Taborda-Bejarano Juan P, Nowak David B, Chaure Fernando, Allen Malika L, Blek Kathryn A, Walterhouse Stephen, Mantsch John R, Garcia-Keller Constanza
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Medical Scientist Training Program, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Cells. 2025 Aug 30;14(17):1354. doi: 10.3390/cells14171354.
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.
小胶质细胞在神经免疫反应中起核心作用，并且在应激和成瘾模型中以及对药物治疗的反应中会经历动态的结构和功能变化。虽然转录组学和蛋白质组学分析能深入了解分子概况，但形态学分析仍然是评估区域特异性小胶质细胞反应的重要指标。然而，仅靠形态学特征往往无法全面捕捉小胶质细胞功能的复杂性，这凸显了标准化方法和补充方法的必要性。在此，我们描述了一种用于分析伏隔核核心（NAcore）中小胶质细胞的标准化成像流程，将无偏共聚焦图像采集与精确的解剖参考点相结合。我们比较了两个广泛使用的图像分析平台——IMARIS和CellSelect-3DMorph——突出了它们的工作流程、输出指标以及在用三磷酸腺苷（ATP）处理后量化小胶质细胞形态方面的效用。这两种工具都能检测到小胶质细胞动态变化中描述清晰的特征，不过它们在自动化程度、分析速度和输出类型方面存在差异。我们的研究结果表明，这两个平台都能提供可靠的形态学数据，CellSelect-3DMorph为高通量分析提供了一种快速、开放获取的替代方法。此外，在主成分分析聚类中使用软件衍生参数已被证明有助于识别按形态区分的不同小胶质细胞亚群。这项工作为形态学分析提供了一个实用框架，并促进了环境和药物干预下小胶质细胞研究的可重复性。
