Lipid membranes, heterogeneous structures surrounding most intracellular organelles, play crucial roles in regulating organelle function. Their morphology, composition, and phase are dynamically regulated during the cell cycle, influencing biophysical properties, membrane protein function, and lipid-protein interactions. However, *in vivo* studies of lipid membranes have been limited by insufficient spatiotemporal resolution, throughput, and long-term stability of existing techniques. This study addresses these limitations by developing a novel imaging technique to study the lipid heterogeneity and dynamics within live cells. The research aims to provide a high-resolution, high-throughput method for visualizing and quantifying lipid membrane properties in various organelles, enabling a deeper understanding of their roles in cellular processes.

Previous studies have investigated lipid membranes using various approaches, but these often suffered from limitations in resolution, throughput, or the ability to simultaneously measure multiple properties. Some methods focused on measuring lipid polarity using ratiometric imaging techniques, while others focused on measuring lipid order using polarization microscopy. However, these techniques often made simplifying assumptions about the relationship between polarity and order, an assumption that is not necessarily valid in the complex environment of a mammalian cell with its diverse lipid composition. Mass spectrometry techniques can quantify molecular lipid compositions, but these are not suitable for live-cell studies. The current study aims to overcome these limitations by developing a new method that combines high resolution with the ability to simultaneously measure both lipid polarity and order in live cells.

The researchers developed a novel super-resolution microscopy technique called Spectrum and Polarization Optical Tomography (SPOT). This technique utilizes Nile Red, a common lipid dye, to universally stain lipid membranes in live cells. SPOT leverages three optical dimensions—intensity, spectrum, and polarization—to obtain high-dimensional information about the lipid membranes. Structured illumination microscopy (SIM) was initially used to improve spatial resolution. However, to further enhance accuracy in measuring lipid polarity and phase, SPOT was developed. SPOT employs structured illumination with multiple phase shifts and the HiLo method to reject out-of-focus background signals. Three pattern directions are used for polarization modulation analysis. The effectiveness of SPOT was validated using simulations and experiments involving actin filaments labeled with fluorophores of known properties. The method was then used to investigate lipid heterogeneity and dynamics in various organelles of U2-OS cells.

SPOT successfully resolved lipid heterogeneity across ten subcellular compartments, including the plasma membrane, nuclear membrane, endoplasmic reticulum (ER), mitochondria, lipid droplets, Golgi apparatus, lysosomes, and endosomes. Quantitative analysis revealed significant differences in both lipid polarity (measured by emission ratio) and phase (measured by polarization modulation depth) among these compartments. For example, nuclear membranes and ER showed high polarity and relatively high order, while plasma membranes and early endosomes showed low polarity and high order. Lipid droplets exhibited the lowest polarity and order. Intra-organelle heterogeneity was also observed, particularly within mitochondria, where the cristae showed higher polarity than the outer membrane. Dynamic changes in lipid properties were observed in endosomes during maturation and in lipid droplets during growth. Time-lapse imaging during cell division revealed significant lipid remodeling during plasma membrane separation, tunneling nanotube (TNT) formation, and mitochondrial cristae dissociation. The observed changes in lipid polarity and order during these processes suggest that lipid remodeling is crucial for these cellular events.

The results demonstrate the power of SPOT in revealing previously unseen details of subcellular lipid organization and dynamics. The ability to simultaneously measure both lipid polarity and phase provides a more complete picture of membrane properties than previous methods. The findings challenge the assumption that higher polarity implies lower order, demonstrating the complexity of lipid interactions in living cells. The dynamic changes observed during cell division highlight the importance of lipid remodeling in cellular processes. SPOT’s high spatiotemporal resolution provides a new approach for studying lipidomics and organelle interactome, opening avenues for further investigation into the relationship between lipid membrane properties and cellular function.

This study presents a significant advance in super-resolution imaging of subcellular lipid membranes. The novel SPOT technique provides high-dimensional, high-throughput imaging, revealing previously unobserved heterogeneity and dynamics within live cells. The findings highlight the importance of lipid remodeling in fundamental cellular processes. Future research could focus on expanding the applicability of SPOT to other cell types and exploring the use of additional dyes to provide more detailed information about lipid composition. Furthermore, integrating SPOT with other multiplexing techniques could provide even richer insights into the interplay between lipid membranes and cellular function.

While SPOT offers significant advantages, some limitations exist. The lateral resolution is still diffraction-limited, although axial resolution is improved. The interpretation of optical properties relies on the characteristics of Nile Red, and potential dye penetration and orientation differences in various membranes might influence measurements. Future calibration studies using *in vitro* lipid vesicles with known composition would further refine the correlation between optical and lipid properties. Automatic segmentation of organelles using AI could enhance data analysis. Despite these limitations, the novel insights gained from SPOT's high-dimensional data outweigh the limitations, offering an important tool for cellular lipidomics.