Characterization of different substrates for Raman spectroscopic imaging of eukaryotic cells

Abstract

For Raman spectroscopic analyses of the cells and other biological samples, the choice of the right substrate material is very important to avoid loss of information in characteristic spectral features because of competing background signals. In the current study, Raman spectroscopy is used to characterize several potential Raman substrates. Raman vibrational bands of the substrate material are discussed. The surface topography is analyzed by atomic force microscopy, and the root mean square surface roughness values are reported. Biocompatibility of the substrates is tested with Hep G2 cells evaluating cellular morphology as well as live/dead staining. Calcium fluoride, silicon, fused silica, borofloat glass, and silicon nitride membranes support cell growth and adherence. Silicon, borofloat glass, and fused silica give rise to Raman signals in the region of interest. Calcium fluoride substrate (UV grade) is suitable for Raman spectroscopic investigation of living cells. Nickel foil is suitable substrate for Raman spectroscopic investigation but cellular adherence and viability depend on the quality of the foil. Silicon nitride membranes coated with nickel chrome is a suitable Raman substrate in closed microfluidic systems.

Recent advances in linear and non‐linear Raman spectroscopy. Part XI

“…The authors emphasize that the right substrate material is very important to avoid loss of information in characteristic spectral features because of competing background signals. In the current study, Raman spectroscopy was used to characterize several potential Raman substrates and the Raman vibrational bands of the substrate material were discussed . Schie and co‐workers provide an estimation of spectra sample size for characterizing single cells using micro‐Raman spectroscopy.…”

“…In the current study, Raman spectroscopy was used to characterize several potential Raman substrates and the Raman vibrational bands of the substrate material were discussed. [62] Schie and co-workers provide an estimation of spectra sample size for characterizing single cells using micro-Raman spectroscopy. Their work demonstrates the importance of adequate spectral sampling and presents an approach for determining the minimum sample size needed to reproduce a Raman spectrum of a whole cell, which are expected to impact future single-cell Raman spectroscopy studies.…”

Spectral decomposition of Raman spectra of mixed‐phase TiO2 thin films on Si and silicate substrates

“…Several computational and experimental approaches such as shifted‐excitation Raman difference spectroscopy are used to remove such noise . An overview and comparison of these approaches can be found in Cordero et al The Raman activity of substrates is an issue in thin‐film Raman spectroscopy . Often, arbitrary linear “backgrounds” are subtracted before Raman peaks are fitted, which is inappropriate for cases in which the intensity of the Raman background characteristic is in the same order of magnitude or higher than the Raman spectra of the analyte.…”

“…[16][17][18] An overview and comparison of these approaches can be found in Cordero et al [19] The Raman activity of substrates is an issue in thin-film Raman spectroscopy. [20,21] Often, arbitrary linear "backgrounds" are subtracted before Raman peaks are fitted, which is inappropriate for cases in which the intensity of the Raman background characteristic is in the same order of magnitude or higher than the Raman spectra of the analyte. That is why the evaluation of thin-film Raman measurements is often very restricted, and they are discussed mainly qualitatively, and often, only the most intense peaks are considered.…”

Micro‐Raman detection of the differentiation state of SH‐SY5Y cells grown on silicon and aluminium substrates

“…Contrary to the common biochemical methods routinely used for the characterization of cells, Raman microspectroscopy provides a chemical fingerprint of the single living cell without fixation, lysis, or the use of labels and other contrast‐enhancing chemicals . In vitro Raman measurement requires growing cells on a substrate as a sample holder, whose Raman signals need to be small and constant and the biocompatibility ensured . It is thus worth to investigate the potential of this noninvasive technique in monitoring the RA‐BDNF treatment effects on SH‐SY5Y cells and analyse concomitantly the role played by different substrates on differentiation outcomes.…”