Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, 76109, United States of America.
Department of Microbiology, Immunology, and Genetics, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX, 76107, United States of America.
Methods Appl Fluoresc. 2021 May 24;9(3). doi: 10.1088/2050-6120/ac0243.
Fluorescence is an established technology for studying molecular processes and molecular interactions. More recently fluorescence became a leading method for detection, sensing, medical diagnostics, biotechnology, imaging, DNA analysis, and gene expression. Consequently, precise and accurate measurements in various conditions have become more critical for proper result interpretations. Previously, in Part 1, we discussed inner filter effect type I, which is a consequence of the instrumental geometrical sensitivity factor and absorption of the excitation. In this part, we analyze inner filter effect type II and discuss the practical consequences for fluorescence measurements in samples of high optical density (absorbance/scattering). We consider both the standard square and front-face experimental configurations, discuss experimental approaches to limit/mitigate the effect and discuss methods for correcting and interpreting experimental results.
荧光是一种用于研究分子过程和分子相互作用的成熟技术。最近，荧光已成为检测、传感、医学诊断、生物技术、成像、DNA 分析和基因表达的主要方法。因此，在各种条件下进行精确和准确的测量对于正确解释结果变得更加关键。在前一部分中，我们讨论了内滤光效应类型 I，这是仪器几何灵敏度因子和激发吸收的结果。在这一部分中，我们分析了内滤光效应类型 II，并讨论了在高光学密度（吸光度/散射）样品中进行荧光测量的实际后果。我们考虑了标准方和前向实验两种配置，讨论了限制/减轻这种影响的实验方法，并讨论了校正和解释实验结果的方法。
