This example involves only one sample it is possible to batch multiple samples thus reducing time and cost per sample. In the example shown in the table it is possible to obtain results in 12 working days. 3D reconstruction itself is very much depend on the size, number and complexity of structures of interest. The post-processing of acquired images from TEM usually takes 2 days. Processing of the sample for TEM will take about 5 days.
#Serial images serial#
How long does it take to serial section sample and to get results of 3D reconstruction?
#Serial images series#
Reconstructing ~150 dendritic spines in this series Imaging area 20um x20um (XY) on each section Sectioning sample to 150 consecutive serial sections Processing 1 sample of brain tissue to TEM Example of cost is shown in the table below: Action We charge £100/hr of microscope time, £18/hr of ultramicrotome time and £50/hr staff time. This will depend on the number of samples we can processed together and volume required to obtain for 3D reconstruction. FAQs What is the cost of the serial sectioning followed by 3D reconstruction? We can offer you expert advice of our resident academics should you need help making a conclusion from your data. If you wish to perform tracing and remodelling yourself, we are happy to send you prepared and aligned stack of images. 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. The last step is tracing structures of interest, which will then be remodelled in 3D. The montages are then aligned between each other in the stack. When images are all acquired, we need montage them first to get high resolution image of the structure. Should your structure of interest is larger than this, please let us know. The maximum size of such imaging + montaging area is 40 µm x 40 µm. 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. Sections are collected onto copper slot grid for TEM.ĭuring imaging on the TEM, a specific area of interest is automatically imaged in each section in the series. Each series may contain anywhere up to 250 sections. During sectioning, a series is prepared where each section is 50 nm thick. The shape of the block face used for sectioning is rectangular with size of about 25-45 µm x 100-500 µm. The solid block or section where the sample is embedded is microsectioned using an ultramicrotome. Your sample should be fixed with 2.5% Glutaraldehyde in either 0.1M phosphate or sodium cacodylate buffer (pH 7.4). Preparation of the sample and choice of fixative are crucial for high quality ultrastructure preservation and further image acquisition.
Depending on the sample, the embedded form may be a solid block or a solid tissue section within 2 sheets of plastic. The alcohols are gradually replaced by resin and the sample is later embedded into epoxy (Epon) resin.
Sample is then dehydrated in alcohols with ascending concentrations. This fixation method preserves the ultrastructure of the sample well for electron microscopy, better than paraformaldehyde. For this purpose we use 2.5% glutaraldehyde in either phosphate or sodium cacodylate buffer, followed with post-fixation in osmium tetraoxide (OsO4). The process your sample goes through Sample preparationįirst, biological sample is chemically fixed to obtain good TEM images. 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 series is imaged on a regular transmission electron microscope (TEM) and images are manually reconstructed for structures of interest. We use a regular TEM sample preparation protocol, we then prepare serial sections of the sample. 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. It gives a researcher information on volume, surface area, number in analysed volume or contact area between different structures. Having 3D model of structure imaged and reconstructed has many advantages. Single 2D TEM images have significant limitations in interpretation of different parameters such as structure’s size, shape, number or relationship to other structures.īut 2D TEM images can be used to recreate and model a structure of interest in 3 dimensions. It is possible to gain understanding of cell interactions and other cellular processes. TEM serial sections 3D reconstruction serviceĮlectron microscopy (EM) technique produces high resolution images of cellular ultrastructures.