Supercritical Angle Fluorescence Characterization Using Spatially Resolved Fourier Plane Spectroscopy,Analytical Chemistry

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Supercritical Angle Fluorescence Characterization Using Spatially Resolved Fourier Plane Spectroscopy
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-03-12 00:00:00 , DOI: 10.1021/acs.analchem.7b04822
Finub James Shirley _{1,

2} , Pieter Neutens ₂ , Rita Vos ₂ , Md. Mahmud-Ul-Hasan _{1,

2} , Liesbet Lagae _{1,

2} , Niels Verellen _{1,

2} , Pol Van Dorpe _{1,

2}

Affiliation

Most fluorescent immunoassays require a wash step prior to read-out due to the otherwise overwhelming signal of the large number of unbound (bulk) fluorescent molecules that dominate over the signal from the molecules of interest, usually bound to a substrate. Supercritical angle fluorescence (SAF) sensing is one of the most promising alternatives to total internal reflection fluorescence for fluorescence imaging and sensing. However, detailed experimental investigation of the influence of collection angle on the SAF surface sensitivity, i.e., signal to background ratio (SBR), is still lacking. In this Letter, we present a novel technique that allows to discriminate the emission patterns of free and bound fluorophores simultaneously by collecting both angular and spectral information. The spectrum was probed at multiple positions in the back focal plane using a multimode fiber connected to a spectrometer and the difference in intensity between two fluorophores was used to calculate the SBR. Our study clearly reveals that increasing the angle of SAF collection enhances the surface sensitivity, albeit at the cost of decreased signal intensity. Furthermore, our findings are fully supported by full-field 3D simulations.

中文翻译：

使用空间分辨傅里叶平面光谱法的超临界角荧光表征

由于大量未结合的（散装）荧光分子的信号通常会被压倒，因此大多数荧光免疫测定法在读出之前需要进行清洗步骤，该信号在通常与基质结合的目标分子信号上占支配地位。超临界角荧光（SAF）传感是用于荧光成像和传感的全内反射荧光的最有前途的替代方法之一。但是，仍然缺乏对收集角度对SAF表面灵敏度（即信噪比（SBR））的影响进行详细的实验研究。在这封信中，我们提出了一种新颖的技术，该技术可以通过收集角度和光谱信息来同时区分游离和结合的荧光团的发射模式。使用连接到光谱仪的多模光纤在后焦平面的多个位置探测光谱，并使用两个荧光团之间的强度差来计算SBR。我们的研究清楚地表明，增加SAF收集角度可以增强表面灵敏度，尽管以降低信号强度为代价。此外，我们的发现得到了全场3D模拟的完全支持。

更新日期：2018-03-12

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