Enhancing the resolution and contrast in CW-STED microscopy

A new method based on the subtraction of images is proposed to enhance the resolution and contrast in CW-STED microscopy. It is realized by the subtraction of a confocal image from a STED image. Detailed theoretical analyses and a series of simulation tests are performed. The validity of this subtraction method in practice is demonstrated by experiments on both fluorescent nanoparticles and microtubule networks in which a better resolution and contrast than the STED image is achieved. Subtractive parameters are discussed in detail and a way of choosing the optimal value is proposed. Compared with other resolution enhancement concepts, this method is much simpler and free from using high intensity lasers.