Polarization gratings aided common-path Hilbert holotomography for high-throughput lipid droplets content assay

Abstract

In this contribution we present a novel polarization gratings aided common-path Hilbert holotomography (CP-HHT) for high-throughput 3D refractive index imaging. Addressing limitations in current holotomography methods, we leverage the extended space-bandwidth product (SBP) through robust phase demodulation using Hilbert spiral transform. Thanks to the application of polarization diffraction gratings our system enables fully tailored holographic settings such as fringe density and shear, thus allowing flexible hologram demodulation, while maintaining simplicity and robustness. The performance is tested on a 3D-printed (using 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 comparable results and resolution to the Fourier transform method in a significantly expanded measurement throughput, bypassing the Kramers-Kronig method. Our CP-HHT approach demonstrates the unique (not possible by fluorescence) high-throughput (especially when compared to cryogenic electron microscopy) capability to differentiate between cholesterol esters vs. triacylglycerol (TAG) rich lipid droplets (LDs), thus has potential for label-free biological research at sub-cellular level. The quantitative analysis of LDs’ refractive index emphasizes the method’s sensitivity in distinguishing between LDs with different neutral lipid content, offering new insights into LD heterogeneity, thus reinforcing the versatility and applicability of our CP-HHT system in broader bioimaging applications.