Non-contact mechanical and chemical analysis of single living cells by microspectroscopic techniques

Abstract

Innovative label-free microspectroscopy, which can simultaneously collect Brillouin and Raman signals, is used to characterize the viscoelastic properties and chemical composition of living cells with sub-micrometric resolution. The unprecedented statistical accuracy of the data combined with the high-frequency resolution and the high contrast of the recently built experimental setup permits the study of single living cells immersed in their buffer solution by contactless measurements. The Brillouin signal is deconvoluted in the buffer and the cell components, thereby revealing the mechanical heterogeneity inside the cell. In particular, a 20% increase is observed in the elastic modulus passing from the plasmatic membrane to the nucleus as distinguished by comparison with the Raman spectroscopic marker. Brillouin line shape analysis is even more relevant for the comparison of cells under physiological and pathological conditions. Following oncogene expression, cells show an overall reduction in the elastic modulus (15%) and apparent viscosity (50%). In a proof-of-principle experiment, the ability of this spectroscopic technique to characterize subcellular compartments and distinguish cell status was successfully tested. The results strongly support the future application of this technique for fundamental issues in the biomedical field.

Imaging mechanical properties of sub-micron ECM in live zebrafish using Brillouin microscopy

“…3E). Compared to previous work [36] which performed similar analysis by subtraction the cell's buffer medium from the overall spectrum, our direct fitting approach also works in heterogeneous tissues and is less susceptible to (laser drift induced) artefacts. Thus, we find that careful spectral analysis allows distinguishing mechanical properties of different mechanical components even in a regime where the structure size approaches the optical resolution of the Brillouin microscope.…”

“…Finally, one has also to consider the fact that if the axial extent of the PSF becomes smaller than the attenuation length of the acoustic modes, only an average mechanical property will be probed, i.e. one would not expect a double-peak in the Brillouin spectrum, and thus one could not distinguish and measure the properties of individual components in the probed focal volume [36]. Our results thus encourage a careful consideration of the spatial scales in Brillouin microscopy with respect to the biological structures of interest, especially when optimizing the relevant optical imaging parameters as well as the sample's relative orientation.…”

On the actual spatial resolution of Brillouin Imaging

“…Moreover, as reported in Fig.1 c), NA hugely affects the angular distribution of the collected wavevectors: the deviation θ from the optical axis reaches an average value of 8.5° for the low NA objective and 32° for the high NA one The q-distribution has two remarkable effects on the Brillouin spectra: on one hand, it lowers the spectral definition, due to the fact that both the frequency and the width of the peak depend on |q| [12][13][14] . In this case, a straightforward, though delicate, deconvolution procedure is needed to extract the relevant material parameters 15 . On the other hand, the q-distribution implies also a distribution in the investigated directions, which in NA=0.9 optics can reach up to 65° from the optical axis.…”

“…In order to substantiate these considerations with experimental data, we measured the evolution of Brillouin spectra across a sharp interface between different materials. The change in intensity of the Brillouin peaks allows reconstructing the Edge Spread Function (ESF), from which we can obtain the spatial resolution of the technique by the width of the Point Spread Function (PSF) 15,24 .…”

“…Moreover, in order to discriminate between optical and acoustic contributions to the ESF, we exploited the unique characteristics of our Raman-Brillouin combined set-up 11 report the measured widths for the two couples of materials and for the two optical setups obtained from Brillouin, 2σB, and Raman, 2σR, measurements. The width of the laser spot, 2σL, as measured when focusing on an optically polished silicon wafer, is also indicated 15 . The Raman width 2σR appears equal or slightly larger than the spot width, taking also into account the small effect at these length scales due to the rugosity of the sample surfaces.…”