Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, 76109, United States of America.
Methods Appl Fluoresc. 2020 Jun 1;8(3):033002. doi: 10.1088/2050-6120/ab947c.
Fluorescence technologies have been the preferred method for detection, analytical sensing, medical diagnostics, biotechnology, imaging, and gene expression for many years. Fluorescence becomes essential for studying molecular processes with high specificity and sensitivity through a variety of biological processes. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biological samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. This is a relatively simple factor to be controlled in any fluorescence experiment. However, many previous approaches have given only approximate experimental methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. Importantly, we present direct experimental results of how the correction works. One considers problems emerging from varying absorption across its absorption spectrum for all fluorophores. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of observed results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide and we show how properly corrected results compare to the initial erroneous results. The procedure is generic and applies to many other applications like quantum yield determination, tissue/blood absorption, or acceptor absorption in FRET experiments.
荧光技术多年来一直是检测、分析传感、医学诊断、生物技术、成像和基因表达的首选方法。通过各种生物过程，荧光在研究具有高特异性和灵敏度的分子过程中变得至关重要。对于实际的荧光应用，一个显著的问题是荧光强度的明显非线性，这是由于内滤效应、样品散射和生物样品固有成分的吸收引起的。样品吸收会导致主要的内滤效应（第一类内滤效应），这是应该首先考虑的因素。这是在任何荧光实验中都可以相对简单地控制的因素。然而，许多先前的方法仅提供了用于校正偏离预期结果的近似实验方法。在这一部分，我们讨论了主要内滤效应的起源，并提出了一种在全吸收范围内校正荧光强度信号的通用方法。重要的是，我们提出了校正效果的直接实验结果。人们考虑了所有荧光团在其吸收光谱上的吸收变化所产生的问题。我们使用若丹明 800 并演示如何在较宽的波长范围内正确校正激发光谱。其次是惰性吸收剂的影响，它会衰减激发光束的强度，因为它穿过比色皿，这会导致观察结果的显著偏差。例如，我们展示了丙烯酰胺对色氨酸类似物 NATA 的荧光猝灭，以及如何正确校正的结果与初始错误结果进行比较。该程序是通用的，适用于许多其他应用，如量子产率测定、组织/血液吸收或 FRET 实验中的受体吸收。
