FLUORESCENCE LIFETIME-BASED SENSING AND IMAGING
FLUORESCENCE LIFETIME-BASED SENSING AND IMAGING
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DOI：
10.1016/0925-4005(95)01658-9
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发表时间：
1995-10-01
影响因子：
8.4
通讯作者：
LAKOWICZ, JR
中科院分区：
文献类型：
--
作者：
SZMACINSKI, H;LAKOWICZ, JR
时间分辨荧光光谱是生物化学、生物物理学和化学物理学的重要研究工具。然而，时间分辨方法也可以用于化学传感。与基于强度的方法相比，基于寿命的感测具有若干优点。由于寿命是独立的总探针强度，它的测量可以提供许多分析物的定量检测，而不需要波长比探针。像氧和卤化物这样的分析物可以通过碰撞猝灭机制来确定。迄今为止，寿命探针分析物识别（结合）已确定为Ca 2+，Mg 2+，K+和pH值。重要的是，寿命的方法提供了一种可能性，扩大灵敏的分析物浓度范围，使用探针光谱位移。荧光寿命方法允许感测没有直接探针的分析物，如葡萄糖、抗原或基于荧光能量转移作为转导机制的任何亲和或免疫测定。仪器、激光技术、光纤，特别是长波长探针的进步可以使时间分辨荧光快速迁移到临床化学、环境传感和工业应用中。我们将描述相位调制仪器，它可以使用简单的光源，其光可以由声光调制器外部调制或由驱动电流内部调制。最后，我们将描述荧光寿命成像显微镜（FLIM），其中图像对比度是从图像的每个点的寿命创建的。时间分辨成像现在是荧光显微镜的现实，并承诺提供各种细胞内分析物和/或细胞现象的化学成像。
Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics and chemical physics. However, time-resolved methods can also be used for chemical sensing. Lifetime-based sensing has several advantages over intensity-based methods. Since the lifetime is independent of the total probe intensity, its measurement can provide quantitative sensing of many analytes without the requirement for wavelength-ratiometric probes. Analytes like oxygen and halides can be determined by the collisional quenching mechanism. To date, lifetime probes for analyte recognition (binding) have been identified for Ca2+, Mg2+, K+ and pH. Importantly, the lifetime method provides a possibility to expand the sensitive analyte concentration range using probes with spectral shifts. The fluorescence lifetime method allows the sensing of analytes for which there are no direct probes, like glucose, antigens, or any affinity or immunoassays based on fluorescence energy transfer as the transduction mechanism. Advances in instrumentation, laser technology, fiber-optics and especially long-wavelength probes can result in the rapid migration of time-resolved fluorescence to clinical chemistry, environmental sensing and industrial applications. We shall describe phase-modulation instrumentation that can use simple light sources for which the light can be modulated externally by acoustooptic modulators or internally by driving current. Finally, we shall describe fluorescence lifetime imaging microscopy (FLIM), in which image contrast is created from the lifetime at each point of the image. Time-resolved imaging is now a reality in fluorescence microscopy, and promises to provide chemical imaging of a variety of intracellular analyte and/or cellular phenomena.