## 2 Materials and Methods
### Processing of Raman Spectra  
Raman spectra of the reference compounds and colon tissue samples were exported to Origin 6.0 (Microcal Origin, Northampton, MA, USA) software as the ASCII data files for further processing (FFT filter smoothing by 5 points, manual baseline correction). The mean \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Characteristics**} & \multirow{2}{*}{**Laboratory**} & \multicolumn{2}{c}{**Portable**} \\ \cline{2-4}  & & **Modular** & **Handheld** \\ \hline Specification & DXR SmartRaman & i-Raman Plus 785H & Ahura FirstDefender \\ Company & Thermo Fisher Scientific & B\&W Tek Inc. & Thermo Fisher Scientific \\ Address & Waltham, MA, USA & Newark, DE, USA & Waltham, MA, USA \\ Size (cm) & 91 \(\times\) 63 \(\times\) 34 & 17 \(\times\) 34 \(\times\) 23.4 & 19.3 \(\times\) 10.7 \(\times\) 4.4 \\ Weight (kg) & \textgreater{}50 & 3 & 0.8 \\ Computer & Desktop, serial port & Laptop, USB & Internal software \\ Excitation wavelength (nm) & 780 & 785 & 785 \\ Laser outlet power (mW) & 90 & 90 (\textless{}300) & 75 \\ Detector & CCD & CCD & CCD \\ Spectral range (cm\({}^{-1}\)) & 100–3409 & 175–3110 & 250–2875 \\ Spectral resolution (cm\({}^{-1}\)) & 2.4–4.4 & 4.5 & 7–10.5 \\ Number of scans & 3–100 & 3–10 & 1 \\ Measuring time (min) & 3–100 & 6–60 & 1–3 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Comparison of laboratory, modular, and handheld Raman instruments used in this work. Figure 1: Laboratory and portable Raman instruments used in the measurement of colon tissue samples. spectra and standard deviations were calculated for 25-30 measurements at independent sites of the samples of colon tissue, adenomatous polyp, and colorectal adenocarcinoma. For all the Raman instruments, the working spectral region of Raman spectra (600-1800 cm\({}^{-1}\)) of calf thymus DNA was analyzed using the normalized least-squares curve-fitting procedure (PeakFit module of Origin 6.0) using multiple Voigt (Gaussian-Lorentzian mix) curves. The best curve-fitting was specified by the lowest possible \(\chi^{2}\) values. The calculation of the bandwidth at half maximum (BWHM) of ten selected bands in the Raman spectra of calf thymus DNA was obtained from the parameters of the corresponding Voigt components in order to compare the effective spectral resolution between instruments. The signal-to-noise ratio (SNR) for the same bands was calculated by dividing the signal intensity in the raw spectra by the noise level in the operating region of the spectrum. The noise level was defined as twice the standard deviation (SD) of the difference between the original and smoothed spectra [25]. These steps were applied to obtain the noise level near each studied Raman band. The individual Raman spectra of tissue samples were imported to Unscrambler X 10.5.1 (CAMO Software AS, Oslo, Norway) software for a statistical evaluation, using discrimination analyses, i.e., hierarchy cluster analysis (HCA) and principal component analysis (PCA), for the working spectral region (600-1800 cm\({}^{-1}\)). This is the region of prominent bands corresponding to stretching and deformation vibrations of the main biological compounds like proteins, nucleic acids, and polysaccharides. Clustering was performed using Ward's method and squared Euclidean distance for 3 clusters. PCA was applied with mean center data for four principal components using singular value decomposition (SVD). The results of the HCA and PCA were represented by dendrograms and component score plots, respectively. Based on the PCA models generated for each class, i.e., normal colon tissue, adenomatous polyps, and adenocarcinoma, represented by 25-30 measurements in independent regions of the sample, the soft independent modeling of class analogy (SIMCA) algorithm was implemented using Unscrambler X 10.5.1.