Table 2.

Examples of published data applying Raman spectroscopy in breast cancer, showing lack of uniformity in sample preparation, Raman spectrometer parameters and spectral pre-processing technique.

Reference	Sample			Instrument settings			Spectral pre-processing
	Tissue / cells	Preparation	Substrate	Laser intensity	Laser wavelength	Microscope	Other
[36]	IDC and adjacent normal tissue. Number nor specified	6μm snap frozen sections	Not defined	10 mW	532 nm; spot size 650 nm	×50 objective	N/A	Spectra were processed (background subtraction, cosmic ray removal, spectral de-mixing) using WITEC Project Plus software (Ulm, Germany).
[143]	Normal (n = 49); fibrocystic change (n = 16); ductal epithelial hyperplasia (n = 3); fibroadenoma (n = 30); infiltrating carcinoma (n = 31)	Snap frozen. Spectra acquired using thawed tissue stored in PBS	Not required	100–150 mW	830 nm; spot size 100μm	×63, water immersion objective Numerical aperture 0.9	Integration time 10–30 s Spectral resolution 8 cm-1	Spectra were Raman shift frequency-calibrated using known spectral lines of toluene. Fluorescence background was removed by fitting the spectrum to a fifth-order polynomial and then subtracting this polynomial from the spectrum. Cosmic rays were removed using a derivative filter.
[65]	Ductal epithelial hyperplasia (n = 3); sclerosing adenosis (n = 19); fibrocystic disease (n = 43); fibroadenoma (n = 5), MCS (n = 4); DCIS (n = 16)	6 μm FFPE sections	MgF2 slides	100–150 mW	830 nm; spot size 2 μm	×63 objective Numerical aperture 0.9	Integration time- 60 s Spectral resolution- 8 cm−1	See ref. [143].
[144]	MCF7; MDA-MB-436; MCF-10A cell lines	20μm sections of cell pellets in agar plugs	Glass slides	10 mW	532 nm	×50 objective	Spectrograph aperture; 50 μm pinhole Exposure time and accumulations; 50 s with 5 accumulations	Baseline corrections and Unit Vector Normalisation were performed on spectra prior to analysis.
[145]	HMEC; HMLE; HMLE-Twist; HMLE-Ras; BT-474; T47D; MDA-MB-231 cell lines	Unfixed or fixed in methanol/acetone (1:1)	MgF2 coverslips	28 mW	785 nm; spot size 6µm2	×50 objective. Numerical aperture 0.5	Diffraction grating 1200 groves/mm Acquisition time: Live cells: 25 s Fixed cells: 15 s	Origin version-8.5 software (Origin Lab Corporation, Northampton, MA, USA) was used for pre-processing. 10-point and 3-point baseline correction methods were applied to spectra in the low and high wavenumber regions, respectively. Spectral noise was reduced using a Savitsky–Golay filter. Spectra were normalised by dividing each point by the norm of the whole spectrum.
[81]	No preparation required- in vivo measurement of breast tissue- normal, fibrocystic change, fibroadenoma and infiltrating carcinoma			82–125 mW	830 nm	Not applicable	1 s exposure	Following spectral acquisition, calibration data (spectrum of 4-acetamidophenol) was collected for spectral corrections. Chromatic intensity variations were corrected by collecting the spectrum of a tungsten white light source diffusely scattered by a reflectance standard (BaSO4). Background signals, generated in the optical fibres, were removed by collecting the scattered excitation light from a roughened aluminium surface and then optimally subtracted from the data in an iterative loop by using a scaling factor related to the tissues’ optical properties. Tissue fluorescence background was modelled with a 6th order polynomial.
[146]	BT-474; MCF-10A cell lines	Cell pellets	Quartz microscope slide	30 mW	785 nm	×50 objective Numerical aperture 0.75	30 s exposure and 2 accumulations Spectral resolution- 3 cm−1	Background spectrum of the quartz slide was subtracted from sample spectrum using an automatic spectral subtraction function (GRAMS/AI spectroscopy software- Thermo Fisher Scientific Inc., Waltham, Massachusetts). Fluorescence background was subtracted using an automated modified polynomial fitting method (MATLAB, MathWorks, Natick, Massachusetts). Spectra were normalised to the mean intensity in the fingerprint range (700–1750 cm−1)
[147]	DCIS (n = 30); IDC (n = 30); normal (n = 7)	FFPE 20μm sections that were subsequently deparaffinised	Glass slides	Low- value not specified	786 nm	Not specified	4 exposures, 16 scans 4 cm−1 spectral resolution	Not specified
[45]	Normal (n = 105); fibroadenoma) (n = 52); malignant (n = 101)	Stored in saline	Not specified	150 mW	785 nm	Not specified	30 s integration time, 20 accumulations	Spectra were calibrated with a cubic fit to known frequencies of Tylenol. Baseline correction, smoothing, calibration, and normalization was carried out based on the ΔCH2 band using algorithms of Grams 32 (Galactic Industries Corporation, Salem, NH, USA).
[148]	4T1 mouse breast cancer cell line	Mice were injected with a suspension of 4T1 cells (1 × 105) and developed tumour nodules at injection site. Mice were sacrificed and tumour, adjacent breast tissue, contralateral mammary gland and its adjacent lymph nodes were removed.	Not required	50 mW	785 nm	20x objective	3 exposures, 10 s exposure time 4 cm−1 spectral resolution	MATLAB (version 6.5) was used to pre-process data. A median filter was applied to the spectra to remove cosmic rays or spikes. Noise was filtered using wavelets. Background fluorescence was subtracted from the denoised spectra using a modified cubic spline algorithm. The spectrum was then normalised so that the minimum and maximum values of the spectrum were 0 and 1, respectively.
[71]	Tissue microarray of normal (n = 79) and breast cancer samples (n = 499)	5um thick and 0.6 mm diameter	Not specified	10 mW	532 nm	×50 long work distance objective	Not specified	Spectra were fluorescence-corrected by employing a six-polynomial algorithm (OmnicTM, Thermo Scientific, Waltham, MA, USA). Pre-processing (baseline correction; Savitzky-Golay smoothing and standard normal variate corrections) was performed using the Unscrambler X 10.2TM software (Camo software, Oslo, Norway)

IDC invasive ductal cancer, PBS phosphate buffered saline, MCS Monckeberg medial calcific sclerosis, DCIS ductal carcinoma in situ, FFPE formalin-fixed paraffin-embedded.