Excitation Spectral Phasor Microscopy (ExSPM) unveils spatiotemporal heterogeneities in polarity of intracellular lipid droplets

• DOI: 10.1101/2025.04.03.647137
• Publication Date: 2025-04-05T21:05:11Z
• Authors: Jiayi Liu; Yi He; Rui Yan; Jinhong Yan; Ruirong Wang; Zhipeng Zhang; Jiankai Xia; Igor Kireev; Ke Xu; Kun Chen

AbstractSpatiotemporal heterogeneities in the polarity of intracellular LDs play essential roles in cellular function but remain largely unexplored. Here, we introduce excitation spectral phasor microscopy (ExSPM), which combines high-throughput excitation spectral imaging with phasor analysis to detect subtle spectral shifts of fluorophores in response to local intracellular environments with exceptional sensitivity and spatiotemporal resolution. Using the lipophilic dye Nile Red, we reveal substantial heterogeneities and dynamic changes in LD polarity within single mammalian cells. We further demonstrate LDs polarity is associated with lipid catabolism through lysosome-dependent acid lipophagy or neutral lipolysis at LDs–mitochondrion contact sites, underscoring its key role in cellular energy homeostasis. Additionally, ExSPM reveals that nutrient deprivation preferentially upregulates neutral lipolysis over acid lipophagy to meet cellular energy demands. Together, ExSPM provides a highly sensitive approach for resolving intricate spatiotemporal heterogeneities in intracellular composition and function, offering broad potential for the investigation of diverse cellular processes.