Systems and methods of improving segmentation and classification in multi-channel images of biological specimens are described herein. Image channels may be arranged and processed sequentially, with the first image channel being processed from an unmodified image, and subsequent images processed from attenuated images that remove features of previous segmentations. For each segmented image channel in sequence, a binary image mask of the image channel may be created. A distance transform image may be computed from the binary mask. Attenuation images computed for all previous channels may be combined with a current attenuation image to create a combined attenuation image. The next image channel in the sequence may then be attenuated to produce an attenuated next image channel, which may then be segmented. The steps can be repeated until all image channels have been segmented.