TITLE: 3D-correlative FIB-milling and Cryo-ET of Autophagic structures in Yeast Cells 
AUTHORS: Cristina   Capitanio, Anna  Bieber, Philipp  S Erdmann, Brenda  A Schulman, Florian  Wilfling, Wolfgang  Baumeister

[DESCRIPTION]
This protocol describes how to plunge-freeze yeast on EM grids and how to target autophagic structures by combining cryo confocal fluorescence data to FIB-milling and tomogram acquisition

[STEPS]
SECTION: YEAST CELL CULTURE
1. Prepare and starve yeast cells before plunging:
SECTION: CRYO-CONFOCAL FLUORESCENCE MICROSCOPY
3. Take fluorescence stacks of target cells on a cryo-confocal fluorescence microscope for correlative focused ion beam milling and cryo-ET:
SECTION: 3D-CORRELATIVE FIB-MILLING
4. 3D-CORRELATIVE FIB MILLING

Target sites corresponding to Atg8 puncta or Ede1 END cargo were 3D-correlated to SEM/IB images in the FIB/SEM microscope (FIB Scios and Aquilos, Thermo Fisher Scientific) using the 3D-Correlation Toolbox (3DCT)20. Lamellae were milled in correlated sites as described in a previously published protocol21. In a few cases (e.g Extended Data Fig. 1a), a widefield microscope integrated in the FIB/SEM chamber (METEOR, delmic) was used to confirm the presence of fluorescence signal in the lamella, as previously published51.
SECTION: TEM DATA ACQUISITION
5. TEM DATA ACQUISITION

