Rhoads, S. N.; Dong, W.; Hsu, C.-H.; Mfulama, N. R.; Ragusa, J. V.; Ye, M.; Henrie, A.; Zanellati, M. C.; Diering, G. H.; Cohen, T. J.; Cohen, S.

Neurons and astrocytes play critical yet divergent roles in brain physiology and neurological conditions. Intracellular organelles are integral to cellular function. However, an in-depth characterization of organelles in live brain cells has not been performed. Here, we used multispectral imaging to simultaneously visualize six organelles - endoplasmic reticulum (ER), lysosomes, mitochondria, peroxisomes, Golgi, and lipid droplets - in live primary rodent neurons and astrocytes. We generated a dataset of 173 Z-stack and 99 time-lapse images accompanied by quantitative analysis of 1418 metrics (the \"organelle signature\"). Comparative analysis revealed clear cell-type speci[fi]city in organelle morphology and interactions. Neurons were characterized by prominent mitochondrial composition and interactions, while astrocytes contained more lysosomes and lipid droplet interactions. Additionally, neurons displayed a more robust organelle response than astrocytes to acute oxidative or ER stress. Our data provide a systems-level characterization of neuron and astrocyte organelles that can be a reference for understanding cell-type-speci[fi]c physiology and disease.