Combining Correlative Cryogenic Fluorescence and Electron Microscopy and Correlative Cryogenic Super‐Resolution Fluorescence and X‐Ray Tomography—Novel Complementary 3D Cryo‐Microscopy Across Scales to Reveal Nanoparticle Internalization Into Cancer Cells

Understanding the intracellular fate of nanoparticles (NPs) is essential for advancing nanomedicine, particularly in targeted drug delivery for cancer therapy. Here, we present a complementary cryogenic microscopy workflow across scales to investigate the uptake and subcellular localization of zirconyl-containing inorganic-organic hybrid nanoparticles (IOH-NPs) in murine breast cancer cells. Our approach integrates cryogenic fluorescence microscopy (cryo-FM), cryo-focused ion beam scanning electron microscopy (cryo-FIBSEM), and cryo-soft X-ray tomography (cryo-SXT), enabling molecular specificity, high-resolution imaging, and volumetric ultrastructural analysis in near-native cellular states. We demonstrate that the cryogenic workflow provides enough contrast and resolution across all modalities for quantifying the IOH-NP uptake: NPs are internalized within 2 h of incubation and progressively accumulate in endolysosomes over time, as confirmed by fluorescence labeling and SXT. Quantitative analysis reveals a marked increase in endolysosomal accumulation of IOH-NPs from 2 to 24 h. Our findings help to establish multimodal cryogenic microscopy as a powerful tool for nanoscale imaging and quantitative analysis of NP uptake within close-to-native cells, offering new insights into NP trafficking and cellular responses relevant to nanomedicine development.