Numerical study of the subtraction threshold for fluorescence difference microscopy

Abstract

Subtraction microscopy has recently been promoted for its compactness and simplicity of enhancing spatial resolution. However, until now, the subtraction factors used in such microscopes to process raw images have been chosen experientially, and it has been impossible to determine whether the resolved structures after subtraction are appropriate or over-processed. Based on vector diffraction theory and two-dimensional convolution, this paper presents numerical investigations of the parameters that may offer the selection criterion of subtraction factors used in subtraction microscopy. It proposes two essential parameters for appropriately evaluating the subtraction factor: the fluorescence peak intensity after subtraction and resolution derivative. (C) 2014 Optical Society of America