Cryo-EM data Acquisition. Tomograms were acquired on a Transmission Electron Microscope (Titan Krios, FEG 300kV, Thermo Fisher Scientific) equipped with an energy filter (Quantum K2, Gatan) and a direct detection camera (K2 Summit, Gatan) at a magnification of 42000x (pixel size 3.52 Å) and defocus ranging from -5 to -3.5 µm. Positions for tomogram acquisition were determined by correlation of fluorescence data to TEM images of the grid squares containing lamellas (3DCT), followed by inspection of low-magnification lamella images. Frames were recorded in dose-fractionation mode, with a total dose of 120 e¯/A² per tilt series using SerialEM52. A dose-symmetric tilt scheme was used with an increment of 2° in a total range of ±60° from a starting angle of 10° (+ or –) to compensate for lamella pre-tilt (mostly around 11°). Frames were aligned using MotionCorr253 and reconstruction was performed in IMOD by using the TomoMAN wrapper scripts54.
SECTION: TEM DATA ACQUISITION
6. 
SECTION: YEAST CELL CULTURE
1.1. Inoculate Yeast from overnight cultures in YPD medium to an OD600 of 0.15.
SECTION: CRYO-CONFOCAL FLUORESCENCE MICROSCOPY
3.2. Acquire a fluorescence and transmitted light grid overview (e.g. using a spiral scan). If parts of the grid are out of focus, acquire a focus map by autofocusing in ~15 points spread over the grid and take acquire a second overview using the focus map. 
SECTION: CRYO-CONFOCAL FLUORESCENCE MICROSCOPY
3.3. Find grid squares suitable for FIB milling, which fulfill the following criteria:
The square contains 1-2 clumps of 4-8 yeast cells close to the center of the grid square
The cells show at least one fluorescence punctum indicating the presence of an autophagic structure. 
The square has at least 8 separate fiducial beads spread across the grid square
The square is located in a position accessible in the FIB/SEM instrument, i.e., no closer than 4 grid squares to the edge of a 200 mesh grid.
Mark the identified grid squares on the overview to allow later identification in the FIB/SEM instrument.
SECTION: CRYO-CONFOCAL FLUORESCENCE MICROSCOPY
3.4. For each grid square, acquire a stack in the appropriate fluorescence channels, with pixel size and focus step appropriate for later deconvolution. On the Leica SP8 system with an NA 0.9 objective, we usually use a xy pixel size of 85 nm and a focus step of 300 nm.
SECTION: CRYO-CONFOCAL FLUORESCENCE MICROSCOPY
3.5. Deconvolve the fluorescence stacks with appropriate software, e.g. using Huygens deconvolution software, and reslice them to isotropic pixel size e.g. with the 3DCT for correlation.
SECTION: 3D-CORRELATIVE FIB-MILLING
4.2. Acquire and SEM overview image of the grid. Use landmarks like holes to perform a 2D correlation with the overview generated in the light microscope and identify the grid squares imaged previously.
SECTION: 3D-CORRELATIVE FIB-MILLING
4.1. Load the grid in the FIB/SEM shuttle with the cut-out facing upwards, and apply a protective organometallic (CpMePtMe3) layer using the Gas Injection System (GIS). 
SECTION: 3D-CORRELATIVE FIB-MILLING
4.3. Visit all target grid squares, find their eucentric height in the FIB/SEM instrument and save the positions. Take high-resolution images with the ion beam and the SEM to use for correlation. Taking all images in the beginning allows you to perform the registration for the next position while milling one position.
SECTION: 3D-CORRELATIVE FIB-MILLING
4.4. Perform the 3D registration using the 3D Correlation Toolbox (3DCT):
Load the resliced deconvolved stacks as first, and the high-resolution ion beam image as second image. To help identifying beads, open the SEM image of the square on the side. Display the fluorescence stack as maximum intensity projection.
Click 4 corresponding beads in both images. Refine the xy position and determine the z height of the beads in the fluorescence stacks using x,y,z gaussian fits. Click "Correlate" to calculate an initial registration.
Add more beads in the fluorescence image, refine their 3D position and click "Correlate" to predict their position on the ion beam image. Click the correct position of the beads on the ion beam image to include them in registration calculations. Continue until you've registered 8-14 beads.
Select the target fluroescence signal in the cells, refine the 3D position with a gaussian fit and predict the position in the ion beam image. A red marker will indicate the position where the lamella should be milled.
SECTION: 3D-CORRELATIVE FIB-MILLING
4.5. Mill the lamella at the target position, milling simultaneously from below and above using ion beam currents of 0.3 nA to 30 pA from rough to fine milling. Optionally, stress release cuts can be added after rough milling to ~800 nm lamella thickness. Mill until a target thickness of 100-150 nm, when the lamella appears transparent in SEM imaging at a voltage of 3 kV.
SECTION: 3D-CORRELATIVE FIB-MILLING
4.6. A
SECTION: CRYO-CONFOCAL FLUORESCENCE MICROSCOPY
3.4.  and fluorescence image stacks were acquired on a cryo-confocal microscope (Leica SP8 with Cryo-Stage) equipped with a 50X/0.9 NA objective (Leica Objective #506520) and two HyD detectors. Stacks (Step size 300 nm, x-y pixel size 85 nm) were acquired using 488 nm and 552 nm laser excitation for eGFP- and mCherry-labelled proteins, respectively. In the case of eGFP only strains (eGFP-Atg8 and eGFP-Ede1/Δypt7), signal from autofluorescentDynabeads was acquired as second channel corresponding to red emission wavelengths to easily distinguish fiducial beads from cellular signal. Stacks were deconvolved using Huygens (Scientific Volume Imaging, http://svi.nl). 
