# Dual-Slope Diffuse Reflectance Instrument for Calibration-Free Broadband Spectroscopy
## 2 Materials and Methods
### Experiment  
The purpose of the experiment was to validate the Dual-Slope broadband Diffuse Reflectance Spectroscopy (DS CW-bDRS) instrument. This was done using a liquid optical phantom which was measured at different dyes concentrations to confirm the DS CW-bDRS's ability to distinguish spectral features and measure absolute absorption. A total of 5 L of the phantom was made and placed in a cylindrical tank (Figures 1a and 2a). The base of the phantom was made of 2% (reduced fat) Milk and Water (MW; 43% milk and 57% water volume fraction) such that the scattering properties were similar to those of biological tissue in the near-infrared. The scattering was assumed to not change as dyes were added since the addition of the dyes is expected to have a negligible effect on scattering. Each time the phantom was measured, the measurement was done by both Multi-Distance Frequency-Domain Near-Infrared Spectroscopy (MD FD-NIRS) and DS CW-bDRS.  
Three dyes were added in the following order: black India-Ink (II; Higgins, Leeds, MA, USA), NIR746A (N7; QCR Solutions, Palm City, FL, USA), and NIR869A (N8; QCR Solutions). II is expected to have a relatively flat or decreasing absorption spectrum with wavelength in the Near-Infrared (NIR) range [35; 36]. By contrast, N7 and N8 are expected to have absorption peaks at around 746 nm and 869 nm, respectively (Figure 3). However, the actual peak wavelength may shift depending on the chemical properties of the solvent [37; 38].  
Figure 2: Broadband diffuse reflectance spectroscopy methods to achieve measurements of absolute absorption spectra. (**a**) Render of diffuse reflectance spectroscopy probe on a diffuse optical phantom. (**b**) Schematic of dual-slope diffuse reflectance spectroscopy device. Acronyms: Universal Serial Bus (USB) and Transistor-Transistor Logic (TTL). (**c**) Schematic of the source (1 and 2) and detector (A and B) positions on the dual-slope diffuse reflectance spectroscopy probe.  
The measurements and dye additions proceeded as follows (measurements meaning both MD FD-NIRS and DS CW-bDRS). First, the MW base phantom was mixed, then measured. Then, II was mixed in and the phantom measured again. Next, N7 was mixed and again the phantom measured. Finally, N8 was also mixed into the phantom and the measurement was done for a final time. The amount of each addition of dye was such that all absorption spectra were expected to stay within typical absorption values of biological tissue (approximately 0.005 to 0.020 mm\({}^{-1}\)) [2].  
The temporal sampling parameters of the DS CW-bDRS system were set to yield a relatively fast sampling rate while still measuring a relevant wavelength range. Along these lines, DS CW-bDRS data were analyzed between 600 nm and 900 nm and the sampling rate was set to 2.5 Hz. For DS CW-bDRS 5 min of data were averaged for analysis. The MD FD-NIRS measurement took 30 s at each step for a total measurement time of 5.5 min (eleven steps; Section 2.1.1), and had a sampling rate of 2.8 Hz. These parameters were chosen such that the rate and time of DS CW-bDRS and MD FD-NIRS were approximately equal.  
This phantom experiment resulted in MD FD-NIRS measurements at two wavelengths and DS CW-bDRS measurements between 600 nm and 900 nm for four phantoms (MW, MW+II, MW+II+N7, and MW+II+N7+N8). Section 2.3 describes the analysis of these data to result in absolute optical properties at two wavelengths and absolute absorption spectra.