25 Aug 2020
KAIST project combines intravital imaging and new labelling dye to show cellular mechanisms.
Longitudinal imaging of the liver in live animals at microscopic resolution, or intravital imaging, would be a valuable step forward in monitoring hepatic steatosis, the progressive build up of fat in liver cells and the progression of the disease.
A project at the Korea Advanced Institute of Science and Technology (KAIST) has now demonstrated a new imaging protocol that could be valuable in understanding the disease, and used it to monitor tiny droplets of fat accumulating in the liver cells of living mice over time. The work was published in Biomedical Optics Express.
"It has been challenging to find a treatment strategy for NAFLD because most studies examine excised liver tissue, that represents just one timepoint in disease progression," said Pilhan Kim of KAIST.
"Our technique can capture details of lipid accumulation over time, providing a highly useful research tool for identifying the multiple parameters that likely contribute to the disease and could be targeted with treatment."
Among the fluorescence dyes used, KAIST employed a recently developed label, SF44, which can selectively identify lipid droplets of interest in vitro. But until now, direct visualization of those droplets in a mouse model had not been achieved.
The optics breakthrough involved a customized intravital confocal and two-photon microscopy system designed to acquire images of multiple fluorescent labels at video-rate with cellular resolution, allowing individual lipid droplets and microvasculature to be imaged simultaneously at 30 Hz frame rate and 512 x 512 pixel field of view.
"With video-rate imaging capability, the continuous movement of liver tissue in live mice due to breathing and heart beating could be tracked in real time and precisely compensated," said Kim. "This provided motion-artifact free high-resolution images of cellular and sub-cellular sized individual lipid droplets."
Real-time changes in cell behavior
A key to the fast imaging system was the ability to achieve Raster-pattern laser-scanning at 30 Hz. According to the team's published paper, this involved a 36-facet polygonal aluminium-coated mirror rotating at 480 revolutions per second for fast-axis scanning, and a galvanomirror scanner operating at 30 Hz for slow-axis scanning.
The researchers also incorporated different lasers, including 488 and 640-nanometer sources, along with four high-sensitivity optical detectors into the setup. This allowed the platform to acquire multi-color images, and capture the different color fluorescent probes used to label the lipid droplets and microvasculature in the livers of live mice.
In trials, the platform successfully visualized subcellular-level features in the model animals, including accumulation of macrovesicular lipid droplets and associated cellular dynamics consequences. Such direct in vivo observation of hepatic lipid droplets could reveal unknown cellular and molecular mechanisms in the pathogenesis of NAFLD or for the assessment of therapeutics.
Further work will now involve studying how the liver microenvironment changes during NAFLD progression by imaging the same mouse over time, and imaging various immune cells and lipid droplets to better understand the complex liver microenvironment in NAFLD progression.
"Our approach can capture real-time changes in cell behavior and morphology, vascular structure and function, and the spatiotemporal localization of biological components while directly visualizing lipid droplet development in NAFLD progression," said Kim. "It also allows the analysis of the highly complex behaviors of various immune cells as NAFLD progresses."