Polarization Gratings Aided Common‐Path Hilbert Holotomography for Label‐Free Lipid Droplets Content Assay

Abstract In this contribution a novel polarization gratings aided common‐path Hilbert holotomography (CP‐HHT) is presented for label‐free 3D refractive index imaging. Addressing limitations in current holotomography methods, the extended space‐bandwidth product is leveraged through robust phase demodulation using the Hilbert spiral transform. The application of polarization diffraction gratings in this system enables fully tailored holographic settings such as fringe density and shear, allowing flexible hologram demodulation, while maintaining simplicity and robustness. The performance is tested using 3D‐printed (fabricated with two‐photon polymerization) brain phantom and fixed HeLa cells supplemented with cholesterol and oleic acids. Reconstruction analysis using the brain phantom indicates that the Hilbert method provides improved spatial resolution to the Fourier transform method in a significantly expanded measurement information content. This CP‐HHT approach demonstrates the unique (not possible by fluorescence) ability to measure the lipid droplets enriched with cholesterol and oleic acid and highlights that they exhibit measurable differences in their refractive index values. These findings suggest that this method is sensitive to variations in neutral lipid content, offering promising insights into lipid droplet heterogeneity and supporting its potential for label‐free sub‐cellular bioimaging applications, thus reinforcing the versatility and applicability of this CP‐HHT system in broader bioimaging applications.