TEM serial sections 3D reconstruction service
Electron microscopy (EM) technique produces high resolution images of cellular ultrastructures. It is possible to gain understanding of cell interactions and other cellular processes. Single 2D TEM images have significant limitations in interpretation of different parameters such as structure’s size, shape, number or relationship to other structures.
But 2D TEM images can be used to recreate and model a structure of interest in 3 dimensions. Having 3D model of structure imaged and reconstructed has many advantages. It gives a researcher information on volume, surface area, number in analysed volume or contact area between different structures.
We provide serial sectioning service as well as three-dimensional (3D) reconstruction of tissues, cultured cells or plants with resolution down to 2 nm x 2 nm x 50 nm (XYZ) of final data set. We use a regular TEM sample preparation protocol, we then prepare serial sections of the sample. The series is imaged on a regular transmission electron microscope (TEM) and images are manually reconstructed for structures of interest. Analysed structure should be in a size range between 5 nm to ~5 µm and its maximum volume would be up to 40 µm x 40 µm x 10 µm.
The process your sample goes through
First, biological sample is chemically fixed to obtain good TEM images. For this purpose we use 2.5% glutaraldehyde in either phosphate or sodium cacodylate buffer, followed with post-fixation in osmium tetraoxide (OsO4). This fixation method preserves the ultrastructure of the sample well for electron microscopy, better than paraformaldehyde. Sample is then dehydrated in alcohols with ascending concentrations. The alcohols are gradually replaced by resin and the sample is later embedded into epoxy (Epon) resin. Depending on the sample, the embedded form may be a solid block or a solid tissue section within 2 sheets of plastic.
Preparation of the sample and choice of fixative are crucial for high quality ultrastructure preservation and further image acquisition. Your sample should be fixed with 2.5% Glutaraldehyde in either 0.1M phosphate or sodium cacodylate buffer (pH 7.4).
Serial sectioning and imaging in TEM
The solid block or section where the sample is embedded is microsectioned using an ultramicrotome. The shape of the block face used for sectioning is rectangular with size of about 25-45 µm x 100-500 µm. During sectioning, a series is prepared where each section is 50 nm thick. Each series may contain anywhere up to 250 sections. Sections are collected onto copper slot grid for TEM.
During imaging on the TEM, a specific area of interest is automatically imaged in each section in the series. During this automatic acquisition we aim to get as much detail as possible, which means imaging the structure in a montage at higher magnification and montaging the images later. The maximum size of such imaging + montaging area is 40 µm x 40 µm. Should your structure of interest is larger than this, please let us know.
Post-processing and 3D reconstruction
When images are all acquired, we need montage them first to get high resolution image of the structure. The montages are then aligned between each other in the stack.
The last step is tracing structures of interest, which will then be remodelled in 3D. In addition to this 3D structure model you will also obtain information on volume, surface area, number in analysed volume or contact area between different structures.
If you wish to perform tracing and remodelling yourself, we are happy to send you prepared and aligned stack of images.
We can offer you expert advice of our resident academics should you need help making a conclusion from your data.
What is the cost of the serial sectioning followed by 3D reconstruction?
This will depend on the number of samples we can processed together and volume required to obtain for 3D reconstruction.
We charge £100/hr of microscope time, £18/hr of ultramicrotome time and £50/hr staff time. Example of cost is shown in the table below:
|Processing 1 sample of brain tissue to TEM
|Sectioning sample to 150 consecutive serial sections
|Imaging area 20um x20um (XY) on each section
|Reconstructing ~150 dendritic spines in this series
How long does it take to serial section sample and to get results of 3D reconstruction?
Processing of the sample for TEM will take about 5 days. The post-processing of acquired images from TEM usually takes 2 days. 3D reconstruction itself is very much depend on the size, number and complexity of structures of interest. In the example shown in the table it is possible to obtain results in 12 working days. This example involves only one sample; it is possible to batch multiple samples thus reducing time and cost per sample.
How many samples can we send at once?
We can receive any number of samples, if they are shipped fixed in 2.5% glutaraldehyde. Several samples may be processed at once. As mentioned above as there is waiting time in between stages, it is possible to do multiple samples along each other, thus reducing time for obtaining results.
Why choose OU EM Suite for your serial sectioning preparation and 3D reconstruction?
We perform a pilot serial sectioning and 3D reconstruction to ensure you are happy with the data quality before proceeding with the rest of the samples, communicating with you any difficulties or options we may discover.
We have nearly 10 years of experience in serial sectioning and 3D reconstruction. We work with residential and external researchers and prepare their series of sections to reconstruct their sample.
See links to a selected few publications done using this 3D reconstruction of serial sections:
Mechanism for long-term memory formation when synaptic strengthening is impaired (PNAS, 2011)
Glia selectively approach synapses on thin dendritic spines (Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 2014)
Hippocampal circuit dysfunction in the Tc1 mouse model of Down syndrome (Nature Neuroscience, 2015)