Microglia are central to neuroimmune responses and undergo dynamic structural and functional changes in models of stress and addiction, and in response to pharmacological treatments. While transcriptomic and proteomic assays provide insights into molecular profiles, morphological analysis remains a valuable proxy for assessing region-specific microglial response. However, morphological features alone often fail to capture the full complexity of microglial function, underscoring the need for standardized methods and complementary approaches. Here, we describe a standardized imaging pipeline for analyzing microglia in the nucleus accumbens core (NAcore), integrating unbiased confocal image acquisition with precise anatomical reference points. We compare two widely used image analysis platforms—IMARIS and CellSelect-3DMorph—highlighting their workflows, output metrics, and utility in quantifying microglial morphology following treatment with adenosine triphosphate (ATP). Both tools detect well described features of microglial dynamics, though they differ in automation level, analysis speed, and output types. Our findings demonstrate that both platforms provide reliable morphological data, with CellSelect-3DMorph offering a rapid, open-access alternative for high-throughput analysis. Additionally, using software-derived parameters in principal component analysis clustering has proven useful for identifying distinct subpopulations of microglia separated by their morphology. This work provides a practical framework for morphological analysis and promotes reproducibility in microglial studies under environmental and pharmacological interventions.

Microglia are central to neuroimmune responses and undergo dynamic structural and functional changes in models of stress and addiction, and in response to pharmacological treatments. While transcriptomic and proteomic assays provide insights into molecular profiles, morphological analysis remains a valuable proxy for assessing region-specific microglial response. However, morphological features alone often fail to capture the full complexity of microglial function, underscoring the need for standardized methods and complementary approaches. Here, we describe a standardized imaging pipeline for ana-lyzing microglia in the nucleus accumbens core (NAcore), integrating unbiased confocal image acquisition with precise anatomical reference points. We compare two widely used image analysis platforms—IMARIS and CellSelect-3DMorph—highlighting their work-flows, output metrics, and utility in quantifying microglial morphology following treat-ment with adenosine triphosphate (ATP). Both tools detect well described features of mi-croglial dynamics, though they differ in automation level, analysis speed, and output types. Our findings demonstrate that both platforms provide reliable morphological data, with CellSelect-3DMorph offering a rapid, open-access alternative for high-throughput analysis. Additionally, using software-derived parameters in principal component analy-sis clustering has proven useful for identifying distinct subpopulations of microglia sepa-rated by their morphology. This work provides a practical framework for morphological analysis and promotes reproducibility in microglial studies under environmental and pharmacological interventions.