Integrated Cryo-Correlative Microscopy
separation plays a key role in a variety of other cellular processes and diseases like cancer and neurodegeneration [11–13]. Wilfling et al. reported a selective autophagy pathway for
phase-separated endocytic protein deposits in yeast [14]. In this pathway Ede1 acts as a selective autophagy receptor and mediates the phase separation of Ede1-dependent endocytic proteins into condensates (END condensates). By employing correlative cryo-ET they were able get a high-resolution understanding of the END condensates.
Materials and Methods METEOR platform. METEOR is a top-down widefield
FLM that can be integrated directly into a cryo-FIB/SEM. Te objective lens of the FLM is placed parallel to the FIB column, allowing for easy switching between FIB milling and FLM imaging. To allow fluorescence imaging inside the FIB/SEM
chamber, the excitation and emission lights need to be guided in and out of the vacuum chamber. In Figure 1, the basic light path and the most important optical components that allow for in-vacuum imaging are depicted. Te excitation beam from a LED light source is first guided to a dichroic mirror. Te dichroic mirror reflects the excitation beam to a vacuum window. Te vacuum window allows light to pass through the vacuum-to-air interface and is designed to fit on one of the existing ports of the FIB/SEM. METEOR can, therefore, be installed on a FIB/SEM without making permanent changes to the chamber. Inside the vacuum, the excitation beam passes through the objective, which is attached to an objective stage that is used for focusing of the objective on the sample and acquiring z-stacks. Te travel range of the stage is 31mm, so the objective can be retracted to above the pole piece when the user has finished using METEOR to allow for full FIB/SEM stage movement. Te emitted light from the sample is guided back through the vacuum window to the dichroic mirror. Te emission beam passes through the dichroic mirror, and an emission filter in the filter wheel blocks unwanted wavelengths from reaching the detector. Te remaining light beam passes
through to the camera, forming an image. Te filter wheel contains four slots, and the filters are customizable according to fluorescence labels of choice. Soſtware. To improve the user-friendliness of the system
we also developed dedicated soſtware named ODEMIS for METEOR. Tis intuitive, open-source soſtware allows the user to control all the FLM settings as well as the FIB/SEM sample stage. Te user can easily navigate the sample, while maintaining focus, and effortlessly switch between SEM, FIB, and FLM imaging modes. Integrated workflow. METEOR is a flexible system that
can be used in a variety of workflows. For example, it can be used to guide on-the-grid lamella milling of a plunge-frozen sample (Figure 2). In this workflow, the user loads the sample in the cryo-FIB/SEM equipped with METEOR. Te microscope stage is moved to the METEOR position, and a stitched overview image is acquired to find ROIs and determine the milling sites. Z-stacks are acquired at these positions for the purpose of correlation. Te sample stage is then moved to the FIB/SEM position to capture a SEM image. Te images are correlated to determine the precise milling location, and the user can start FIB milling a lamella. During and aſter lamella milling, the stage can be moved to the FLM position to check if the fluorescence from the ROI is still present in the lamella. To demonstrate this integrated workflow, we prepared
targeted cryo-lamellae from Saccharomyces cerevisiae (yeast). Sample preparation. We used a yeast strain where eGFP-
tagged Ede1 was overexpressed to enable targeted lamella milling. To enhance correlation efficiency between FLM and FIB/SEM imaging, we supplemented the cell suspension with fiducial markers (Dynabeads™). Te cells were then applied to a TEM grid and plunge-frozen. Correlative cryo-FIB milling. To obtain an electron- cell section containing the ROI,
transparent correlative
cryo-FIB milling was used. Te grids were transferred to a Scios FIB/SEM equipped with a cryo-stage (Termo Fisher Scientific). To protect the samples during the milling pro- cess, the samples were first coated with an organometallic
Figure 1: A) Illustration of METEOR (in black) installed on a FIB/SEM (in blue). B) Schematic representation of the light path of METEOR. The excitation light (blue) is guided from the light source to the dichroic, through the objective and to the sample. The emission light is guided back from the sample through the objective, then the vacuum window, and through the filter wheel to finally reach the camera and form an image.
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