In situ cryo-FIB/SEM Specimen Preparation Using the Waffle Method

Oleg Klykov, Daija Bobe, Mohammadreza Paraan, Jake Johnston, Clinton Potter, Bridget Carragher, Mykhailo Kopylov, Alex Noble

Automation in Cryo-FIB Preparation, from Cellular to Tissue Structural Biology

Patrick Cleeve, Monica Pia Caggiano, David Dierickx, James Whisstock, Alex de Marco

Joshua Mendez

Rohit Kannachel

Edward Eng

Daija Bobe

Gabriella Angiulli

Alex de Marco

Director, Electron Microscopy

Cryo-EM Research

de Marco LabThe de Marco Lab, led by the Simons Electron Microscopy Center (SEMC) Director Alex de Marco, develops cryo-electron tomography (cryo-ET) into a routine, high-throughput tool for studying molecular structure directly inside cells. The group’s goal is to make in situ structural biology accessible and automated through every stage of the workflow—from sample preparation and cryo-FIB lamella fabrication through data collection and analysis—so that native cellular environments can be imaged with the scale and reproducibility that purified-sample cryo-EM already enjoys.The lab focuses on developing methods and instrumentation: automated cryo-FIB milling and correlative targeting; new devices and software for sample preparation and microscope control; and subtomogram-averaging pipelines that recover high-resolution structures from crowded cellular interiors. Building these tools within SEMC’s high-volume service environment lets the lab test new approaches on real user workflows and disseminate them to the broader structural biology community. These methods are applied to questions that require structural information across scales: how cells establish and maintain polarity; how molecular machines organize cell division; and how dynamic macromolecular assemblies change structure and composition during cellular processes. By pairing technology development with biological discovery, the group studies molecular events in their native context, bridging isolated complexes and intact cells.MethodsOptimizing cryo-EM data acquisition: SEMC researchers develop and refine strategies that improve the efficiency, reproducibility, and information content of cryo-EM and cryo-ET data collection. This includes work on acquisition workflows, dose management, targeting, throughput, and approaches that make high-resolution imaging more robust across different specimen types. Cryo-FIB methods and automation: The program advances cryo-focused ion beam methods for in situ structural biology, including lamella preparation, lift-out workflows, plasma-FIB approaches, correlative targeting, and automation designed to increase throughput while preserving native cellular architecture. Sample preparation and freezing technologies: SEMC develops and applies preparation workflows that support a wide range of biological samples, including plunge freezing, blotless vitrification, high-pressure freezing, cryo-CLEM-guided targeting, micropatterning, and other approaches that improve specimen quality for single-particle cryo-EM and cellular cryo-ET. Improving cryo-EM and cryo-ET data analysis: Research in data analysis focuses on extracting biological information from heterogeneous and flexible samples. Areas of emphasis include single-particle analysis of flexible RNA and ribonucleoprotein assemblies, methods for resolving conformational heterogeneity, subtomogram and tomographic analysis, and segmentation approaches that connect molecular structure to cellular context. Ultra-sensitive proteomics and multiomics approaches: SEMC also integrates structural biology with ultra-sensitive proteomics and emerging multiomics strategies. These approaches enable researchers to connect molecular structure, protein composition, and cellular state, supporting studies where limited sample material or single-cell measurements are essential.Biological ApplicationsSEMC research applies these methods to biological questions that require structural information across scales, from purified molecular assemblies to native cellular environments. Current application areas include understanding how cells establish and maintain polarity, how molecular machines organize cell division, and how dynamic macromolecular assemblies change structure and composition during cellular processes.

Cryo-FIB (Focused Ion Beam)

Cryo-focused ion beam (cryo-FIB) milling enables researchers to prepare thin, electron-transparent lamellae directly from whole cells, tissues, or larger specimens while preserving native structure. By removing excess material while keeping the region of interest intact, cryo-FIB transforms thick biological samples into tractable imaging experiments for high-resolution tomography.NYSBC operates complementary FIB instruments: the Aquilos2 for high-precision conventional FIB milling and the Hydra and Arctis plasma-FIB systems for faster throughput and advanced workflows. Both platforms support iterative workflows that pair light microscopy targeting with cryo-FIB milling, enabling researchers to locate specific cellular features and prepare them for tomographic imaging. Staff-supported service, hands-on training, and collaborative projects are all available as needed.