## 2 Materials and Methods
### Measurement Setup: Requirements and Procedures  
Diffuse reflectance spectra require a specific optical setup for accurate and reproducible measurements. While it is possible to assemble it from scratch, yielding appreciable results, several dedicated spectrometers are commercially available at the time of writing this paper for higher precision measurements (cf. Table 1). Naturally, successor or predecessor models are, depending on their specifications and measurement options, also suitable. Figure 2: Photograph of the sample preparation components to compress powders into a pellet. 1: recoil-free hammer, 2: piece of paper, 3: powder sample, 4: spatula, 5: tappet, 6: sample holder, 7: razor blade, 8: glass/microscope slide. Since double-beam spectrometers measure sample and white standard simultaneously, the determination of the diffuse reflectance of non-luminescent samples becomes facile, fast and reliable. Due to the parallel acquisition method, lamp intensity fluctuations pose no problem. However, in case of fluorescence, extraneous light emission at wavelengths distinct from the excitation creates spurious additional signals that render an unfiltered diffuse reflectance spectrum inaccurate. Using a fluorescence spectrometer instead yields the specific advantage to perform diffuse reflectance measurements on any material at relative ease. For studies on biological samples, however, light is frequently focused either into a cuvette or onto a surface, making such a setup highly susceptible to measurement artifacts as will be explained in Section 2.2.1. Such a scenario is henceforth omitted in favor of a collimated (or, optionally, defocused) beam. In the following, we will explain the principal scheme of a dedicated fluorescence spectrometer with the _FLS980_ (Edinburgh Instruments) as an example whose layout is sketched in Figure 3.  
For fluorescent samples, a setup utilizing two individual monochromators is essential: The excitation monochromator selects an wavelength region for sample illumination while the emission monochromator transmits the linear diffuse reflectance of the sample, thereby canceling additional \begin{table}
\begin{tabular}{l l} \hline \hline
**Fluorescence Spectrometer** & **Ref.** \\ \hline _FLS1000_, Edinburgh Instruments, Ltd. & [42] \\ _RF-6000_, Shimadzu Corp. & [43] \\ _K2 Fluorometer_, ISS, Inc. & [44] \\ _Fluorolog_, HORIBA Scientific, Ltd. & [45] \\ _FluoroxMax_, HORIBA Scientific, Ltd. & [46] \\ _F-7000_, Hitachi High-Technologies Corp. & [47] \\ _OmniTilmo_, Zolix Instruments Co., Ltd. & [48] \\ _FluoTilmo 300_, PicQquant GmbH & [49] \\ _FP-8000 Series_, JASCO Deutschand GmbH & [50] \\ _FL 8500_, PerkinElmer, Inc. & [51] \\ _Cary Eclipse_, Agilent Technologies, Inc. & [52] \\ \hline
**Double-Beam Spectrometer** & **Ref.** \\ \hline _LAMBDA 950 UV/VIS_, PerkinElmer, Inc. & [53] \\ _UV-3600 Plus UV-VIS-NIR_, Shimadzu Corp. & [54] \\ _UH5300_, Hitachi High-Technologies Corp. & [55] \\ _Cary 5000 UV-Vis-NIR_, Agilent Technologies, Inc. & [56] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: A non-exhaustive selection of spectrometers capable of performing diffuse reflectance measurements. Figure 3: Sketch of a fluorescence spectrometer for diffuse reflectance measurements. BP: band-pass filter, LP: long-pass filter, ND: neutral density filter. fluorescence emission. Hence, this method of operation is frequently termed as a _synchronous scan_, i.e., both monochromators are set to the same wavelength. Apart from monochromators, additional spectral filters remove potential higher diffraction orders transmitted through grating-based monochromators and possible fluorescence of the sample. In case only monochromatic illumination is required, the light source/monochromator combination can be replaced by an appropriate laser or via bandpass filtering behind a broadband emission source.