Following infiltration, samples are embedded in polymerised JB-4 dye mix.
- A stereomicroscope with very bright illumination from the base. (Image 1)
- This enables samples to be visualised during the embedding procedure, despite the dark red colour of the JB-4 mix.
- As an example, we use a Schott KL 2500 LCD fibre optic lamp, with a 250 watt bulb, providing a large bundle diameter up to 15 mm.
- Glass capillaries to manipulate samples, e.g. glass Pasteur pipette lengths that have been heated to produce blunt ends.
- Although fine forceps can be used to manipulate samples, there is less danger of damage using glass capillaries.
- Moulds for embedding the samples. (Image 2)
- Plastic chucks to attach to the base of the samples. (Image 3)
- Custom-made chucks are available from Indigo Scientific. These are not listed on the website, but can be ordered by emailing firstname.lastname@example.org
- If samples are to be attached directly using the JB-4 mix, it is best to use chucks with both a central hole and cross grooves. This is more convenient than using traditional 2-step procedures in which an adhesive resin is used to attach the polymerised block to the chuck.
Basic Embedding Procedure
- At room temperature, add Solution B to cold JB-4 dye mix and mix vigorously. We recommend 0.6 ml of Solution B per 10 ml of JB-4 dye mix.
- We use glass scintillation vials, with up to 20 ml of mix per vial, and add twice the manufacturer’s recommendation of Solution B as the dyes appear to have some inhibitory effect on polymerisation.
- Fill the mould with the polymerising mix and add the sample, ensuring no air bubbles are trapped on its surface.
- Between approximately 10 and 30 minutes later, there will be an optimum period of a few minutes during which the viscosity of the mix increases. At this point, samples can be realigned and will be held in their new positions.
- If Solution B is added to a room temperature JB-4 mix, polymerisation will occur more quickly and there will only be a very brief period of increasing viscosity. If the mix does not polymerise fully, or is taking longer to polymerise, check your components by testing polymerisation of JB-4 without dyes. Incomplete removal of methanol during sample infiltration also appears to inhibit polymerisation.
- Adjust samples as necessary, but avoid excessive movement as this can cause unsightly variations in the background of HREM images.
- Polymerisation proceeds from the bottom upwards. As a result, samples will be held more firmly at the bottom and can be easily damaged by realignments in very viscous mix.
- Place a plastic chuck on top of the mould, ensuring that enough mixture is present to fill up the central hole in the chuck and the cross grooves.
- Note that only some of this will polymerise, so it’s essential that there is enough to attach the block adequately to the chuck.
- Leave the samples to polymerise completely in an oxygen-free atmosphere. It is sufficient to cover the exposed surface of JB-4 with a thin layer of mineral oil.
- Samples will polymerise in a couple of hours or less, but are best left overnight at room temperature.
Samples embedded using this method will rest on the bottom of the mould (i.e. the top surface of the block). As a result, after polymerisation, the sample will often protrude slightly through the block surface and this will be visible in the first HREM images.
Embedding with a cushion
If it is important to obtain a complete image series (e.g. for complete 3D models), samples should be embedded after first forming a thin cushion of polymerised JB-4 at the base of the mould. (Since JB-4 polymerisation is oxygen-sensitive, it is extremely difficult to polymerise a layer thinner than 1-2 mm).
- Add sufficient polymerising mix to the mould to obtain a layer 2-3 mm thick and completely cover with mineral oil.
- Leave to polymerise for 30-60 minutes before following the Basic Embedding Procedure. (It is not necessary to remove the mineral oil from the cushion before proceeding).
Note that the cushions will have a slightly convex surface, which can make stable positioning of samples difficult. One solution is to wait until increasing viscosity of the mix holds the sample in position. Alternatively, after filling the mould with polymerisation mix, but before adding the sample, it is possible to flip the cushion upside down with fine forceps. This ensures that there is a flat surface for the sample to rest on. Note also that cushion thickness may vary from sample to sample, despite identical polymerisation conditions.
The cushion will be visible in the raw HREM images, since the interface between the cushion and the remainder of the JB-4 block generally appears much brighter than the rest of the plastic. This effect appears to get worse, the longer the time gap between polymerisation of the cushion and embedding the sample. It is not, therefore, advisable to leave cushions overnight before use.
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