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.
Highlights
In the vast research area of cellular biology, a largely followed approach consists in attempting to connect cell function with its complex architectural structure and the intricate hierarchy of its dynamic processes
The unprecedented contrast joined with the high-frequency resolution and the extreme statistical accuracy allow the detailed study of the shape of the Brillouin spectrum in a living cell: a micro-system rich in water immersed in a buffer environment
Our results significantly extend to the bulk, the previous analysis performed by atomic force microscope (AFM), which probed the system at lower frequencies and primarily on the surface[54]
Summary
In the vast research area of cellular biology, a largely followed approach consists in attempting to connect cell function with its complex architectural structure and the intricate hierarchy of its dynamic processes. Confocal laser scanning microscopy, immunohistochemistry and fluorescence techniques[1,2,3,4,5] allow for the acquisition of functional images that are pivotal for the characterization of the processes and distribution of molecular species in cells and overcome the diffraction limit Despite their impact, these approaches require chromophores and/or fluorescent dyes that selectively bind subcellular components and could interfere with the natural biological structures and processes under examination. A preferable label-free approach is offered by spectroscopic methods, such as Raman and Brillouin, based on inelastic light scattering processes Based on the interaction of light with spontaneous acoustic phonons in the GHz frequency range, Brillouin spectroscopy is sensitive to the viscoelastic properties of the materials that probe the collective dynamics mediated over the acoustic phonon wavelength
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