Systems and methods for flattening the image across the entire field by correcting the image for both the fluorescence and scatter spatial variations. Images of a stable fluorescence target and a scattering target are separately acquired in an imaging system. From these target images, a pixel remapping function, e.g., including two correcting pixel slopes, is calculated for subsequent image pixel remapping. An image of a sample under investigation is then acquired by the imaging system and the sample image is remapped based on the pixel remapping function for the imaging system to form a corrected (field flattened) image. Which correction pixel slope to be used is determined based on whether a sample image pixel value is higher or lower than a threshold value.