Raman spectral imaging of single living cancer cells: a preliminary study

Abstract

Raman microspectroscopy allows probing subcellular compartments and provides a unique spectral fingerprint indicative of endogenous molecular composition. Although several spectroscopic cell studies have been reported on fixed samples, only few attempts concern single growing cells. Here, we have tested different optical substrates that would best preserve cell integrity and allow direct measurement of Raman spectra at the single living cell level. Calu-1 lung cancer cells were used as a model and their morphology and growth were assessed on Raman substrates like quartz, calcium fluoride, and zinc selenide. Data show that quartz was the most appropriate taking into consideration both cell morphology and proliferation rate (47% on quartz vs. 55% of BrdU-positive cells on conventional plastic). Using quartz, 40 cells were analysed and Raman spectra were collected from nuclei and cytoplasms using a 785 nm laser excitation of 30 mW at the sample, in the spectral range of 580-1750 cm(-1), and an acquisition time of 2 x 10 sec/spectrum. Discriminant spectral information related to nucleus and cytoplasm were extracted by multivariate statistical methods and attributed to nucleic acids, lipids, and proteins. Finally, Raman spectral imaging was performed to show the distribution of these components within the cell.

Improved protocols for vibrational spectroscopic analysis of body fluids

“…Whereas dry samples are often required for FTIR analysis of tissue sections, Raman microspectroscopy is perfectly compatible with water immersion measurement which enhances the signal to background ratio and the collection of higher quality data [36,37]. The weak contribution from water offers the possibility to study cells in an aqueous environment and thus to keep them alive for the duration of the measurement [2,31,38], whereas the strong absorption from water means that such measurements in infrared can only be performed with high brightness sources such as synchrotrons [39].…”

“…They are routine techniques for fingerprinting and identifying chemicals and act as standard methods in analytical chemistry and pharmacy [1]. Both techniques being truly label-free, since the inherent vibrational signature of the biochemistry of the cell or tissue is being observed [2], their potential for di- …”

Identifying and localizing intracellular nanoparticles using Raman spectroscopy

“…Using a 785 nm laser as source, a 10 s acquisition gives a signal to noise ratio more than acceptable for the identification of the different spectral features present. 22,38 However, two accumulations are required by the software to remove any spurious spikes in the spectra recorded, and thus the total acquisition time per spectrum was 20 s (2x10 s). A mild smoothing was applied to further improve the spectral quality, as described in Section 2.8.…”

“…In comparison to infrared spectroscopic analysis, the weak contribution from water offers the possibility to study the cells in an aqueous environment, maintaining viability for the duration of the measurement. 22 The specific information contained in the Raman spectrum of the cells or subcellular compartments provides a signature of the sample studied which can be related to molecular content or changes to the physiology as a result of external stimuli. [23][24][25][26][27][28] In confocal microscopic mode, the spatial resolution is of the order of ≤1 µm, depending on source wavelength, providing access to the subcellular organisation of the cells.…”

Three dimensional collagen gels as a cell culture matrix for the study of live cells by Raman spectroscopy

“…Furthermore, the spatial resolution is diffraction limited to ~10m, and thus IR absorption is not the favoured technique for live cell analysis. The weak scattering efficiency of water and the high spatial resolution of the visible or near -IR wavelengths employed for Raman microspectroscopy place this technique as the favoured tool for the study of living cells 13,14 , whereby live cells in a physiological solution can be mapped with an immersion lens giving sub cellular resolution of order of 1m 15 .…”