) as our sample and investigated the optical properties of IGNCs, such as its property of STED, optical stability, and evaluating its application potential in STED microscopy. We examine the optical stability of IGNCs in the process of continuous excitation and depletion. It took about 120 seconds for IGNCs to reach 20 % photobleach with 105 W/cm2 excitation and 109 W/cm2 depletion. Its stability is good and comparable to that of Atto565 (about 210 seconds for 20 % photobleach with 101 W/cm2 excitation) when considering the excitation intensity. We also found that under the illumination of depletion beam, an apparent fluorescence depletion phenomenon occurred in the excited IGNCs film. However, the fluorescence depletion efficiency can reach only 57 %, and the saturation intensity (Is) of IGNCs is 2.75 GW/cm2, which is much larger than that of common fluorescence dyes (~ 106 - 107 W/cm2). After studying the reason in detail, we found that the low depletion efficiency and high Is can be explained by the mechanism of intersystem crossing (ISC) and reverse intersystem crossing (RISC) in the process of electron transition. In order to increase depletion efficiency and decrease photobleach of IGNCs, we adopted time-gated detection technique and used low laser pulse repetition rate. The results showed 14 % improvement in depletion efficiency and less damage than previous one. With large Is of IGNC, its fluorescence is not easy to be depleted, so we think that IGNC is not suitable for STED microscopy except combining outstanding time-gated detection technique and optimizing the used width, delayed time, wavelength of pulses.