Multimodal imaging of cells and tissues: All photons are welcome

Research groups around the world are studying the spatial location and distribution of molecules within cells using an increasing number of analytical techniques such as infrared (IR), Raman and X-ray fluorescence (XRF) spectroscopy. The information obtained from these techniques in terms of lipids, proteins and the general metabolome is complementary, but commonly the analysis of the data is performed individually on each technique. These three techniques are based on different interactions of the sample with light with different energy and wavelengths, leading to dissimilarities in the spectral features offered by each technique. Table 1 summarises the main features of Raman and IR microspectroscopy, both representing vibrational spectroscopic methods. Raman spectroscopy is a scattering technique, in which energy is transmitted from a photon to a molecule, resulting in a shift in the wavelength of the incident light beam. Fourier transform (FT)-IR spectroscopy, on the other hand, is an absorbance technique where the molecule absorbs a photon and gains energy moving from a lower to a higher vibrational energy state. These methods are complementary in terms of providing molecular information on samples, as molecules or functional groups that tend to be strong Raman scatters are usually weak IR absorbers and vice versa. The techniques also complement each other in terms of their advantages and disadvantages for the investigation of biological systems. FT-IR spectroscopy is a non-destructive method with a good signal-to-noise (S/N) ratio and a high efficiency. Raman may lead to a thermal destruction of a cell or tissue due to the high output power of the light source and has a considerably lower S/N ratio, unless the energy of the incident light is close to an electronic transition of the analyte. In that case, resonance Raman enhances the S/N by several orders of magnitude. Surface enhanced Raman spectroscopy (SERS) can also be used for increasing the S/N ratio of Raman spectroscopy. However, as the light source in Raman is a typically a laser with wavelength ranging from the ultraviolet to near IR (240–1064 nm), the achievable spatial resolution is higher depending on the wavelength of the incident photons. Furthermore, as water is a weak Raman scatterer, cells and tissues can be studied using Raman spectroscopy under physiological conditions.