# Label-free imaging throughout adipocyte differentiation by stimulated Raman microscopy

Ferrara M. A., Filograna A., Ranjan R., Corda D., Valente C., Sirleto L.

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Lipid droplets (LDs) are dynamic organelles that play crucial roles in regulating the storage and the turnover of neutral lipids. Furthermore, extensive studies underline the relevant connection between lipid storage defects and the pathogenesis of several metabolic diseases, including obesity, diabetes, atherosclerosis as well as cancer. Nevertheless, many aspects of LD biology are still unrevealed, probably due to the current use of invasive imaging methods that often affect the formation and the maturation of LD. Thus, it becomes increasingly urgent to develop reliable imaging tools to unambiguously examine the dynamics of LD accumulation within their cellular context. Here, we used Stimulated Raman Scattering (SRS) microscopy as a label-free method to image LDs in 3T3-L1 adipocyte cells. This non-invasive technique allows us to acquire images with high spectral and spatial resolution along with three-dimensional sectioning and resolving capabilities of lipids and proteins during adipocyte differentiation. Our results show an increased number of large $(>15 \mu$ m${}^{2}$\$) LDs during adipocyte differentiation. Furthermore, we provide a detailed separation of cellular distribution and spatially resolved maps of lipids and proteins at different stages of adipocyte differentiation. Our data demonstrate that stimulated Raman imaging is an advanced label-free approach that allows to examine the physiology of intracellular lipids and opens up new avenues for the diagnosis of LD-associated pathologies.