Contrast reversal confocal microscopy

We propose a novel method to improve the lateral resolution of a general confocal laser scanning microscopy (CLSM) by 10–35%, which can be readily realized by manipulating the shape of the excitation focal spot to a doughnut one. As the contrast of image is reversed synchronously, the raw data is further handled by the deconvolution algorithm to convert the doughnut point spread function (PSF) to a peak-centered distribution, so that a more intuitive vision will be given. Depending on the strategies like saturation excitation or doughnut size shrinkage, the resolution can be further optimized. This setup is valid for diverse systems and may find use in a variety of applications.