Figure 1.
Imaging the visceral adipose tissues (VATs) of lean and obese mice with NLO microscopy.
(A) Histology of lean (upper panel) and obese (lower panel) VATs. (B) CARS imaging of lipid droplets of adipocytes (red) and two-photon fluorescence (TPF) imaging of preadipocytes (green) immunolabeled with FITC-conjugated antibodies to Pref-1 of a lean VAT. (C) CARS imaging of lipid droplets (red) and two-photon autofluorescence imaging of unidentified cells (green) surrounding an adipocyte of an obese VAT.
Figure 2.
Lipid-rich adipose tissue macrophages (ATM) identified with Oil Red O (ORO) staining and immunofluorescence imaging.
(A) Phase contrast and bright field images of isolated VAT stromal cells stained with ORO. (B) Immunofluorescence images of isolated VAT stromal cells stained with conjugated antibodies to CD4 and CD68 cell surface markers for macrophages.
Figure 3.
Potential source of autofluorescence and lipid in macrophages.
(A) Widefield fluorescence images of RAW 264.7 macrophages (left panel) and RAW264.7 macrophages in co-culture with explanted VATs (right panel). (B) Peptide array to identify adipose tissue secreted pro-inflammatory cytokines. (C) Immunofluorescence (IF) imaging and (D) Western blot analysis revealed increased expression of iNOS in RAW264.7 macrophages co-cultured with explanted VATs as compared to control RAW264.7 macrophages. (E) RAW264.7 macrophages co-cultured with explanted VATs accumulated intracellular lipid droplets observable with ORO staining (upper right panel) and CARS imaging (lower right panel). (F) Real time PCR analysis of gene expression of fatty acid binding proteins (encoded by Fabp1-6 genes) and fatty acid transport proteins (encoded by Slc27a1-6 genes) in control RAW264.7 macrophages and RAW264.7 macrophages co-cultured with VATs.
Figure 4.
Evidence for lipid mobilization from adipocyte and uptake of exogenous lipid by ATMs.
(A) Free fatty acids (FFA) concentration of the VAT conditioned medium as a function of incubation time. Error bars represent standard deviation across triplicate measurements. (B) Evidence of lipid droplet microvesiculation revealed by CARS imaging. (C) An autofluorescent and lipid-rich ATM was observed in proximity of adipocytes with lipid droplet microvesiculation. (D) Monitoring the trafficking of exogenous deuterated palmitic acid with spontaneous Raman microspectroscopy. Absence and presence of C-2H peak in the lipid droplets of adipocytes and ATMs, respectively, were observed.
Figure 5.
Imaging the kinetics of lipid accumulation in ATMs of explanted VATs.
Steady accumulation of lipid droplets in ATMs was observed with CARS microscopy as a function of time in explanted VATs. Collagen fibrils visualized with second harmonic generation were displayed in blue.
Figure 6.
Quantitative analysis of VAT composition in lean and obese mice.
(A) Average number of lipid-rich ATMs per analysis volume with xyz dimensions of 250 µm×250 µm×50 µm. (B) Average diameter of lipid droplets of ∼200 adipocytes for lean VATs and ∼100 adipocytes for obese VATs. (C) Normalized level of collagen fibrils type I, as measured by SHG intensity, in the VATs of lean and obese mice. Data was normalized to 1 for lean VATs and respectively for obese VATs. Error bars represents standard deviation across 81 volumes analyzed.