采用表达嵌合抗原受体（CAR）的T细胞的移植转移已经显示出对B细胞恶性肿瘤有显著的临床疗效。然而，CAR T细胞领域目前正在面临着几个主要的挑战。特别是，当针对实体肿瘤时，CAR T细胞策略尚未产生有利的临床反应。在这种情况下，了解限制T细胞免疫治疗效果的决定因素具有至关重要的意义。对CAR T细胞的表征通常基于流式细胞术和全转录组分析。这些方法对于确定调节T细胞增殖和扩张能力的固有因素非常有价值。但是，它们没有考虑到T细胞反应的空间和动力学方面。特别是，为了控制肿瘤生长，CAR T细胞需要进入肿瘤，迁移到复杂的肿瘤环境中，并与其靶标形成有益的结合体。先进的成像技术结合创新的临床前模型是揭示CAR T细胞动态的有希望的工具。在本综述中，我们将讨论使用实时成像显微镜获得的改造T细胞的生物学最新结果。这些基于成像的研究已经出现了一些重要的概念，例如CAR T细胞的多杀伤潜力。最后，我们将强调如何将成像技术与其他工具结合起来解决在改造T细胞领域中尚未解决的问题。 © 2023年，作者在Springer-Verlag GmbH Germany，Springer Nature的独家许可下发布。

Adoptive transfer of T-cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies. Nonetheless, the field of CAR T-cells is currently facing several major challenges. In particular, the CAR T-cell strategy has not yet produced favorable clinical responses when targeting solid tumors. In this context, it is of paramount importance to understand the determinants that limit the efficacy of T-cell-based immunotherapy. Characterization of CAR T-cells is usually based on flow cytometry and whole-transcriptome profiling. These approaches have been very valuable to determine intrinsic elements that condition T-cell ability to proliferate and expand. However, they do not take into account spatial and kinetic aspects of T-cell responses. In particular, in order to control tumor growth, CAR T-cells need to enter into the tumor, migrate within a complex tumor environment, and form productive conjugates with their targets. Advanced imaging techniques combined with innovative preclinical models represent promising tools to uncover the dynamics of CAR T-cells. In this review, we will discuss recent results on the biology of engineered T-cells that have been obtained with real-time imaging microscopy. Important notions have emerged from these imaging-based studies, such as the multi-killing potential of CAR T-cells. Finally, we will highlight how imaging techniques combined with other tools can solve remaining unresolved questions in the field of engineered T-cells.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.