共時無色相差雙色擴展超解析顯微技術

Abstract

超解析顯微技術（super-resolution microscopy）作為光學顯微技術領域裡的一類重要技術，能夠讓科研人員打破繞射極限的約束，在極微小的尺度上進行細胞或分子領域的研究。超解析顯微技術發展至今在各種基本方法中衍生出了諸多不同的技術，在解析度或是易用性上都已經有了程度不一的改良。其中，單分子定位顯微鏡（SMLM）更是能夠在解析度上達到十至二十奈米的卓越尺度。在當前的方法研究中，更多的目光被聚焦在了不同類別技術和概念的整合上，以彌補各項技術中存在的性能缺陷並且進一步提升成像性能。多色顯微影像在超解析影像，乃至普通的光學顯微影像中都是十分常見以及被大量使用的工具，在單分子定位顯微技術中的使用也對醫學及生物中的分子共定位研究起到了很大的推動。目前，對於實現多色單分子影像已經有多個方法被提出，而其中大部分仍然使用不同波長的鐳射進行激發，以多個波段進行熒光訊號的接受，如此的實現方法會在系統中無可避免地帶來色相差，對於十奈米甚至十奈米以下的定位準確性造成巨大幹擾。 本研究提出了一項共時無色相差雙色擴展超解析顯微方法，將單一波長激發，單一波段接收訊號並實現雙色影像的方法引入EX-dSTORM (Expansion Direct Stochastic Optical Reconstruction Microscopy)這一強大的混合超解析技術，從而在物理上徹底消除色相差的存在，並且大大簡化拍攝傳統雙色EX-dSTORM影像的流程。通過對特定熒光染料光動力學特征甄別，能夠在原有單色影像光學組件下實現雙色單分子影像，結合擴展顯微技術帶來的樣品物理上的放大，這項方法能夠在盡可能消除人為引入誤差的狀況下達到十奈米的影像解析度。

Super-resolution microscopy, as an important technological breakthrough in optical microscopy, enables researchers to overcome the diffraction limit and conduct studies at extremely small scales in cellular or molecular domains. Over time, super-resolution microscopy has evolved into various techniques with different improvements in resolution and ease of use. Among these, single molecule localization microscopy (SMLM) stands out for achieving resolutions of 10 to 20 nm. Current research increasingly focuses on integrating different techniques and concepts to address existing limitations and further enhance imaging performance. Multicolor microscopy imaging is a common and widely used tool in both super-resolution and conventional optical microscopy, significantly aiding molecular co-localization studies in biology and medicine. Several methods have been proposed to achieve multicolor single-molecule imaging, most of which still rely on multiple lasers in different wavelengths for excitation and multiple bands for fluorescence signal reception. This approach inevitably introduces chromatic aberration into the imaging system, significantly impacting localization accuracy, especially at resolutions of 10 nanometers or below. This study introduces a simultaneous chromatic aberration-free dual-color expansion single molecule localization method. It integrates the single-wavelength excitation and single-band signal reception approach into EX-dSTORM (Expansion Direct Stochastic Optical Reconstruction Microscopy), a powerful super-resolution microscopy technique. This unique method physically eliminates chromatic aberration and significantly simplifies the conventional process of acquiring dual-color EX-dSTORM images. By identifying specific photo-switching kinetic characteristics of fluorescent dyes, dual-color single-molecule imaging can be achieved using the existing monochromatic optical setups without changing components. Combined with the physical magnification brought by expansion microscopy, this approach can achieve an image resolution of 10 nanometers while minimizing artificially introduced errors.