荧光团
光漂白
显微镜
荧光
荧光显微镜
人口
荧光寿命成像显微镜
共焦显微镜
生物物理学
仪表(计算机编程)
校准
化学
纳米技术
材料科学
光学
计算机科学
物理
生物
医学
环境卫生
量子力学
操作系统
作者
Gerone A Gonzales,Johnathan Canton
标识
DOI:10.1007/978-1-0716-3338-0_11
摘要
Dual-wavelength and dual-fluorophore ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole-population methods of being able to resolve individual cells and even individual organelles. In this chapter, we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.
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