Image based phenotype analysis methods, such as the widely used Cell Painting analysis, use high connotation imaging and multi parameter outputs to study biological, genetic, and chemical disturbances in cells. This increasingly popular method is being applied to everything from drug discovery projects to genome screening research. In standard Cell Painting experiments, various organelles and structures (nucleus, cytoplasm, mitochondria, Golgi apparatus, endoplasmic reticulum, cytoskeleton, cytoplasmic RNA, and nucleolus) are collected in five imaging channels. This extensive cell staining can monitor and quantify various forms of disturbances at single-cell resolution. The limitations of published Cell Painting analyses include limited availability of suitable dyes and imaging systems providing sufficient spectral separation capabilities. Specifically, the Golgi apparatus and actin are imaged through the same channel, which poses a challenge for distinguishing these two structures in image analysis. This restriction may interfere with the selection of hits or mask the effects of certain compounds, especially those with mechanisms of action (MOA) that affect the Golgi apparatus (biosynthetic pathway) or cytoskeleton network.