GUIDE – Hints and Tips on Using CMC’s
1. Use with Fluorescent Microscopes. These new correlative microscopy coverslips (CMC’s) are not designed to replace the glass or quartz coverslips, which have superior optical properties, and so are far better for fluorescent microscopy.The film itself has a structure which is visible under certain light conditions, especially with fluorescence. The CMC’s are designed to satisfy the needs of correlative microscopy where an initial, general analysis is performed using light or fluorescent microscopy and then the specimen is further processed for analysis by SEM, TEM or cryofixation.
If you need to perform detailed analysis using fluorescent microscopy then the CMC’s are not the solution. If however you want to perform a comprehensive analysis using different analysis techniques then the CMC’s are the only solution. The key advantage of the CMC’s is that they are made on special film that can be cut or punched (for cryo applications), something that is not possible with glass coverslips. The grid image is also transferred to any embedded specimen making cell location far easier.
2. Best Types of Resin to Use. Because this is new technology it often requires a change in the customers preparation method. The method that the customer has traditionally used for glass coverslips may not be suitable for these film ones. There will be a website up and running soon (www.correlative-microscopy-coverslips.com) that will provide all technical information and blogs so that customers can share experiences and contribute to method development.
The key thing to successful use of these CMC’s seems to be the type of resin and the preparation. We have already mentioned about the need to ensure a hermetic seal between the Beem capsule and the CMC film. The preparation works perfectly with low viscosity epoxy resins (eg Spurr,EPON). Sufficient polymerisation is obtained in 24 hours at 60C – it does not normally require 48 hours.
3. Always clean the coverslips before applying the specimen. This can be done either with UV light or alcohol. After using alcohol it is best to immerse the coverslip in polysyline to give better adhesion (just using alcohol can leave the surface ‘too clean and too smooth’).
4. Film Type. The CMC’s are made on a special polyester base film which is 0.18mm thick. There is no absolute tolerance specified on the thickness of the film.
We are gathering information all the time from users as we develop the preparation methods, so all feedback is very useful.