Jul 21, 2025
Ultrastructural analysis of cultured neurons using TEM
- Merja Joensuu1
- 1Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia
Protocol Citation: Merja Joensuu 2025. Ultrastructural analysis of cultured neurons using TEM. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l218k4g1y/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: July 18, 2025
Last Modified: July 21, 2025
Protocol Integer ID: 222819
Keywords: Electron microscopy, Flat embedding, ultrastructural analysis of cultured neuron, cultured neuron, neuronal culture, transmission electron microscopy, image quantification for transmission electron microscopy, using tem, ultrastructural analysis, tem, imaging
Abstract
This protocol describes the sample preparation, imaging and image quantification for transmission electron microscopy (TEM) of neuronal cultures.
Materials
- 2% FA
- 2% GA
- 0.1 M NaCac buffer pH 7.4
- Reduced 1% OsO4
- 1.5% potassium ferrocyanide
- Mq (Milli-Q water)
- 2% Uranyl Acetate (aq)
- EtOH (50%, 60%, 70%, 90%, 100%)
- EPON or LX112 resin
- Beem capsules
Troubleshooting
Flat Embedding protocol using BioWave tissue processing system (Pelco)
29m
Fix neurons with 2% glutaraldehyde (Electron Microscopy Sciences, 16210) diluted in 0.1 M sodium cacodylate (Sigma-Aldrich, C0250) buffer pH 7.4, at room temperature for 20 min
20m
Wash 2 x 3 min with 0.1 M sodium cacodylate buffer
9m
Osmicate using 1% Osmium tetroxide (v/v, Electron Microscopy Sciences, 19100) reduced with 1.5% potassium ferrocyanide (v/v, Electron Microscopy Sciences, 25154-10) in Biowave using the following settings: MW 80 W, 2 min ON – 2 min OFF – 2 min ON, Vac: on
6m
Repeat osmication step using 1% Osmium tetroxide (v/v, Electron Microscopy Sciences, 19100) reduced with 1.5% potassium ferrocyanide (v/v, Electron Microscopy Sciences, 25154-10) in Biowave using the following settings: MW 80 W, 2 min ON – 2 min OFF – 2 min ON, Vac: on
6m
Wash 1 x Milli-q water in Biowave using the following settings: MW 250 W, 40 s, VAC: off
1m
Contrast using 2% Uranyl Acetate (aq, Electron Microscopy Sciences, Cat. no. 22400) diluted in Milli-q in Biowave using the following settings: MW 150 W, 1 min ON – 1 min OFF – 1 min ON, VAC: on
3m
Dehydrate in 50% EtOH at room temperature for 1 min in fume cupboard
1m
Dehydrate in 60% EtOH at room temperature for 1 min in fume cupboard
1m
Dehydrate in 70% EtOH at room temperature for 1 min in fume cupboard
1m
Dehydrate in 90% EtOH at room temperature for 1 min in fume cupboard
1m
Dehydrate twice 100% EtOH at room temperature for 1 min in fume cupboard
Embed cells in 1:1 (v/v) of 100% EtOH:EPON (Electron Microscopy Sciences, 14120 for neurons grown on glass coverslips or glass-bottom dishes) or LX-112 resin (Ladd, 21210 - LX 112 for neurons grown on plastic dishes) in Biowave using the following settings: MW 250 W 3 min, VAC: on
3m
Embed cells twice in 100% EPON or LX-112 resin in Biowave using the following settings: MW 250 W 3 min, VAC: on
6m
Place a beem capsule filled with Epon or LX-112 upside down on top of the cells
Polymerise resin at 60°C for 48 h
2d
Thin sectioning
Trim 1 mm x 1 mm pyramid, and thin section (80-90 nm) samples using an ultramicrotome (such as Leica Biosystems, UC6FCS)
TEM imaging
3d
Image samples with a transmission electron microscope (such as JEOL USA, Inc. model 1101 and 1400) equipped with cooled charge-coupled device camera (Olympus; Morada CCD Camera). For the quantification of presynaptic vesicle numbers, endosomal numbers and size, and presynaptic size, and mitochondrial size and subcellular localisation, as well as for imaging the ultrastructure of ERGIC, Golci complex and endoplasmic reticulum, EM images are acquired randomly (i.e an image is taken when an organelle or region of interest is identified).
1d
For HRP-analysis, images are acquired when HRP signal is observed.
1d
For secretory pathway organelle analysis, somatic area of neurons is selected for imaging.
1d
Quantification
4w
Number of synaptic vesicles and endosomes is counted from electron micrographs using Adobe Photoshop (Adobe, 22.4.3 release) Count Tool and related to the presynaptic area (µm2) which are manually segmented and measured using ImageJ/Fiji (https://imagej.nih.gov/ij/) measure tool. Same tool is also used to measure the size of endosomes (µm2). Vesicles with sectional area ≤0.003 µm2 are classified as synaptic vesicles, and those >0.003 µm2 as endosomes. Presynapses are identified as rounded structures enriched with synaptic vesicles, typically positioned adjacent to postsynaptic densities and connected to axons.
1w
Mitochondrial size is measured using Fiji/ ImageJ measure tool and assigned to either axons (i.e. elongated structures often containing microtubules, presynaptic connections, and having a diameter >200 nm), presynapses (as described above) or soma (i.e. the region of a neuron that includes the cell body of cell soma and dendrite(s), but excludes the axon, and often has a visible nucleus and postsynaptic densities). The subcellular location of mitochondria is calculated by counting the number of mitochondrial cross-sections per presynapses. It is worth noting that this quantification does not present the absolute presynaptic numbers of presynaptic mitochondria, as the EM sections only capture a thin section of the synapse (e.g. elongated mitochondria could span the sections multiple times) but the quantification rather reflects the overall mitochondrial presence in the presynapses.
1w
Quantification of number of presynaptic glycogen deposits is done using Adobe Photoshop Count
Tool.
1w
The quantification of the number of HRP-containing synaptic vesicles and endosomes is quantified with Adobe Photoshop Count Tool, and the size (sectional area, µm2) of the HRP-stained endosomes is quantified manually using ImageJ/Fiji.
1w
Protocol references
This protocol is an adaptation of
1) Jokitalo E, Cabrera-Poch N, Warren G, Shima DT. Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum. J Cell Biol. 2001 Jul 23;154(2):317-30. doi: 10.1083/jcb.200104073. PMID: 11470821; PMCID: PMC2150754.
2) Vihinen H, Jokitalo E. Studying Golgi Structure and Function by Thin Section TEM. Methods Mol Biol. 2023;2557:141-159. doi: 10.1007/978-1-0716-2639-9_11. PMID: 36512215
3) Wenzel M, Dekker MP, Wang B, Burggraaf MJ, Bitter W, van Weering JRT, Hamoen LW. A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline. Commun Biol. 2021 Mar 8;4(1):306. doi: 10.1038/s42003-021-01809-8. PMID: 33686188; PMCID: PMC7940657.