Optimized workflow of site-specific cryo-lamella preparation for cryo-ET using integrated light and electron microscope.

Cryo-electron tomography (cryo-ET) is an essential tool for resolving cellular structures in their native state. However, achieving site-specific sample preparation remains a significant challenge, particularly for deeply buried or rare cellular targets. Focused ion beam (FIB) milling is commonly used to prepare thin lamellae from vitrified samples, but traditional FIB methods often lack the abilities to target specific regions of interest. Correlative light and electron microscopy (CLEM) overcomes this limitation by combining light microscopy (LM) with scanning electron microscopy (SEM), enabling the identification and localization of structures of interest within the specimen. This targeted approach enhances the accuracy and efficiency of FIB milling by ensuring that lamellae are thinned at precisely the right locations. Recent advances in integrated cryo-CLEM workflows have streamlined this process, offering enhanced precision and reproducibility in sample preparation for cryo-ET. Here, we present an optimized protocol that utilizes this integrated approach to identify and target specific cellular structures, such as the contact sites between lipid droplets (LD) and mitochondria. This protocol facilitates the precise preparation of cryo-lamellae and enhances the efficiency of data acquisition in cryo-ET, offering a promising strategy for high-resolution structural biology studies.