## 2 Materials and Methods  
### Sample Preparation and Collection  
Bacterial cultures were isolated and purified from broiler chicken carcass rinses at the U.S. National Poultry Research Center by the Poultry Microbiological Safety and Processing Research Unit and were stored in 20% glycerol at \(-80\)\({}^{\circ}\)C, except for the _Campylobacter_ species, which were obtained from the American Type Culture Collection (Manassas, VA, USA). Stock cultures were removed from the freezer as needed and were inoculated onto the organism's appropriate growth media, then incubated for the necessary time-temperature relationship [12]. A list of the microorganisms and abbreviations used can be found in Table 1.  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Microorganism** & **Microorganism** \\ \hline _Campylobacter coli_ (Cc) & _Salmonella_ Enteritidis (SE) \\ _Campylobacter fetus_ (Cf) & _Salmonella_ Heidelberg (SH) \\ _Campylobacter jejuni_ (Cj) & _Salmonella_ Infants (SI) \\ _Enterobacter cloane_ (Ecl) & _Salmonella_ Javiana (SI) \\ _Enterococcus faecalis_ (Ef) & _Salmonella_ Kentucky (SKe) \\ _Escherichia coli_ (Eco) & _Salmonella_ Kiambu (SKi) \\ _Klebsiella exydoca_ (Ko) & _Salmonella_ Mbandanka (SMb) \\ _Listeria innocuai_ (Li) & _Salmonella_ Montevideo (SMo) \\ _Listeria monocytogenes_ (Lm) & _Salmonella_ Muenchen (SMu) \\ _Macrococcus assolvititics_ (Mc) & _Salmonella_ Seftenberg (SE) \\ _Penaibacillus polynyma_ (Ppo) & _Salmonella_ Typhimuriumium (ST) \\ _Pseudomonas putida_ (Ppu) & _Salmonella_ Typhimurium-NAL (STN) \\ _Staphylococcus aureus_ (Sa) & _Salmonella_ Weltevreden (SW) \\ _Staphylococcus simulans_ (Ss) & _Salmonella_ Seftenberg (SE) \\ _Staphylococcus aureus_ (Sa) & _Salmonella_ Seftenberg (SE) \\After incubation, the cultures were stored at 4 \({}^{\circ}\)C with sample slides prepared, and the HMI was collected within 24 h. Bacterial cultures were sampled as mentioned in [PERSON] et al. [13]. In brief, the method calls for an inoculation loop to pick a typical colony from an agar plate, then it is inoculated into 100 \(\mu\)L of deionized water, vortexed, followed by placing 3 \(\mu\)L of the bacterial suspension on a common glass microscope slide, then allowing it to air dry under a biosafety cabinet for 15 min. A coverslip was applied, and the glass slide was placed on the HMI system's sample stage and viewed under a 100\(\times\) oil objective (Olympus, Tokyo, Japan). This effectively affixes the cells to the slide for hypercube image collection, without damaging the microorganisms, resulting in HMI of individual live cells obtained without the use of reagents, tags, or dyes.  
The HMI system consists of an acousto-optic tunable filter (AOTF; Gooch and Housego, Ilminster, UK), 16-bit electron multiplying charge coupled device (EMCCD) (Andor Technology, Belfast, Northern Ireland), optimized darkfield condenser (Cytoviva, Auburn, AL, USA), 24 W tungsten halogen (TH) light (Osram, Munich, Germany), and a digital upright microscope (i80 Nikon, Lewisville, TX, USA). The TH light source was offset from the HMI system in a lamp house connected underneath the sampling stage via a fiber optic cable, which prevents heat damage to bacterial cells generated from the lamp. The HMI system collected 89 TIFF files in 4-nm increments in the range of 450-800 nm, stacking files together to form a hypercube. Hypercubes were 1000 \(\times\) 1000 \(\times\) 89, resulting in 89 million data points per hypercube from one sample.