Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

过氧化物酶体是真核细胞器，对于多种代谢途径至关重要，包括脂肪酸氧化、氨基酸降解和醚脂生物合成。因此，过氧化物酶体功能障碍会导致儿童发病的神经退行性疾病，包括过氧化物酶体生物发生障碍（PBD）。由于其生物发生和功能的动态性、组织特异性和背景依赖性，过氧化物酶体的活细胞成像对于研究过氧化物酶体调节以及PBD相关异常的诊断至关重要。然而，过氧化物酶体成像工具包在许多方面都存在缺陷，没有用于底物导入的报告基因，也没有可以对功能失调的过氧化物酶体进行染色的细胞渗透性探针。在这里，我们报告 BODIPY-C12 荧光脂肪酸探针对活哺乳动物细胞中功能性和功能性过氧化物酶体进行染色。然后我们继续改进 BODIPY-C12，生成过氧化物酶体特异性试剂 PeroxiSPY650 和 PeroxiSPY555。这些探针结合了高过氧化物酶体特异性、红色和远红光谱中的明亮荧光以及快速非细胞毒性染色，使其成为过氧化物酶体活细胞、整个生物体或组织成像的理想工具。最后，我们证明 PeroxiSPY 能够诊断 PBD CRISPR/Cas9 细胞模型和患者来源的细胞系中的过氧化物酶体异常。