Department of Medicine, University of Tartu, Tartu, Estonia; Institute of Physics, University of Tartu, Tartu, Estonia.
Cytometry A. 2011 Oct;79(10):775-88. doi: 10.1002/cyto.a.21110. Epub 2011 Jul 27.
A handful of research teams around the world have recently begun to utilize multiphoton techniques in cytometry, especially for in vivo applications. These approaches offer similar enhancements to flow cytometry as the multiphoton phenomenon brought to the field of microscopy at the turn of the 20th century, with at least six advantages over single-photon excitation. Here, we review the published literature on multiphoton cytometry in vivo or in vitro from the initial experiments in 1999 to present. Multiphoton cytometry instrumentation set-ups vary from adapted multiphoton microscopy to a dedicated system, with laser pulse power and repetition rate serving as important variables. Two-beam geometry enables quantitation of cell size. Labeling strategies include conjugated fluorophore targeting, with folate and/or dendrimer platforms. With two-color measurement, ratiometric labeling is also possible, where one dye serves as a trigger to indicate the amount of excitation a cell receives, and another informs of cellular function. With two-color labeling, geometric fluorophore distribution proves important in theory and experiment for detection sensitivity curves and detected event intensity correlation. The main biological achievements to date using this young technology are reviewed, with emphasis on real-time monitoring of minute-by-minute and long-term cell dynamics as well as the clinically significant surveillance of circulating tumor cells. For this goal, minimally invasive two-photon flow cytometry with a fiber probe may overcome the primary issue of sample volume. The technique of multicolor, multiphoton flow cytometry greatly enhances the capabilities of flow cytometry to investigate the dynamics of circulating cells in cancer and other important diseases, and may in the future benefit from advances in microscopy such as super-resolution imaging, coherent control, and bioluminescence.
世界上有少数几个研究小组最近开始在细胞术领域中应用多光子技术，特别是用于体内应用。这些方法为流式细胞术提供了与 20 世纪之交多光子现象为显微镜领域带来的类似增强，与单光子激发相比至少有六个优势。在这里，我们回顾了从 1999 年的最初实验到现在发表的关于体内或体外多光子细胞术的文献。多光子细胞术仪器设置从适应多光子显微镜的设置到专用系统变化，激光脉冲功率和重复率是重要变量。双光束几何形状可定量测量细胞大小。标记策略包括缀合荧光团靶向，使用叶酸和/或树突状平台。通过双色测量，还可以进行比率标记，其中一种染料用作触发器，以指示细胞接收到的激发量，另一种染料则指示细胞功能。通过双色标记，几何荧光团分布在理论和实验中对于检测灵敏度曲线和检测事件强度相关性都很重要。迄今为止，使用这项新技术取得的主要生物学成就得到了回顾，重点是实时监测每分钟和长期的细胞动力学以及循环肿瘤细胞的临床重要监测。为此目的，使用光纤探头的微创双光子流式细胞术可能会克服样品体积的主要问题。多色多光子流式细胞术技术极大地增强了流式细胞术调查癌症和其他重要疾病中循环细胞动力学的能力，并且将来可能会受益于显微镜技术的进步，例如超分辨率成像、相干控制和生物发光。
