## 2 Materials and Methods
### Hyperspectral Imaging System  
A line-scan (push-broom) hyperspectral imaging (HSI) system and LED-based violet fluorescence excitation light were used in this study. The HSI system consists of a 14-bit electron-multiplying charge-coupled-device (EMCCD) camera (MegaLuca R EMCCD camera, Andor Technology PLC, Belfast, Northern Ireland), an imaging spectrograph (Hyperspec VS imaging spectrograph, Headwall Photonics, Fitchburg, MA, USA) with 60 \(\upmu\)m slit, a lens (Schneider-Xenoplan 1.4/23 C-mount lens, Schneider Optics, Hauppauge, NY, USA) and a motorized positioning table (Velmex, Bloomfield, NY, USA). Figure 1 shows a schematic illustration and a photo of the HSI system which can acquire visible or near-infrared reflectance images by using quartz-tungsten halogen lights as well as fluorescence images by using either ultraviolet (UV) or violet (V) excitation lights. The two UV/violet LED-based line lights were built in-house and each incorporated four 365 nm and four 405 nm 10 W light-emitting diodes (LEDs; LedEngin, CA, USA). A 480 nm long wavelength pass filter (Kodak Wratten Gelatin Filter, No. 8) was placed in front of the object lens to remove the tail-end of the excitation light and second-order effect. In this study, the HSI system was used only with the 405 nm violet excitation light because previous research found that fluorescence from the violet excitation (e.g., 410 nm) enhanced the detection of fecal matter on produce to a greater degree when compared to fluorescence from UV excitation or visible/near-infrared reflectance [33].  
Total 81 layers of hyperspectral images were collected across the range of 480 to 800 nm at approximately 4 nm intervals. The EMCCD has 1004 \(\times\) 1002 pixels (spatial \(\times\) spectral), which include spectral bands below and above the 480 to 800 nm spectral region of interest. Of the 1002 spectral pixels, only the 405 pixels within the spectral region of interest were acquired. In addition, pixel binning (averaging) was used to reduce the data volume by two horizontally and by five vertically, resulting in 502 spatial pixels and 81 spectral wavebands. Hyperspectral images were acquired using line-scan incremental steps of 1 mm with 0.09 s exposure time for pure fecal, soil-fecal matrix, and compost samples.  
Figure 1: Schematic illustration and color photo of hyperspectral imaging (HSI) system. Only the violet (405 nm) LEDs were used as excitation source for fluorescence imaging application in the current study.  
HSI system data acquisition was carried out using software developed in-house in Visual Basic (Version 6, Microsoft, Seattle, WA, USA). Sample images were corrected with subtraction of EMCCD dark current and adjustment for spatial flat-field responses using two additionally acquired images, respectively. A dark current image was acquired with the light source turned off and the camera lens covered. A flat-field image of a uniform fluorescent target sample (i.e., white plotter paper) was used to correct for heterogeneous illumination and responses of the HSI system. Image processing and analyses, such as dark current and flat-field corrections, spectral data extraction, and principal component analysis (PCA), were accomplished with combined use of in-house software written in Visual Basic and Matlab (Version R2007b, The MathWorks, Inc., Natick, MA, USA, 2007).