Increased optical pathlength through aqueous media for the infrared microanalysis of live cells

Abstract

The study of live cells using Fourier transform infrared spectroscopy (FTIR) and FTIR microspectroscopy (FT-IRMS) intrinsically yields more information about cell metabolism than comparable experiments using dried or chemically fixed samples. There are, however, a number of barriers to obtaining high-quality vibrational spectra of live cells, including correction for the significant contributions of water bands to the spectra, and the physical stresses placed upon cells by compression in short pathlength sample holders. In this study, we present a water correction method that is able to result in good-quality cell spectra from water layers of 10 and 12 μm and demonstrate that sufficient biological detail is retained to separate spectra of live cells based upon their exposure to different novel anti-cancer agents. The IR brilliance of a synchrotron radiation (SR) source overcomes the problem of the strong water absorption and provides cell spectra with good signal-to-noise ratio for further analysis. Supervised multivariate analysis (MVA) and investigation of average spectra have shown significant separation between control cells and cells treated with the DNA cross-linker PL63 on the basis of phosphate and DNA-related signatures. Meanwhile, the same control cells can be significantly distinguished from cells treated with the protein kinase inhibitor YA1 based on changes in the amide II region. Each of these separations can be linked directly to the known biochemical mode of action of each agent. Graphical abstract.

Keywords: Cancer; Drug-cell interactions; Fourier transform infrared spectroscopy (FTIR); Infrared microspectroscopy (IRMS); Single cell; Synchrotron radiation (SR).

Graphical abstract

Fig. 1

Schematic to show data processing workflow for the water correction algorithm

Fig. 2

Vector-normalised mean spectra of 65 LNCaP cells from three repeat loadings of (a) a 6 μm spacer and (b) a 12 μm spacer. The 12 μm loadings show comparable quality and reproducibility, despite the significantly increased water contribution to be removed

Fig. 3

Normalised mean spectra of 120 cells, from three replicates, overlaid for PL63-treated cells after 1 and 20 h of drug treatment (a) and after 20 h of incubation with DMSO and drug (b). The corresponding second derivative spectra are shown in (c) and (d) to enhance spectral features corresponding to biological changes with drug treatment. The standard deviation of each mean spectrum is shown by the shaded area

Fig. 4

Normalised mean spectra of 120 cells, from three replicates, overlaid for YA1-treated cells after 1 and 20 h of drug treatment (a) and after 20 h of incubation with DMSO and drug (b). The corresponding second derivative spectra are shown in (c) and (d) to enhance spectral features corresponding to biological changed with drug treatment. The standard deviation of each spectrum is shown by the shaded area

Fig. 5

Enlarged region of mean spectra overlaid for PL63-treated cells after 1 and 20 h of drug treatment. Apparent drug-induced changes can be observed particularly at 1217 and 1244 cm⁻¹ as well as from 1180 to 1210 cm⁻¹

Fig. 6

Enlarged region of mean spectra overlaid for YA1-treated cells after 1 and 20 h of drug treatment. Apparent drug-induced changes can be observed across the 1480–1560 cm⁻¹ range, covering the amide II region

Fig. 7

CVA score plot describing 95% of the variance of the second derivative data, showing grouping of DMSO-treated control cells (black) and cells treated with PL63 and YA1 (blue and red, respectively) after 20 h of incubation time