May 08, 2025
iFLM-guided Cryo-FIB milling
- kevin rose1
- 1UC Berkeley
Protocol Citation: kevin rose 2025. iFLM-guided Cryo-FIB milling. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4zey8vo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 24, 2025
Last Modified: May 08, 2025
Protocol Integer ID: 124865
Keywords: ASAPCRN
Abstract
Defective mitochondrial quality control in response to loss of mitochondrial membrane polarization is implicated in Parkinson’s disease by mutations in PINK1 and PRKN. Application of in situ cryo-electron tomography (cryo-ET) made it possible to visualize the consequences of mitochondrial depolarization at higher resolution than heretofore attainable. Parkin-expressing U2OS cells were treated with the depolarizing agents oligomycin and antimycin A (OA), subjected to cryo-FIB milling, and mitochondrial structure was characterized by in situ cryo-ET. Phagophores were visualized in association with mitochondrial fragments. Bridge-like lipid transporter (BLTP) densities potentially corresponding to ATG2A were seen connected to mitophagic phagophores. Mitochondria in OA-treated cells were fragmented and devoid of matrix calcium phosphate crystals. The intermembrane gap of cristae was narrowed and the intermembrane volume reduced, and some fragments were devoid of cristae. A subpopulation of ATP synthases re-localized from cristae to the inner boundary membrane (IBM) apposed to the outer membrane (OMM). The structure of the dome-shaped prohibitin complex, a dodecamer of PHB1-PHB2 dimers, was determined in situ by sub-tomogram averaging in untreated and treated cells and found to exist in open and closed conformations, with the closed conformation is enriched by OA treatment. These findings provide a set of native snapshots of the manifold nano-structural consequences of mitochondrial depolarization and provide a baseline for future in situ dissection of Parkin-dependent mitophagy.
Notched base-clipped EM grids were loaded into an Aquilos 2 with integrated fluorescence (iFLM, ThermoFisher) with the notch facing upwards.
Grids were first screened using SEM to identify potential lamellae sites and assess general grid quality. Grids with 20-40 cells in the centers of grid squares were used for lamellae generation.
Cells were then screened iteratively using iFLM to target specific regions of clustering mCherry or BFP signal for lamellae site placement. Adjust laser power and exposure time as necessary. Z-stacks were taken at each site cover 5x5 grid squares per Z-stack. These images were mainly used to determine where mitochondria were clustered to in untreated or OA treated cells.
Grids were then sputter coated for 15 seconds using 30mA current and 10Pa pressure, and subsequently GIS coated for 1 minute.
Auto-TEM was then used to generate a lamellae of approximately 200 nm thickness at each site using the following step-wise protocol for lamellae generation:
0.3-0.5 nA of current was used to ablate cell material to 3 μm in thickness.
100 pA current thinned cells to 1 μm.
50 pA was used to thin cells to 500 nm.
30 pA was used to thin lamellae to their final 200 nm thickness.
Grids containing lamellae were immediately retrieved from the Aquilos and moved to liquid nitrogen for no longer than 3 days, or loaded directly into a krios with the notch perpendicular to the tilt axis for data collection.