SECTION: CORRELATIVE FOCUSED-ION BEAM MILLING
4. Mill lamellas containing the fluroescently labelled target structures for tomogram acquisition:
SECTION: CRYO-CONFOCAL FLUORESCENCE MICROSCOPY
3.1. Clip/mount grids on modified autogrids with cut-out for FIB-milling and load the grid into the cryo-fluorescence microscope. 
SECTION: CORRELATIVE FOCUSED-ION BEAM MILLING
4.1. Load grid into the FIB/SEM shuttle with the cutout facing upwards. Apply a protective organometallic layer (CpMePtMe3) on the grid using the Gas Injection System (GIS).
SECTION: CORRELATIVE FOCUSED-ION BEAM MILLING
4.2. Acquire an SEM overview image of the whole grid. Using holes or grid bar junctions as landmarks, perform a 2D correlation with the grid overview acquired in the lfluorescence microscope to find all target squares.
SECTION: CORRELATIVE FOCUSED-ION BEAM MILLING
4.3. Visit each target grid square and save its position at eucentric height. Take high-resolution SEM and ion beam (IB) images at the milling angle for correlation. On a Scios/Aquilos FIB/SEM instrument, we usually use a milling angle of 18°, corresponding to a lamella pre-tilt of 11° relative to the grid. Taking the images of all the squares first allows for an easier parallelization of image registration and FIB milling and thus a more efficient use of instrument time.
SECTION: CORRELATIVE FOCUSED-ION BEAM MILLING
4.4. For each square, perform 3D correlation with the 3D correlation toolbox (3DCT) as follows:
Load the resliced deconvolved fluorescence stack as first, and the ion beam image as second image. For colocalization experiments, load additional fluorescence stacks as extra channels. Display fluorescence data as maximum intensity projections. 
Identify and mark four corresponding beads in the fluorescence and ion beam image. Refine the bead positions in the fluorescence data by performing an x,y,z gaussian fit, then click "Correlate" to calculate the initial registration.
Click further beads in the fluorescence image, refine their 3D positions and predict their positions in the IB image by clicking "Correlate". Click the correct bead positions in the IB image and repeat.
Iterate step 3.4.3 until reaching a consistent registration with 8-14 beads.
Select the target fluorescence punctum, refine its 3D position with a Gaussian fit and predict its position in the ion beam. The red marker displayed on the ion beam image indicates the target position for the lamella.
SECTION: CORRELATIVE FOCUSED-ION BEAM MILLING
4.5. Mill the lamella at the target position, milling simultaneously from above and below. The ion beam currents and target thicknesses of our usual milling steps (2xrough and 2xfine) are:
0.3 nA - 1.2 µm
0.1 nA - 800 nm
Optional: Add stress release cuts in the grid square on both sides of the lamella.
50 pA - 250 nm
30 pA - 100-150 nm, or until transparency at SEM voltage of 3 kV.
SECTION: CORRELATIVE FOCUSED-ION BEAM MILLING
4.6. After milling, acquire an SEM and IB image of the grid square with the final lamella and perform 3D correlation with the pre-milling fluorescence data as described in step 3.4 to verify that the lamella is at the correct position, and to identify the target position for tomogram acquisition.
SECTION: CORRELATIVE FOCUSED-ION BEAM MILLING
4.7. After milling all lamellas, take an SEM overview of the whole grid to allow easy recognition of all lamellas in the TEM.
SECTION: CORRELATIVE CRYO-ET TOMOGRAM ACQUISITION
5. Acquire tomograms of target structures in correlatively milled lamellas:
SECTION: CORRELATIVE CRYO-ET TOMOGRAM ACQUISITION
5.1. Load grids into the TEM with the previous milling direction perpendicular to the tilt axis.
SECTION: CORRELATIVE CRYO-ET TOMOGRAM ACQUISITION
5.2. Acquire an overview image of each grid square containing a lamella, and a montage of each lamella.
SECTION: CORRELATIVE CRYO-ET TOMOGRAM ACQUISITION
5.3. To determine positions for tomogram acquisition, register the pre-milling fluorescence data with the TEM grid square overview using 3DCT as described in step 3.4. Check the identified positions in the lamella overview to center the target structure during tilt series acquisition.
SECTION: CORRELATIVE CRYO-ET TOMOGRAM ACQUISITION
5.4. Run tilt-series at the correlated positions, using e.g. batch acquisition in SerialEM Navigator. We usually use a dose-symmetric tilt scheme with 2° steps starting from a lamella pretilt of 10° to +70° and -50°. Images are acquired in dose-fractionation mode and a final target dose of 120 e-/Å2.
SECTION: YEAST CELL CULTURE
1.2. Let cultures grow at 30°C, 200 rpm, till OD600 is 0.8. At this point, switch to starvation medium SD-N. 
Incubate cells in starvation medium at 30°C for a time span of 0.5 - 3 hours. 
SECTION: YEAST CELL CULTURE
1.3. Before plunging, add 1 μm beads to the cell solution, at a dilution of 1:20. 
The beads are necessary in the next steps to perform 3D-correlation on the grid.
SECTION: PLUNGE FREEZING
2. Prepare Vitrobot and grids for plunging:

Set up Vitrobot as follows: Blotforce = 8, Blottime = 10s, room temperature.

Plasma clean grids (200 Mesh Cu SiO2 R1/4, Quantifoil) for 30 s, just before plunging.
SECTION: PLUNGE FREEZING
2.1. Apply 4 µl of starved cell solution with beads on the grid, blotted and plunged in ethane-propane with a Vitrobot Mark IV and plunge. 
SECTION: PLUNGE FREEZING
2.2. Store the grids in a grid box in liquid nitrogen. 
SECTION: CORRELATIVE CRYO-ET TOMOGRAM ACQUISITION
5.5.