Subtraction threshold for an isotropic fluorescence emission difference microscope

Isotropic fluorescence emission difference microscopy proposed recently provides a simple method to enhance the spatial resolution in three-dimensions (3D) for fluorescence imaging. However, the subtraction threshold to achieve the condition for appropriately resolving the sample in 3D have not been studied. Then the subtraction factors used in this type of microscopes are still experientially chosen. Based on vector diffraction theory and a 3D numerical model developed here, the subtraction threshold is numerically investigated for the isotropic fluorescence subtraction microscopy. The subtraction factors and peak intensities at the threshold are obtained and comparied both in lateral and axial planes for achieving most appropriate subtraction and inspecting the isotropic characteristic. The effects of radius ratios of implemented 0-π annular phase plate for generating three dimensional donut spot on the subtracted resolution, peak intensity and negative sidebands are also discussed. (paper)