Micro-Raman spectroscopy detects individual neoplastic and normal hematopoietic cells

Abstract

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific, slow, biologically perturbing, or a combination thereof. Here, we show that single-cell micro-Raman spectroscopy averts these shortcomings and can be used to discriminate between unfixed normal human lymphocytes and transformed Jurkat and Raji lymphocyte cell lines based on their biomolecular Raman signatures. We demonstrate that single-cell Raman spectra provide a highly reproducible biomolecular fingerprint of each cell type. Characteristic peaks, mostly due to different DNA and protein concentrations, allow for discerning normal lymphocytes from transformed lymphocytes with high confidence (p << 0.05). Spectra are also compared and analyzed by principal component analysis to demonstrate that normal and transformed cells form distinct clusters that can be defined using just two principal components. The method is shown to have a sensitivity of 98.3% for cancer detection, with 97.2% of the cells being correctly classified as belonging to the normal or transformed type. These results demonstrate the potential application of confocal micro-Raman spectroscopy as a clinical tool for single cancer cell detection based on intrinsic biomolecular signatures, therefore eliminating the need for exogenous fluorescent labeling.

FIGURE 1

Average Raman spectra of individual Raji, B-, Jurkat, and T-cells in the fingerprint range from 600 cm⁻¹ to 1700 cm⁻¹. Each spectrum was obtained by averaging the spectra of several (∼16–86) individual spectra. The solid lines indicate the average spectra and the shaded lines delineate one standard deviation.

FIGURE 2

(Top) Autofluorescence image of a Jurkat T-cell adhered to a glass coverslip. The cells were excited at 632.8 nm wavelength and scanned in confocal microscopy mode to provide a spatial map of the location and size of the cells. Raman spectra were then obtained by repositioning the laser beam to positions 1–5 as indicated in the image. (Bottom) Raman spectra obtained from the different locations within the Jurkat T-cell as shown in top panel. Each spectrum was acquired within 180 s integration time with a laser power of 10 mW at 632.8 nm wavelength.

FIGURE 3

(a) Overlay plot of the Raman spectra obtained from T (black line) and Jurkat (red line) cells. Also shown below is the difference spectrum. (b) Scatter plot showing the intensity value of select peaks with the most distinguishable differences from individual T- and Jurkat cell spectra and their average peak strengths and standard deviations. (c) Overlay plot of the Raman spectra obtained from B- (black line) and Raji cells (red line). The difference spectrum is shown below. (d) Scatter plot showing the intensity value of select peaks from individual B- and Raji cell spectra and the average peak strengths and standard deviations.

FIGURE 4

Principal component analysis of all individual spectra of Raji, Jurkat, T-, and B-cells. Plots of the first principal component (PC1) versus the second principal component (PC2) for (a) Jurkat cells and T-cells, (b) Rajicells and B-cells, and (c) Jurkat, Raji, T-, and B-cells.

FIGURE 5

Comparison of the difference spectra between (a) T-Jurkat and (b) B-Raji with the first principal component for each comparison presented in Fig. 4. (c) The T-cell versus Jurkat cell. (d) B-cell versus Raji cell. (e) T-cell versus B-cell versus Jurkat cell versus Raji cell. Increased positive or negative deviation from baseline indicates greater contribution to the PC.