Communication Dans Un Congrès Année : 2024

Spontaneous Raman and Stimulated Raman spectroscopy for the analysis of adipocytes

Spectroscopie Raman spontanée et stimulée pour l'analyse des adipocytes

Olaia Naveiras
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Résumé

Performing in-situ analyses of biomolecules in living cells is crucial in biomedical research. There is growing interest in developing techniques to characterize adipocyte maturation rapidly and non-invasively. Multiple methods are available to assess the composition of adipocytes. However, these methods are destructive or invasive. Spontaneous Raman spectroscopy (SpRS) is a label-free method to assess adipocytes in-vitro. This technique provides molecular composition information at a micrometer scale without damaging the sample. The Raman spectrum assesses the molecular composition within the probed micro-volume (~2 µm). SpRS suffers from a low signal-to-noise ratio (SNR), leading to long acquisition times of Raman images. Recent development like Stimulated Raman Spectroscopy (SRS) has improved SNR, enabling fast 3D Raman images (100×100×40 µm). This study compares SpRS and SRS for adipocyte analysis. For this work, lipid composition of a murine bone marrow derived stromal cell line was assessed in two differentiation conditions, either through classical induction of adipogenesis (medium supplemented with dexamethasone, 3-isobutyl-1-methylxanthine, and insulin) or through spontaneous adipogenesis (non-supplemented, serum-containing medium) after 17 days in culture. SpRS were done with an HR800 (HORIBA). SRS were done on a Stellaris 8 CARS/CRS (Leica Microsystems). Both SpRS and SRS allows the analysis of individual lipids droplets down to 2µm (LD) within adipocytes. Raman spectra from SpRS are obtained within the fingerprint region (containing characteristics Raman bands) with a high spectral resolution (0.5 nm). The lipid composition can be imaged (Figure A). LD diameters are evaluated by analysis of the optical image. The tradeoff with SpRS is a long acquisition time for 2D Raman images up to 2 adipocytes (~ 3h/image). SRS analysis allows fast acquisition of 3D Raman images of up to 3 adipocytes within 30 min (Figure B). From 3D images, diameter, volume, number of neighbors and unsaturation ratio are evaluated by image analysis. The tradeoff with SRS is the acquisition of one individual band per image with a low spectral resolution (4 nm). SpRS and SRS analyses found similar results. Classical adipogenic induction led to the preferential accumulation of unsaturated lipid species, as opposed to lower unsaturation ratio on spontaneously differentiated adipocytes coupled to differential lipid droplet size. In conclusion, SpRS and SRS are powerful tools for adipocyte analysis in vitro. SpRS is suitable for detailed spectral analysis while SRS is ideal for faster 3D imaging of multiple cells.
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Dates et versions

hal-04938951 , version 1 (10-02-2025)

Identifiants

  • HAL Id : hal-04938951 , version 1

Citer

Hélène Rougé-Labriet, Jérôme Delattre, Guillaume Falgayrac, Magalie Lacaze, Josefine Tratwal, et al.. Spontaneous Raman and Stimulated Raman spectroscopy for the analysis of adipocytes. 8th International meeting on Bone Marrow Adiposity, BMAS, Sep 2024, Montreal (Canada), Canada. ⟨hal-04938951⟩
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