In situ compositional profiling for endoplasmic reticulum by super-resolution-guided stimulated Raman scattering imaging

The endoplasmic reticulum (ER) is an organelle consisting of a network of membranous structures essential for various cellular activities, making its homeostasis critical for proper cell function. The composition of its membrane can be easily affected by various cellular stressors, triggering ER stress response. Therefore, conducting a detailed structural and compositional analysis of ER is crucial. However, due to resolution limits, analyzing the ER composition in situ remains difficult. Here, we propose a dual-modality imaging and analysis method integrating stimulated Raman scattering (SRS) and structured illumination microscopy (SIM) for imaging the lipid and protein contents of ER structures. With super-resolved structural guidance provided by SIM, the protein/lipid ratio was quantified for ER using multispectral SRS imaging. The spatial mapping of ER compositions in a single cell revealed subcellular diversity in the protein and lipid ratios in the ER structures, which significantly altered under ER stress.