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Super-resolution fluorescence microscopy is an important tool in biomedicalresearch for its ability to discern features smaller than the diffractionlimit. However, due to its difficult implementation and high cost, theuniversal application of super-resolution microscopy is not feasible. In thispaper, we propose and demonstrate a new kind of super-resolution fluorescencemicroscopy that can be easily implemented and requires neither additionalhardware nor complex post-processing. The microscopy is based on the principleof stepwise optical saturation (SOS), where $M$ steps of raw fluorescenceimages are linearly combined to generate an image with a $\sqrt{M}$-foldincrease in resolution compared with conventional diffraction-limited images.For example, linearly combining (scaling and subtracting) two images obtainedat regular powers extends resolution by a factor of $1.4$ beyond thediffraction limit. The resolution improvement in SOS microscopy istheoretically infinite but practically is limited by the signal-to-noise ratio.We perform simulations and experimentally demonstrate super-resolutionmicroscopy with both one-photon (confocal) and multiphoton excitationfluorescence. We show that with the multiphoton modality, the SOS microscopycan provide super-resolution imaging deep in scattering samples.

Super-resolution fluorescence microscopy by stepwise optical saturation