Raman active components of skin cancer

Abstract

Raman spectroscopy (RS) has shown great potential in noninvasive cancer screening. Statistically based algorithms, such as principal component analysis, are commonly employed to provide tissue classification; however, they are difficult to relate to the chemical and morphological basis of the spectroscopic features and underlying disease. As a result, we propose the first Raman biophysical model applied to in vivo skin cancer screening data. We expand upon previous models by utilizing in situ skin constituents as the building blocks, and validate the model using previous clinical screening data collected from a Raman optical fiber probe. We built an 830nm confocal Raman microscope integrated with a confocal laser-scanning microscope. Raman imaging was performed on skin sections spanning various disease states, and multivariate curve resolution (MCR) analysis was used to resolve the Raman spectra of individual in situ skin constituents. The basis spectra of the most relevant skin constituents were combined linearly to fit in vivo human skin spectra. Our results suggest collagen, elastin, keratin, cell nucleus, triolein, ceramide, melanin and water are the most important model components. We make available for download (see supplemental information) a database of Raman spectra for these eight components for others to use as a reference. Our model reveals the biochemical and structural makeup of normal, nonmelanoma and melanoma skin cancers, and precancers and paves the way for future development of this approach to noninvasive skin cancer diagnosis.

Keywords: (170.1610) Clinical applications; (170.1790) Confocal microscopy; (170.3880) Medical and biological imaging; (170.6510) Spectroscopy, tissue diagnostics; (300.6450) Spectroscopy, Raman.

Fig. 1

Schematic of confocal Raman and confocal laser-scanning microscope used for skin measurements. The flip mirror and CMOS camera were used for bright-field imaging. ISO, isolator; D1, D2: dichroic mirror; P1 – P3: pinhole; L1 – L6, lens; GM, galvanometer mirror; FM, flip mirror.

Fig. 2

Extracting cellular components from a BCC lesion. Raman images of nucleus (a) and cytoplasm (b) and Raman substrate (c) are compared with bright-field image (d), CLSM image (e) and histopathology image (f). The boxes on (e) and (f) mark the location of Raman imaging. The contrast of the CLSM image was provided by the relative difference in refractive index of cells and the surrounding stroma. Plots on the right show Raman spectra of in situ nucleus, synthetic DNA and their difference spectrum. Also in situ cytoplasm, synthetic actin and their difference spectrum. Scale bar: 10 μm.

Fig. 3

Extracting the epidermal component from a normal skin section. Raman images of in situ keratin (a) and Raman substrate (b) are compared with bright-field image (c) and histopathology image (d). Plots on the right show Raman spectra of in situ, synthetic keratin and their difference spectrum. Scale bar:10 μm.

Fig. 4

Extracting dermal components from a BCC skin section. In situ collagen (a) and elastin (b) are resolved from the image. The dye used by the dermatologist to mark the orientation of the tissue was also detected (c). Raman images are compared with the bright-field image (d), CLSM image (e) and histopathology image (f). The box on (e) marks the location of Raman imaging. The arrow in (f) points to a thin blue-gray elastic fiber. Plots on the right displays Raman spectrum of in situ collagen, synthetic collagen and the difference spectrum. Also Raman spectrum of in situ elastin, synthetic elastin, and their difference spectrum. Scale bar: 10 μm.

Fig. 5

Extracting lipids within a hair follicle from a SCC skin section. In situ ceramide (a), triolein (b) and Raman substrate (c) are resolved from the image. Raman images are compared with the bright-field image (d), CLSM image (e) and histopathology image (f). Some lipids in (f) were lost during the staining processing. The box on (e) and (f) marks the location of Raman imaging. Difference spectrum between in situ ceramide and palmitic acid and difference spectrum between in situ ceramide and triolein are also shown. Scale bar: 10 μm.

Fig. 6

Extracting melanin from a MM skin section. Raman image of melanin (d) are compared with bright field image (a) CLSM image (b) and histopathology image (c). Basis spectra of melanin and beta carotene are shown on the right. Scale bar: 50 μm.

Fig. 7

Basis Raman spectra of water, calcium hydroxyapatite (CaH), hemoglobin (Hb), hair follicle (HF) and keratin pearl (KP) collected in situ are displayed. The difference spectrum between HF and KP is also shown.

Fig. 8

Basis spectra used in the biophysical model of skin. Model components include collagen (a), elastin (b), triolein (c), nucleus (d), keratin (e), ceramide (f), melanin (g), water (h). See Data File 1 for underlying values.

Fig. 9

Model fitting results for in vivo human skin spectra categorized as Normal, BCC, SCC, AK, PL and MM. Mean Raman tissue spectra (solid lines), model fits (dotted lines), residuals are plotted on the same scale. Fit coefficients in percentage are listed on the right. The arrow indicates the most characteristic changes for each lesion type.