以共軛焦顯微鏡觀察介電質微米球之光學超解析特性之研究與應用

Abstract

自光學顯微鏡被發明以來，就被廣泛的運用在物理、化學、醫學等各項領域中。然而由於光本身的繞射特性，使得光學解析度存在著最小的極限，及為著名的繞射極限。這使得光學顯微鏡無法用於觀察奈米等級的微觀世界。為了生物醫學上的需要，在過往的十幾年內，光學超解析(optical super-resolution)顯微技術被蓬勃的發展。然而，雖然這些技術已在實驗上獲得顯著的成效，但由於這些技術本身工作原理，大多技術都需要高強度的雷射照射以及螢光特性的樣本。使得這些技術在使用上面臨著重重的限制。 近年來，微米球透鏡白光超解析現象的發現，使得人們可以用更為簡單的製程直接藉由可見光觀察奈米尺度的微觀世界。然而，此項技術依然存在著幾項缺點。例如影像的光學對比度很差，光學超解析影像的視野範圍被微米球大小限制，以及未知機制使在這項技術在應用上顯得左支右絀。雖然隨之發現直徑稍大的微米球依然也能夠提供超解析影像，使得視野範圍提升了不少，然而依然還是受限於微米球的尺度。 在我們的工作中，嘗試著對以上三項缺點作研究與改進。對於光學對比度而言，我們結合微米球透鏡與共軛焦顯微鏡(confocal microscopy)改善其光學度比度的問題。並藉由微米球成像的特性，發現了一種新的簡易的方法，使得微米球透鏡可以自由的移動，如此微米球的視野範圍就不再受到尺寸的限制。最後，我們也列舉了幾種可能的微米球成像機制，並利用實驗的結果對於這些機制作一一的討論。希望能夠進一步的瞭解其成像的機制。

Optical microscopy has made significant impacts in lots of fields since its invention, especially in biology and material sciences. Due to the diffractive nature of light, the resolution of optical microscopy is constrained to about half wavelength, known as Abbe limit. To conquer this limit, some ground-breaking techniques have been demonstrated in the past decades, including far-field photo-activated localization microscopy (PALM), stimulated emission depletion (STED) microscopy and so on. However, for most of these super-resolution techniques, fluorescent samples are required, and high laser intensities are necessary to achieve higher resolution. Recently, a simple method has been demonstrated to break diffraction limit by adopting a microsphere lens under a microscope objective. 50-nm resolution was claimed by this microsphere-assisted imaging. Comparing to the other super-resolution techniques, the microsphere technique is free from constraints of fluorescence, high intensity, and surface flatness. However, it still has some constrains in application, such as bad optical contrast, small super-resolution field of view and unknown mechanism. It is known that confocal microscopy provides higher optical contrast and spatial resolution than bright-field microscopy. In this work, we combined confocal microscopy with microsphere lens, and demonstrated super-resolution confocal microscopy without the need of fluorescence. And then we make a moveable microsphere film with PMMA, a thermoset material, so that it provides unlimited field of view by moving the microsphere. At last we discuss some possible mechanism with our experiment result, and exclude some theory. Our technique not only can be implemented easily in common confocal systems, but will greatly augment the observation of biological cells and material science.