Lipids are critical for several cellular functions, and any change in homeostasis of cellular lipid content is associated with several metabolic diseases, such as heart disease, diabetes, and fatty liver. Observation of lipid droplets in cells is vital to metabolic dynamics studies in vivo.

To visualize the location of lipid content, lipid-specific fluorescence dyes are used for observation under light microscopes. However, they have technical limitations, such as fluorophore instability. Another alternative is Raman spectroscopy but it limits the observation of real-time dynamics due to low capture speed.

Advantages of 3D Holotomography

Cellular lipids are aggregated as droplets due to their hydrophobic nature. Large droplets have distinctively high refractive index (RI) compared to cell organelles. Holotomography (HT) allows 3Dlocalization of individual lipid droplets without the need for a stain or other preparation. The freedom from any pre-treatment allows easy measurement of the distribution of cellular lipid in live cellsandany changes over time.

TomoStudio™ retrieves quantitative information, like mass, concentration, volume, and surface area of the lipid droplets, based on the 3D RI tomograms. The time-lapse imaging allows tracking the changes of lipid droplets and metabolic studies of cellular lipid content.

There are two major advantages for adapting 3D HTimaging technique to study lipid metabolism invariousorganisms.

High-resolution label-free imaging

Conventionallive-cell lipid content imaging requires staining the cell. However, the stain can disrupt the native condition of the culture environment.

Holotomography detects the absolute RI of each voxel by measuring the phase delay, caused by the

lipid molecules in each compartment. The RI differences between the medium, fatty acids, cytoplasm, or other cell organelles, provides enough contrast to distinguish lipids from other cellular compartments.

Higher specificity

To avoid fast photobleaching or non-specific staining with fluorescence dyes, researchers need additional wash-out procedures and quick observation of the target. This makes it difficult to design a long-term time-lapse observation of lipid droplets. In addition, the low intensity of the fluorescence probes may cause an increase in number and size of the droplets of several fatty acids. Simply put, conventional methods may potentially change the behavior of normal cells.

If you are looking for an efficient 3D label-free imaging at high resolution, check out Tomocube holotomography at Nexus Scientific or contact [email protected].The HT-X is Tomocube’s first high-throughput Holotomographic microscope, capable of 3D label-free imaging at high resolution and 3D fluorescence imaging.