Practical progress of a project
The preliminary meeting
A preliminary meeting allows to precisely define the requests, the questions to be answered, the protocols used, etc. It will also allow a first contact with electron microscopy for those who do not know the technique. Planning is done for the first series of samples and/or for training.
Important notice: users are required to fix their biological material themselves, for safety reasons (except special experiments such as deposits for negative contrast).
- The sample to be studied is easily moved after fixation, and ready for the electron microscopy process.
- Immediate and rapid fixation is essential for the preservation of the cellular ultrastructure, and therefore for observation by electron microscopy.
- Fixation allows us to work on all types of biological materials without confinement restrictions.
The Electronic Microscopy Platform reserves the right to refuse to work with samples that have not been processed according to the protocols given (the same applies to samples that will be offered to us at other more advanced stages, i.e. already included, cut and colored).
The initial steps are essential to obtaining usable results, and errors made at this precise moment are difficult to correct. Orientation, size and quantity are particularly critical parameters to be monitored.
Fixation protocols (specific to electron microscopy)
Below is a summary of the most commonly used fixation methods:
For a morphological study:
- 1 hour in 3% glutaraldehyde diluted in 0.1M phosphate buffer, at room temperature.
For immunological labeling on frozen cells, there are two possible protocols:
- 1 hour in 1% glutaraldehyde diluted in 0.1M phosphate buffer, at room temperature
- 1 hour in 4% PFA + 0.2% glutaraldehyde diluted in 0.1M phosphate buffer, at room temperature.
The choice between these two methods will be made according to the sensitivity of the antibodies. The use of PFA leads to poorer morphology but will better preserve the antigens and therefore the sensitivity of the labeling for some antibodies.
The electron microscope is accessible from Monday to Friday by appointment. Observation slots are usually from 10 a.m. to 12:30 and from 2 p.m. to 5:30 p.m.
The work of observation and shooting is carried out in the presence of the engineer in charge, who handles the microscope, or independently after training.
We provide training for each step of our services. This is intended for projects taking place over a long period of time or with a large number of samples.
- User training on the different equipment available
- Training in sample preparation techniques:
- inclusion (epoxy resins or freezing),
- pre- and post-inclusion immunocytochemistry: detection of proteins, labeling with peroxidase or colloidal gold
- ultra-thin cuts
Data acquisition and processing
The acquisitions are in the form of an image in dm4 format, which is the proprietary format of the acquisition software. This format allows retention of microscope operating data during observation, as well as image analysis using Digital Micrograph and to some extent ImageJ. The Digital Micrograph software is available free of charge on the site of the manufacturer of our acquisition cameras. It just requires registration to be used. http://www.gatan.com/products/tem-analysis/gatan-microscopy-suite-software
Data can be converted to Tiff format after acquisition and collaborators can retrieve them immediately. They can also be converted to JPEG, but this is a destructive compression format and therefore there is loss of information.
The most practical way is to bring a USB key or an external hard drive when observing. It is difficult to predict the size of the data collected but it is not uncommon for the image to exceed one gigabyte.
We keep the acquisitions on our side, but in the absence of a decentralized storage solution on the platform, we cannot guarantee this conservation 100%.