Multispectral Raman Differentiation of Malignant Skin Neoplasms In Vitro: Search for Specific Biomarkers and Optimal Wavelengths

Abstract

Confocal scanning Raman and photoluminescence (PL) microspectroscopy is a structure-sensitive optical method that allows the non-invasive analysis of biomarkers in the skin tissue. We used it to perform in vitro diagnostics of different malignant skin neoplasms at several excitation wavelengths (532, 785 and 1064 nm). Distinct spectral differences were noticed in the Raman spectra of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), compared with healthy skin. Our analysis of Raman/PL spectra at the different excitation wavelengths enabled us to propose two novel wavelength-independent spectral criteria (intensity ratios for 1302 cm^-1 and 1445 cm^-1 bands, 1745 cm^-1 and 1445 cm^-1 bands), related to the different vibrational "fingerprints" of cell membrane lipids as biomarkers, which was confirmed by the multivariate curve resolution (MCR) technique. These criteria allowed us to differentiate healthy skin from BCC and SCC with sensitivity and specificity higher than 95%, demonstrating high clinical importance in the differential diagnostics of skin tumors.

Keywords: basal cell carcinoma; confocal Raman and photoluminescence microspectroscopy; diagnostic biomarkers; optical biopsy; signal processing; skin cancer; squamous cell carcinoma.

Figure 1

The skin cross-section showing dermal penetration by different wavelengths of laser excitation in the visible–near-infrared range (532, 785 and 1064 nm).

Figure 2

Raman and photoluminescence (PL) spectra of (a) healthy skin (normal skin), (b) basal cell carcinoma (BCC) and (c) squamous cell carcinoma (SCC) at 532 nm laser excitation in 900–3100 cm⁻¹ range.

Figure 3

Raman/PL spectra (original spectra), Raman spectra (with background subtracted) and fluorescence background of (a) normal skin, (b) BCC and (c) SCC at 532 nm laser excitation.

Figure 4

Normalized mean Raman spectra of (a) normal skin, (b) BCC and (c) SCC at 532 nm laser excitation in the range of 1200–1800 cm⁻¹.

Figure 5

(a) Normalized mean Raman spectra with (b) standard deviation intervals (solid lines with grey space) of normal skin, BCC and SCC (10–20 independent sites) at 532 nm laser excitation.

Figure 6

Raman/PL spectra of (a) normal skin, (b) BCC and (c) SCC at 785 nm laser excitation in 900–3100 cm⁻¹ range.

Figure 7

Raman/PL spectra (original spectra), Raman spectra (with background subtracted) and fluorescence background of (a) normal skin, (b) BCC and (c) SCC at 785 nm laser excitation.

Figure 8

Normalized mean Raman spectra of (a) normal skin, (b) BCC and (c) SCC in the range of 1200–1800 cm⁻¹ at 785 nm laser excitation.

Figure 9

(a) Normalized mean Raman spectra with (b) standard deviation intervals (solid lines with grey space) of normal skin, BCC and SCC (10–20 independent sites) at 785 nm laser excitation.

Figure 10

Raman/PL spectra of (a) normal skin, (b) BCC and (c) SCC at 1064 nm laser excitation in 900–3100 cm⁻¹ range.

Figure 11

Normalized mean Raman spectra of (a) normal skin, (b) BCC and (c) SCC in the range of 1200–1800 cm⁻¹ at 1064 nm laser excitation.

Figure 12

(a) Normalized mean Raman spectra with (b) standard deviation intervals (solid lines with grey space) of normal skin, BCC and SCC (10–20 independent sites) at 1064 nm laser excitation.

Figure 13

Classification of normal skin, BCC and SCC samples in vitro at 532 nm laser excitation by means of Raman band ratios using linear and quadratic discriminant analysis. (a,b) Band ratios for determination of various skin tumors: the red line separates normal skin from BCC and SCC, the black line BCC from SCC; (c,d) ROC curves with AUC values for normal skin, BCC and SCC; (e,f) confusion matrix of classification in percent for all data (Raman spectra with fluorescent background).

Figure 13

Classification of normal skin, BCC and SCC samples in vitro at 532 nm laser excitation by means of Raman band ratios using linear and quadratic discriminant analysis. (a,b) Band ratios for determination of various skin tumors: the red line separates normal skin from BCC and SCC, the black line BCC from SCC; (c,d) ROC curves with AUC values for normal skin, BCC and SCC; (e,f) confusion matrix of classification in percent for all data (Raman spectra with fluorescent background).

Figure 14

Classification of normal skin, BCC and SCC samples in vitro at 785 nm laser excitation by means of Raman band ratios using linear discriminant analysis. (a) Band ratios for determination of various skin tumors: the red line separates normal skin from BCC and SCC, and the black line BCC from SCC; (b) ROC curves with AUC values for normal skin, BCC and SCC; (c) confusion matrix of classification in percent for all data (Raman spectra with fluorescent background).

Figure 15

Classification of normal skin, BCC and SCC samples in vitro at 1064 nm laser excitation by means of Raman band ratios using linear and quadratic discriminant analysis. (a) Band ratios for determination of various skin tumors: the black line separates normal skin from all tumors (BCC together with SCC); (b) ROC curves with AUC values for normal skin and tumors; (c) confusion matrix of classification in percent for all data (Raman spectra with fluorescent background).

Figure 15

Classification of normal skin, BCC and SCC samples in vitro at 1064 nm laser excitation by means of Raman band ratios using linear and quadratic discriminant analysis. (a) Band ratios for determination of various skin tumors: the black line separates normal skin from all tumors (BCC together with SCC); (b) ROC curves with AUC values for normal skin and tumors; (c) confusion matrix of classification in percent for all data (Raman spectra with fluorescent background).

Figure 16

(a) Eight components obtained by MCR-ALS analysis of in vitro Raman/PL spectra (Raman spectra with fluorescent background) of skin neoplasms at 532 nm laser excitation (lower part; dashed line denotes the spectra of triolein as given in [20])—original and fitted example spectra of normal skin sample (upper part); ROC curves with AUC values and confusion matrices (classification rate in percent) for normal skin and tumors using (b) linear and (c) quadratic discriminant analysis.

Figure 17

(a) Eight components obtained by MCR-ALS analysis of in vitro Raman/PL spectra of skin neoplasms at 785 nm laser excitation (lower part; dashed line denotes the spectra of triolein as given in [20])—original and fitted example spectra of normal skin sample (upper part); (b) ROC curves with AUC values and confusion matrices (classification rate in percent) for normal skin and tumors using linear and quadratic discriminant analysis.

Figure 18

(a) Seven components obtained by MCR-ALS analysis of in vitro Raman/PL spectra of skin neoplasms at 1064 nm laser excitation (lower part)—original and fitted example spectra of normal skin sample (upper part); ROC curves with AUC values and confusion matrices (classification rate in percent) for normal skin and tumors using (b) linear discriminant and (c) quadratic discriminant analysis.