title: Polarized THG microscopy identifies compositionally different lipid droplets in mammalian cells.
abstract: Cells store excess lipids as two major compounds, triacylglycerols (TAGs) and cholesteryl esters (CEs), inside lipid droplets (LDs). The degree of lipid ordering is considered to play a major role in the mobility and enzymatic processing of lipids in LDs. Here, we provide evidence that polarized third-harmonic generation (THG) microscopy distinguishes between native TAG- and CE-enriched LDs in cells due to the different ordering of the two lipid species. We first demonstrate that the responses from synthetic TAG- and CE-enriched LDs using THG microscopy with linear and circular polarizations differ according to their different intrinsic ordering. We then employ simulations to dissect how polarization effects influence the THG from an isotropic LD. Finally, we induce TAG- and CE-enriched LDs in murine macrophages and demonstrate that polarized THG responses increase in a nonlinear fashion with increasing CE/TAG ratio. This suggests that with an increasing CE content, there is a rather sharp transition toward increased LD ordering. Our results demonstrate that polarized THG microscopy enables label-free quantitative analysis of LD ordering and discriminates between compositionally different LDs in intact mammalian cells.
authors.affiliation: Department of Physics, Tampere University of Technology, Tampere, Finland. Electronic address: godofredo.bautista@tut.fi.
authors.forename: Godofredo
authors.initials: G
authors.lastname: Bautista
authors.affiliation: Institute of Biomedicine, Anatomy, University of Helsinki, Helsinki, Finland; Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Simon G
authors.initials: SG
authors.lastname: Pfisterer
authors.affiliation: Department of Physics, Tampere University of Technology, Tampere, Finland; COMP Centre of Excellence and Department of Applied Physics, Aalto University, Aalto, Finland.
authors.forename: Mikko J
authors.initials: MJ
authors.lastname: Huttunen
authors.affiliation: Institute of Biomedicine, Anatomy, University of Helsinki, Helsinki, Finland; Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Ranjan
authors.affiliation: Institute of Biomedicine, Anatomy, University of Helsinki, Helsinki, Finland; Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Kristiina
authors.initials: K
authors.lastname: Kanerva
authors.affiliation: Institute of Biomedicine, Anatomy, University of Helsinki, Helsinki, Finland; Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Elina
authors.initials: E
authors.lastname: Ikonen
authors.affiliation: Department of Physics, Tampere University of Technology, Tampere, Finland.
authors.forename: Martti
authors.initials: M
authors.lastname: Kauranen
mesh_terms: D000818:Animals; D002460:Cell Line; D066292:Lipid Droplets / Q000737:chemistry*; D008264:Macrophages / Q000166:cytology; D051379:Mice; D008853:Microscopy