Multicolor Two-Photon Microscopy Imaging of Lipid Droplets and Liver Capsule Macrophages In Vivo

Lipid droplets (LDs) store energy
and supply fatty acids and cholesterol.
LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD).
Recently, studies have focused on the role of hepatic macrophages
in NAFLD. Green fluorescent protein (GFP) is used for labeling the
characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such
as the NAFLD model. Here, we have developed a tool for two-photon
microscopic observation to study the interactions between LDs labeled
with LD2 and liver capsule macrophages labeled with GFP
in vivo. LD2, a small-molecule two-photon excitation
fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence
upon excitation at 880 nm, high cell staining ability and photostability,
and low cytotoxicity. This probe can clearly observe LDs through two-photon
microscopy (TPM) and enables the simultaneous imaging of GFP+ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1+ LCMs and LDs increased with the progress
of fatty liver disease, and spatiotemporal changes in LCMs were observed
through intravital 3D TPM images. LD2 will aid in studying
the interactions and immunological roles of hepatic macrophages and
LDs to better understand NAFLD.