PinCorr: A high-pressure freezing carrier with intrinsic landmarks for cryo-correlative light and electron microscopy

Abstract

Cryo-correlative light and electron microscopy methods enable targeted structural analysis of fluorescently labelled features in vitrified specimens. However, correlative workflows on high-pressure frozen samples often remain challenging due to the lack of persistent landmarks for reliable sample tracking and image registration between different microscopes. Standard high-pressure freezing carriers provide little intrinsic reference information, as the exposed sample surface is often smooth and rotationally ambiguous, complicating localisation of regions of interest across imaging platforms. Here, we introduce PinCorr, a 3-mm high-pressure freezing carrier with an integrated coordinate system formed by four asymmetrically arranged pillars with distinct geometries. These built-in landmarks remain visible after freezing and provide a stable, sample-independent reference frame for orientation and correlation between cryo-fluorescence microscopy and electron microscopy. We show that PinCorr supports fluorescence-guided cryo-volume imaging, serial lift-out for cryo-electron tomography and freeze-substitution workflows followed by room-temperature on-section correlation. PinCorr thus provides a hardware-based approach to establishing a persistent spatial reference frame in HPF-based correlative imaging workflows for thick and multicellular specimens.