Integrated Cryo-Correlative Microscopy
Figure 2: An example of a workflow where METEOR is used to guide on-the-grid lamella milling of a plunge-frozen sample.
platinum layer using a gas injection system needle. Next, clusters of yeast cells suitable for FIB milling and containing END condensates were identified using METEOR. Te END condensates appear as bright green puncta (Figure 3). Fluo- rescent image z-stacks of promising regions were acquired and correlated with SEM images. Lamellae were cut at corre- lated sites as described in published protocols [5,15]. During milling, lamellae were inspected using METEOR to check if the END condensates were still present. Aſter the final pol- ishing step, the lamellae were inspected using METEOR to confirm the presence of END condensates before transferring the sample to the cryo-TEM. TEM imaging. Tomograms were recorded on a Titan
Krios 2 cryo-TEM (FEG 300kV, Termo Fisher Scientific) with a post-column energy filter (20eV slit, BioQuantum™, Gatan), and a direct detector camera (K2 Summit®, Gatan) operated in dose fractionation mode. Te TEM images of milled grid squares were overlaid on the maximum intensity projection (MIP) of the FLM stacks acquired on the final lamellae using the Landmark Correspondences plug-in in Fiji [16]. Te correlation was used to determine locations for tomogram acquisition. Tilt series were acquired with a dose-symmetric tilt scheme (2° steps, between -50° and 70° to compensate for lamella pre-tilt (11°)). Total dose was ∼120 e- / Å2
) at a pixel
size of 3.52Å using SerialEM [17]. Te data were processed with TOMOMAN [18] using MotionCor2 for frame alignment [19]. Tilt series alignment and tomogram reconstruction were performed in IMOD [20], and tomograms were denoised using CryoCARE [21].
2021 November •
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Results Guided lamella milling. METEOR was used to guide
lamella milling from the yeast strain overexpressing eGFP-Ede1 previously characterized by Wilfling et al. [14]. Since we used an integrated FLM we were able to monitor the fluorescence level of the ROI throughout the milling process. In Figure 3 we show electron beam, ion beam, and FLM images at different stages of lamella milling, starting with the identification of the ROI (Figure 3A, 3D, 3G), a control during milling (Figure 3B, 3E, 3H), and the final image aſter polishing (Figure 3C, 3F, 3I). Te pink arrows indicate that the fluorescent signal is maintained during milling, indicating that the ROI is present in the final lamella. We were thus able to confirm that the END condensates were present, and the lamella was good enough to transfer to the cryo-TEM. Guided tomography. Te grid was transferred to the cryo-
TEM, and the lamella was found using a low-magnification EM setting. To determine the region of tomogram acquisition, a FLM/TEM overlay was performed (Figure 4A). Based on this overlay the region of tomogram acquisition was chosen. Te ROIs were vitreous, proving that in situ FLM imaging, even aſter fine milling, did not devitrify the lamella. Te tomograms also revealed several structural properties of the END condensates and their surroundings, allowing for a better understanding of their structural organization. Te condensates form a distinct assembly with a teardrop-like shape close to the plasma membrane. Tere is a striking exclusion of ribosomes, and the endoplasmic reticulum (ER) surrounds the END condensate (Figure 4B). For further reading on the END condensates and the role of Ede1, we refer to the study of Wilfling et al. [14].
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