Apr 02, 2026
3D FIBSEM CLEM Mouse Brain Tissue
- Yumei Wu1,2,
- Pietro De Camilli1,2
- 1Departments of Neuroscience and of Cell Biology, Program in Cellular Neuroscience, Neurodegeneration, and Repair, Howard Hughes Medical Institute, Yale School of Medicine, New Haven, CT 06510.;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network.
Protocol Citation: Yumei Wu, Pietro De Camilli 2026. 3D FIBSEM CLEM Mouse Brain Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl84x49g2w/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: April 02, 2026
Last Modified: April 02, 2026
Protocol Integer ID: 314452
Keywords: ASAPCRN
Abstract
This protocol details the procedure of Correlative Light Microscopy and Electron Microscopy (CLEM) with 3D Focus Ion Beam Scanning Electron Microscopy (FIBSEM) technique for mouse brain tissue in Zeiss Crossbeam 550 FIBSEM system.
Materials
- 12-mm glass coverslips
- Epon resin
- Sputter coater (Ted Pella, Inc.)
- Crossbeam 550 FIBSEM workstation
- SmartSEM (Carl Zeiss Microscopy GmbH, Oberkochen, Germany)
- Atlas engine 5 (Fibics incorporated, Ottawa, Canada)
- Platinum GIS (gas injection system)
- Carbon GIS
- Inlens detector
- ESB detector
- DragonFly Pro software [Object Research Systems (ORS) Inc., Montreal, Canada]
- EMS (Electron Microscopy Sciences), Hatfield, PA
Troubleshooting
General preparation
Mouse is anesthetized with a Ketamine/Xylazine anesthetic cocktail (16 mg/ml Ketamine and 0.8 mg/ml Xylazine) injection.
Perfused transcardially with 37 °C pre-warmed 4% PFA and 0.05% Glutaraldehyde in 0.1M Phosphate buffer (PB) (0.1M Na2HPO4+0.1M NaH2PO4, PH7.4) at 5-6 ml/min with a pump.
Dissect the brain out and keep in the same fixative overnight at 4 °C.
Wash 3x20min in 0.1M PB buffer.
Sagittally section the brain at 50 µm thickness using a vibratome.
Mount the sections on glass slides in 0.1 M PB, coverslipped.
Acquire the fluorescence images using an Olympus SliceView VS200 slide scanner equipped with a Hamamatsu Orca-Fusion camera and a 40x Olympus UPlanXApo objective. Sections containing abundant tdTomato puncta is post-fixed in 2.5% glutaraldehyde with 2 mM CaCl2 (J.T. Baker) in 0.1 M sodium cacodylate buffer for 2–3 hours on ice.
Incubate in 2% osmium tetroxide, 1.5% potassium ferrocyanide (K4Fe(CN)6) (Sigma-Aldrich), and 2 mM CaCl2 in 0.1 M sodium cacodylate buffer for 1 hour on ice.
Treat the sections with thiocarbohydrazide (TCH) for 20 minutes at room temperature.
Second osmication in 2% osmium tetroxide in water for 30 minutes at room temperature.
Incubate the sections in 2% aqueous uranyl acetate overnight at 4 °C.
Dehydration in 20% etoh for 5 min
Dehydration in 50% etoh for 5 min
Dehydration in 70% etoh for 5 min.
Dehydration in 90% etoh for 5 min
Dehydration in 100% ethanol for 5 minutes x3.
Dehydration in propylene oxide for 10 minutes at room temperature.
Infiltration in 25% Epon resin (Embed 812) in propylene oxide several hours
Infiltration in 50% Epon in propylene oxide for several hours.
Infiltration in 75% Epon in propylene oxide overnight.
Infiltration in 100% Epon in propylene oxide overnight.
Change to fresh 100% resin for several additional hours.
Mount the sections onto 12-mm glass coverslips in a drop of Epon, remove excess resin, and polymerized at 60 °C for 48 hours.
Glue the coverslip with epon-embedded brain section onto the SEM (scanning electron microscopy) sample mounting aluminum.
en bloc coating with 20nm thick layer of platinum on the sample surface was carried out with the sputter coater (Ted Pella, Inc.).
Load the sample into a Crossbeam 550 FIBSEM workstation operating under SmartSEM (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) and Atlas engine 5 (Fibics incorporated, Ottawa, Canada).
Relocate the dystrophic DAergic axon (large tdTomato puncta) on fluorescence light microscope image on SEM (scanning electron microscope) stage based on the land markers of blood vessels, cell bodies and axonal bundles on the dorsal striatum with SE2 detector (EHT, 5kV; probe current, 2nA) at working distance (WD) of 10 nm.
Incline the stage (to 54 degree) and lifted up (to the WD of 5 nm) to adjust the eucentric height and get the coincident point.
Define the imaging area of 80x50 µm covering the region of interest (ROI).
Deposit the ROI’s surface with 1 µm thick protective platinum pad with inserting platinum GIS (gas injection system) and applying FIB probe at 30kV:7nA.
Mill and fill the autotune marks and 3D tracking marks into the platinum pad with FIB probe at 30kV:50pA and carbon GIS.
Deposit 1 µm thick upper protective carbon pad onto the top of these marks-containing platinum pad with inserting carbon GIS and using the FIB probe at 30kV:7nA.
FIB probe at 30kV:30nA is used for the coarse trench (40 µm deep).
FIB probe at 30kV:3nA is used for the fine trench.
FIB probe at 30kV:700pA is used for milling the imaging surface during the 3D tracking.
Inlens detector is used for FIBSEM imaging (EHT, 1.5 kV; probe current, 0.5 nA).
The filtering grid bias voltage of ESB detector is set at 1000 V, the collector bias voltage is set at 300 V.
Dwell time is set at 0.5 µs, line averaged by 2.
The imaging resolution is set at 8 nm/pixel at X, Y axis, slicing by 4 nm/slice along Z axis (binned down by 2 when images was exported). The final data set can get an isotropic resolution at 8 nm/voxel by this setting.
The images are aligned and exported with Atlas 5 (Fibics incorporated, Ottawa, Canada), further processed and 3D segmented with DragonFly Pro software [Object Research Systems (ORS) Inc., Montreal, Canada]. Except noted all reagents are from EMS (Electron Microscopy Sciences), Hatfield, PA.