Hyperspectral Raman microscopy can accurately differentiate single cells of different human thyroid nodules

Abstract

We report on the use of line-scan hyperspectral Raman microscopy in combination with multivariate statistical analyses for identifying and classifying single cells isolated from clinical samples of human thyroid nodules based on their intrinsic Raman spectral signatures. A total of 248 hyperspectral Raman images of single cells from benign thyroid (n = 127) and classic variant of papillary carcinoma (n = 121) nodules were collected. Spectral differences attributed to phenylalanine, tryptophan, proteins, lipids, and nucleic acids were identified for benign and papillary carcinoma cells. Using principal component analysis and linear discriminant analysis, cells were identified with 97% diagnostic accuracy. In addition, preliminary data of cells from follicular adenoma (n = 20), follicular carcinoma (n = 25), and follicular variant of papillary carcinoma (n = 18) nodules suggest the feasibility of further discrimination of subtypes. Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an objective approach for analyzing single cells from fine needle aspiration (FNA) biopsies to enable the minimally invasive diagnosis of "indeterminate" thyroid nodules and other challenging cases.

Fig. 1

Brightfield and Raman images of (a-f) papillary thyroid carcinoma cell and (g-l) benign follicular cell for select Raman bands.

Fig. 2

Average Raman spectra of (a) 127 benign (blue line) and (b) 121 PTC classic variant (red line) cells. The gray shadows represent ± 1 standard deviations (SD) over the average values. (c) The difference spectrum (benign – CVPTC) highlights the peaks that are associated with benign cells (blue shade) and CVPTC cells (red shade).

Fig. 3

(a) 3D PCA score plot of all individual Raman spectra for benign (blue circles) and CVPTC (red diamonds) cells. (b) Offset plot of the first three PCs coefficients.

Fig. 4

(a) 2D and (b) 3D PCA-LDA score plots of benign (blue) and papillary carcinoma (red) cells with 95% interval confidence covariance error ellipses/ellipsoids.

Fig. 5

Average Raman spectra of (a) 20 FTA (green line), (b) 20 FTC (magenta line) and (c) 18 FVPTC (dark gray line) cells. The light gray shadows represent ± 1 standard deviations (SD) over the average values.

Fig. 6

(a)-(b) 2D and (c)-(d) 3D PCA-LDA score plot from 20 FTA (green plot), 25 FTC (magenta plot), 18 FVPTC (dark gray plot) and 121 CVPTC (red plot) cells.(e)-(f) Confusion matrices.