The methods and systems disclosed herein include obtaining a first plurality of images of a sample, where each image in the first plurality of images corresponds to a different spectral band of illumination light incident on the sample or emission light from the sample, obtaining a second plurality of images of the sample, where each image in the second plurality of images corresponds to a different spectral band of illumination light incident on the sample or emission light from the sample, aligning the first and second pluralities of images based on information from a first image from the first plurality of images and a second image from the second plurality of images, where the first and second images correspond to a shared spectral band, and combining at least some members of the first plurality of images and at least some members of the second plurality of images to form an image stack.