The substrate matters in the Raman spectroscopy analysis of cells

Abstract

Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research.

Figure 1

(a) Sketch of a yeast cell and (b) different components of the cell identified by RS under 514.5 nm laser excitation. Image was drawn by the author Raul D. Rodriguez.

Figure 2

(a) Raman spectra of the five substrates used for the Raman spectroscopy analysis of yeast cells: silicon with native 2–3 nm layer of SiO_x; 100 nm SiO₂ on Si; objective glass slide; highly oriented pyrolytic graphite (HOPG), and gold. A 514.5 nm laser was used at 2 mW power, acquisition time 10 min. (b) Raman spectroscopy results of the yeast deposited on the different substrates. The grey boxes show the spectral regions overlapping with the (subtracted) substrate signal. Trp refers to tryptophan. (c) Maximum intensity of the band at 1310 cm⁻¹ for the different substrates.

Figure 3. Numerical simulations using finite element method of electric field distribution in a yeast cell and its surroundings deposited on different substrates:

(a) air, (b) gold, and (c) HOPG substrate (d) 3D image of electric field distribution in the yeast cell on gold. The simulations were performed under 515 nm excitation.

Figure 4

(a) Raman spectra of a silver SERS substrate before and after cleaning in a diluted HNO₃ solution; (b) the AFM visualization of the nanostructured silver substrate; (c) Selective single-cell attachment on the nanostructured Ag substrate is observed as compared to (d) the multiple cell aggregate formation on the glass substrate. (e) Raman spectra comparison of yeast cells deposited on a smooth Ag surface, glass, and on Ag particles. The inset shows a SEM image of some Ag nanocrystals.

Figure 5

(a) Atomic force microscopy image of a single cell on the silver nanoparticle substrate. (b) Raman line scan showing the spatial distribution of the CH₂ vibration at 1446 cm⁻¹ related to proteins and lipids. (c) Raman spectrum of a single cell using SERS, the high enhancement even makes the bands visible at the same level as the silicon substrate allowing the observation of the phenyl band that was previously masked. No substrate background subtraction was performed in this case.