LM - Correlative Microscopy Coverslips Hints and Tips

EMS66108-01, EMS66108-02, EMS66108-03, G493-01, G493-05, G493-10

Physical and Chemical Characteristics 

• Resistant to normal chemicals used in electron microscopy 
• No oxygen retention and compatible resin LR White 
• Good optical quality in brightfield & UV fluorescence 
• Excellent transparency 
• Does not deform at temperatures (positive 100°C and negative liquid N2) 
• Rigid, does not float in the middle of culture 
• Easy to handle and cut with a knife or micro-punch Simple sterilisation using alcohol or UV
• Detaches easily from resin after polymerisation 
• Low cost

How to use the Correlative Microscopy Coverslips 

1. Sterilise the coverslip with alcohol, then dry and add the culture. 
   Ensure that the grid is positioned correctly so that the text is readable. 
2. Observe your cell culture using light microscopy (transmitted and/or fluorescence) and identify the area of interest (Fig. 1 and 2). 
3. Record the images needed, and note the co-ordinates of the squares where there are cells of interest (Fig. 1 and 2 show co-ordinate 8C). 
4. Fix, dehydrate and embed with resin for examination by transmission electron microscopy. 
5. At the end of the embedding procedure, invert a BEEM type capsule filled with resin onto the coverslip covering the selected cells of interest (Fig. 3). 
6. Cure and detach the coverslip (Fig. 4), the footprint of the grid (Fig. 5) allows location of the position. Trim the block (Fig. 6) in the selected area then make cuts using an ultra microtome.

Technical Advice

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 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 customer's preparation method. The method that the customer has traditionally used for glass coverslips may not be suitable for these film ones.

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 (e.g. Spurr, EPON). Sufficient polymerization is obtained in 24 hours at 60°C – 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.