Liebsch G, Klimant I, Krause C, Wolfbeis O S
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Germany.
Anal Chem. 2001 Sep 1;73(17):4354-63. doi: 10.1021/ac0100852.
We present a referenced scheme for fluorescence intensity measurements that is useful for imaging applications. It is based on the conversion of the fluorescence intensity information into a time-dependent parameter. A phosphorescent dye is added in the form of approximately 10-microm particles to the sample containing the pH-sensitive fluorescent indicator. Both the reference dye and the pH probe are excited simultaneously by a blue LED, and an overall luminescence is measured. In the time-resolved imaging method presented here, two images taken at different time gates were recorded using a CCD camera. The first image is recorded during excitation and reflects the luminescence signal of both the fluorophore (pH) and the phosphor (reference). The second image, which is measured after a certain delay (after switching off the light source), is solely caused by the long-lived phosphorescent dye. Because the intensity of the fluorophore contains the information on pH, whereas phosphorescence is pH-independent, the ratio of the images displays a referenced intensity distribution that reflects the pH at each picture element (pixel). The scheme is useful for LED light sources and CCD cameras that can be gated with square pulses in the microsecond range. The fundamentals and potential of this new method, to which we refer as time domain dual lifetime referencing (t-DLR), are demonstrated.
我们提出了一种用于荧光强度测量的参考方案，该方案对成像应用很有用。它基于将荧光强度信息转换为一个随时间变化的参数。以大约10微米颗粒的形式向含有pH敏感荧光指示剂的样品中添加一种磷光染料。参考染料和pH探针同时由蓝色发光二极管激发，并测量总的发光强度。在此提出的时间分辨成像方法中，使用电荷耦合器件（CCD）相机记录在不同时间门拍摄的两张图像。第一张图像在激发期间记录，反映了荧光团（pH）和磷光体（参考）的发光信号。第二张图像在一定延迟后（关闭光源后）测量，仅由长寿命磷光染料引起。由于荧光团的强度包含pH信息，而磷光与pH无关，图像的比值显示出反映每个像素处pH值的参考强度分布。该方案适用于可通过微秒级方波脉冲选通的发光二极管光源和电荷耦合器件相机。展示了这种我们称为时域双寿命参考（t-DLR）的新方法的基本原理和潜力。
