BAM Federal Institute for Materials Research and Testing, Richard-Willstaetter-Strasse 11, 12489 Berlin, Germany.
Anal Chem. 2012 Feb 7;84(3):1345-52. doi: 10.1021/ac2021954. Epub 2012 Jan 24.
There is an increasing interest in chromophores absorbing and emitting in the near-infrared (NIR) spectral region, e.g., for applications as fluorescent reporters for optical imaging techniques and hence, in reliable methods for the characterization of their signal-relevant properties like the fluorescence quantum yield (Φ(f)) and brightness. The lack of well established Φ(f) standards for the NIR region in conjunction with the need for accurate Φ(f) measurements in transparent and scattering media encouraged us to built up an integrating sphere setup for spectrally resolved measurements of absolute fluorescence traceable to radiometric scales. Here, we present the design of this setup and its characterization and validation including an uncertainty budget for the determination of absolute Φ(f) in the visible and NIR. To provide the basis for better measurements of Φ(f) in the spectral window from ca. 600 to 1000 nm used, e.g., for optical imaging, the absolute Φ(f) of a set of NIR chromophores covering this spectral region are measured and compared to relative values obtained using rhodamine 101 as Φ(f) standard. Additionally, the absolute Φ(f) values of some red dyes that are among the most commonly used labels in the life sciences are presented as well as the absolute quantum yield of an optical probe for tumor imaging.
人们对近红外（NIR）光谱区域内吸收和发射光的发色团越来越感兴趣，例如，将其作为荧光报告物用于光学成像技术，因此，需要可靠的方法来表征其与信号相关的特性，如荧光量子产率（Φ(f)）和亮度。由于缺乏适用于近红外区域的既定Φ(f)标准，并且需要在透明和散射介质中进行精确的Φ(f)测量，这促使我们建立了一个积分球装置，用于对绝对荧光进行光谱分辨测量，该测量可追溯到辐射测量刻度。在这里，我们介绍了该装置的设计及其特性和验证，包括确定可见和近红外区域绝对Φ(f)的不确定度预算。为了更好地测量约 600nm 至 1000nm 光谱窗口内的Φ(f)，例如用于光学成像，我们测量了一组涵盖该光谱区域的近红外发色团的绝对Φ(f)，并将其与使用罗丹明 101 作为Φ(f)标准获得的相对值进行了比较。此外，我们还展示了一些在生命科学中最常用的红色染料的绝对Φ(f)值，以及用于肿瘤成像的光学探针的绝对量子产率。
