Integrated Cryo-Correlative Microscopy for Targeted Structural Investigation In Situ
Marit Smeets,1 * Anna Bieber,2†
Andries Effting,1 Éric Piel,1 1Delmic B.V., Delſt, Te Netherlands
Cristina Capitanio,2† Bassim Lazem,1
2Max Planck Institute of Biochemistry, Martinsried, Germany 3Fondazione Human Technopole, Milan, Italy †Equal contribution
*
smeets@delmic.com Oda Schioetz,2† Philipp Erdmann,2,3 Thomas van der Heijden,1 and Juergen Plitzko2
Abstract: Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET.
Keywords: structural biology, FIB/SEM, CLEM, cryo-ET, cryo-FIB milling
Introduction Cryo-electron tomography (cryo-ET) is an extremely
powerful technique that allows studies of the cellular landscape at high resolution in a near-native state [1]. In cryo-ET, a vitrified cellular sample is imaged in a transmission electron microscope (TEM) as it is tilted from approximately -600 to +600. Tis results in a series of 2D images that can be combined to reconstruct a 3D volume referred to as a tomogram. Tis technique is powerful enough to resolve 3D structures of intracellular organelles and protein complexes within their cellular environment with sub-nanometer resolution [2,3]. Resolving structures in their cellular context at this level of detail is a groundbreaking advancement for structural and cellular biology. Te combined knowledge of the location, interactions, and structure of a biomolecule is crucial to the understanding of its cellular function. Cryo-FIB milling. In order to acquire cryo-ET data,
samples should be thin enough to let electrons pass through without excessive scattering, as this can result in a drop in signal-to-noise ratio and, consequently, image blurring [4]. Sample thickness should ideally be between 100 and 300nm, therefore, most cellular samples for in situ studies need to be thinned down. To do so, the use of a cryogenic focused ion beam with a scanning electron microscope (cryo-FIB/SEM) has become the gold standard. Te FIB is used to mill away the surrounding material and create a thin cryo-lamella [5,6]. Cryo-CLEM. When studying a specific cellular structure, identifying the region of interest (ROI) is a crucial step to mill
20 doi:10.1017/S1551929521001280
a final lamella, which contains the structure of interest. To achieve this, cryo-fluorescence light microscopy (cryo-FLM) is oſten used to precisely localize the ROI and avoid off-target milling [2,7,8]. Using fiducial beads or cellular features as markers, the fluorescence signal can then be correlated to the FIB/SEM or TEM images in an approach commonly known as cryo-correlative light and electron microscopy (cryo-CLEM). Te cryo-CLEM workflow, however, brings about numerous challenges. One of these challenges is sample transfer. Te sample is usually imaged by cryo-FLM before FIB milling and can be inspected again with the cryo-FLM aſter milling to check for the presence of the ROI [9]. Both steps require sample transfer to and from the fluorescence microscope, increasing the risk of ice contamination and damage to the sample, as well as adding days of work to the already laborious process of cryo-ET sample preparation. Moreover, the process of correlative FIB milling is not an easy task. First, it requires markers for navigating to the correct spot on the sample and, even then, the correlation of the fluorescence and SEM images may not always be accurate enough, and the ROI could be missed. Second, sample movement and lamella bending can occur during FIB milling, which can result in the initial correlation no longer holding true and, in turn, the production of lamellae lacking the ROI despite a correct initial correlation. Integrated cryo-CLEM. To overcome these challenges, in
recent years, fluorescence imaging systems that can be directly integrated in the FIB/SEM chamber have been developed [10]. Here we present METEOR, a commercially available widefield FLM designed to be integrated directly into the FIB/SEM chamber. Having an FLM inside the cryo-FIB/SEM greatly reduces the amount of handling steps required, resulting in less contamination, less damage, and a more streamlined cryo-ET workflow. Furthermore, it allows for the easy inspection of the sample with FLM aſter the milling process, thereby improving the yield of the samples containing the ROI that end up in the cryo-TEM. Application example: phase-separated compartments.
Biochemical processes inside of the cell take place in a highly crowded and complex environment. For these processes to be executed correctly, cells need compartments and organelles to organize biological matter. Many compartments are separated from the cellular environment by membranes. Tere are also many membrane-less compartments that are known to form via protein phase separation. Recent work has revealed that phase
www.microscopy-today.com • 2021 November
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