现有的光学超分辨显微成像技术主要依赖于特殊的荧光标记物,其对于大多数非荧光样品的超分辨成像就变得无能为力。因此我们提出将光学相减显微技术应用到非荧光样品的成像当中,利用普通共聚焦光斑和面包圈型光斑分别激发样品的散射光成像,从而得到样品同一区域的两幅图像,再通过图像相减的方法提高了图像空间分辨率。不同于一般的超分辨成像方法,这种光学相减显微镜不需要特殊的样品预处理过程,同时两次成像的激发光强度可以保持在一个较低水平,避免了样品损伤的影响。随后金纳米小球和有机聚合物微丝的散射成像实验证明了光学相减显微镜可以将空间分辨率提高到215 nm (0.33λ, 1λ = 650 nm),并且通过探测散射信号得到更多的样品细节信息。
The existing form of super-resolution microscopy based on specific fluorescent tagging is unable to obtain super-resolution images of non-fluorescent samples. Hence, we have developed optical subtraction microscopy for obtaining super-resolution imaging in such cases. This method is based on image subtraction between the two optical scattering images from general confocal excitation and doughnut-shaped excitation, respectively. Unlike super-resolution fluorescence microscopy, subtraction microscopy requires no preprocessing of the sample, and the excitation power can be kept low to avoid sample damage. The non-fluorescent imaging of gold nanobeads and polymer nanofibers has been realized to demonstrate the feasibility of super-resolution subtraction microscopy. The lateral resolution decreases to 215 nm (0.33λ, 1λ = 650 nm) in subtraction imaging, and greater imaging detail of the sample is achieved via optical scattering