Numerical calibration of the spatial overlap for subtraction microscopy

Abstract

Recently proposed subtraction microscopy provides a simple and compact approach to enhance the spatial resolution. However, inevitable imperfect overlap of the Gaussian and donut spots could deteriorate the quality of the subtracted images, while this effect is rarely investigated quantitatively. Based on vector diffraction theory, the effects of spatial displacements on the image resolution, signal intensity, and subtraction threshold are studied with several microscope parameters here. The peakposition shift is found to be a useful and convenient parameter to inspect and calibrate the spatial overlap of excitation spots in such microscopes. (C) 2015 Optical Society of America