TITLE: Correlative Light-Electron Microscopy of α-synuclein Aggregates in Primary Neurons
AUTHORS: Felix Kraus, vtrinkaus , Ruben Fernandez Busnadiego

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details methods for correlative light-electron microscopy of α-synuclein aggregates in primary neurons.</div></div>

[STEPS]
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Grow primary neurons on EM grids (SiO2, R1/4 or 1.2/20, Quantifoil):
Carbon Au grids may also be used, but they should be coated with an additional layer of carbon to stabilize the grids and to allow the neurons attach better on the grids.
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Before using the EM grids, glow discharge them for  at highest voltage (PDC-3XG Harrick plasma cleaner) and subsequently coat with  and .
[poly-D-lysine]
[laminin]
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To have a sufficient number of neurons on the grids, plate 120,000 neurons per well of a 24-well plate.
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Transduce neurons with vector / do seeding experiment.
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Plunge freeze the neurons:
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First, exchange the medium to neuronal medium containing 10 % glycerol to ensure good vitrification (see also Baeuerlein et al., Cell, 2017).
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Hold the grids carefully with plunging tweezers and insert the tweezers in the holder of the Vitrobot (Thermofisher). Make sure grids are blotted from the front as well as from the back with Whatman paper. Use a humidity of 80 % and a temperature of , a blot force of 10 and blot time of .
37 °C
Prepare the Vitrobot prior to exchanging the neuronal medium to ensure fast plunge-freezing and only short incubation of the neurons in 10 % glycerol (around 5 min). For preparing the Vitrobot, turn it on, apply the settings you want to use, add liquid nitrogen (LN2) into the LN2container and liquefy the ethane / propane gas in the cavity that is cooled by LN2).
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After plunging, store EM grids in appropriate EM grid boxes in LN2.
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For cryo-fluorescence microscopy, clip EM grids in autogrid holders with cut-out to enable FIB-milling under LN2. Place clip ring on top of EM grid in the back of the autogrid frame, cells facing downwards to enable FIB-milling of the cells later on.
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Transfer clipped grids into shuttles of the CorrSight (Thermofisher) microscope. Cells facing again downward.
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Transfer shuttles into CorrSight shuttle boxes and bring the grids to the microscope.
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Move the shuttle into the cryo-box of the cooled-down CorrSight microscope.
For cooling down, the tank in the back of the cryo-box is filled with LN2 and the slowly evaporating LN2 gas is led through the box to the outside, cooling the atmosphere in the box to a temperature of -180 °C.
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Open the LA software and the MAPS software, create a new project in MAPS and find areas of interest in the specimen at 5 x magnification. Take overview images of the EM grids in DIC mode as well as in fluorescence mode (choose channel depending on your fluorophore).
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Find cells that look promising (they should be close to the center of the grid and should be located in the middle of a square but not on the EM grid bar) and acquire 20 x or 40 x images, depending on the size of the feature of interest. The MAPS project will be saved automatically.
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After imaging of the grids, take them carefully out of the cryo-box and place them in LN2.
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Move the MAPS project to the computer of the FIB instrument (Scios or Quanta, Thermofisher).
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Transfer the EM grids from the CorrSight shuttles into the FIB shuttle under LN2. Grids should be inserted into the shuttle with cells facing towards the experimenter and the cut-out should face upright. Transfer the EM grids subsequently under vacuum into the FIB chamber and place them onto the stage which is cooled by nitrogen gas, which is led through a liquid nitrogen container and cooled to LN2 temperature.
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Coat the grids with organic platinum to avoid damage of the sample by out-of-focus gallium ions. Tilt the stage to 18 ° to allow milling at a shallow angle.
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Find the eucentric height of the stage, by correlating the SEM and FIB image and moving the stage iteratively.
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Open MAPS software at the FIB computer and take an overview image of the EM grid with the SEM beam.
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Load the SEM image into the MAPS software (on some instruments where MAPS and the FIB can communicate directly, the image will be automatically uploaded into the software). Apply the three-point-method of correlation to the grid (see also Guo et al., Cell, 2018).
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For the three-point-method click with the right mouse button onto ‘Align’. Select three alignment markers and place them on the SEM as well as on the fluorescence image on points that can be easily recognized (e.g. broken carbon or corners of grid bars). Click ‘finish’ when you are done and the two images will be overlaid.
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Then find the cells of interest and zoom in with the SEM. Take again a SEM image and correlate that with the fluorescence image (take care to be in eucentric height and now use the 20 x or 40 x image). Use again the three-point-method to make sure you find the correct cell and region of interest (Figure 1 b, Figure 2 a-c). Do that iteratively until fluorescence image and SEM image match perfectly.
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Image the cell in the FIB beam and start with the milling process (Figure 1 c). For milling start with a high current of 0.5 nA at 30 kV and go down to 50 pA for fine milling.
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Mill as long as you get a thin transparent lamella, which is roughly 100 to 150 nm in height. Double check the thickness of the lamellas by imaging it at 3 kV in the SEM. It should be transparent at 3 kV to be sufficiently thin. Every time the stage is moved, repeat the three-point-method, to make sure you still target the correct area.
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After the milling, transfer the grids out of the FIB under vacuum and store the grids in suitable EM grid boxes.
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During your next TEM session: Transfer the grids from the boxes into the TEM and acquire tomograms at areas of interest (Figure 1 e and f).