Once embedded, samples are sequentially imaged using an HREM microtome with optics and camera.
- Stereo dissecting microscope with a strong fluorescence light source
- HREM microtome with optics filters and camera
A commercially available HREM setup is shown in Image 1.
- First check that the block is sufficiently hard for use.
- For all blocks, a simple test is to see how easily the block surface can be distorted by pressure from a fingernail. A hard block will show a minimal, discrete indentation or scratch whilst a soft block will distort to give a broad groove. For larger blocks, also test the response to pressure across the sides of the block, especially near the chuck. Hardened blocks resist pressure whilst with soft blocks a slight compression is discernable. Blocks that have previously polymerised hard (or have been baked hard) can become slightly soft during prolonged storage, either at room temperature or in the cold. We are not sure what causes this and in the UK, this seems to be more of a problem in the summer months. It can be solved by baking the blocks again.
- Illuminate the sample from the base and use a stereomicroscope to view the specimen perpendicularly, through the block surface.
- Scratch a bounding box on the block surface, containing the entire sample.
- Because of the stereo optics, for an accurately positioned box it is essential to choose one side to draw and then repeatedly rotate the specimen by 90°, drawing the same orientation mark each time. Otherwise the images are likely to clip the sample on at least one of its edges.
- Turn the block so that the microtome blade makes contact with a narrow part of the block first, rather than a wide block edge. This minimises the chance of block breakage and reduces any section chattering. (Image 2)
- Use the bounding box to set up the image magnification for the HREM system. The bounding box should fill the image frame to ensure the maximum possible magnification.
- Ensure the surface of the block is at the level of the blade before setting the magnification.
- For the highest quality images it is advisable to use a new section of blade for each sample.
- Blade quality can be variable and you may need to avoid regions that give excessive scratch marks on the block surface.
- We use tungsten carbide Leica Disposable Blades TC-65 (barcode: 14021626379). Although disposable they still cost around £100 each, so we use them as much as possible.
- Trim the remainder of the cushion using the HREM microtome until the sample can just be visualised.
- Samples can be detected in the live camera image, a few microns before they reach the block surface, appearing as a faint but distinctive shadow. Alternatively, with direct visualisation of the block surface under GFP excitation wavelength light, the top of the sample can be seen as a distinct dark region immediately prior to its appearance at the sectioned surface.
- Ensure the focus is accurate using the imperfections in the surface of the cut block.
- Before imaging, it 's often hard to see the sample as it is slightly below the surface, which makes it difficult to check if the optics are focussed correctly. However, by looking carefully it 's possible to find a focus plane that reveals very slight irregularities in the polymerised block surface; this is a fairly accurate focus position.
- Establish optimum illumination and exposure settings.
- These can vary from block to block, and cannot be standardised accurately. Remember that the cushion/block interface can appear much brighter than the rest of the block.
- Set the desired section thickness.
- We do not recommend sectioning thicker than 6 μm and usually choose to discard captured data from analysis rather than increasing section thickness. Typically we section E9.5 embryos at ~ 1 μm, E14.5 hearts at ~ 2 μm, E14.5 embryos, E18.5 hearts and adult tissues at ~ 3 μm.
- Use a small fan to ensure that sections or section fragments do not remain on the block to obscure subsequent images.
- Whilst sectioning ensure there are no fluctuations in lighting, as this can cause exposure changes that will be evident in the image data.
- In our experience LED light sources give reliably reproducible exposures even with triggering. The ambient lighting should be kept constant if possible as changes will be detected in the image stack.
- Review focus position
- After a reasonable portion of the sample has been imaged (eg 20 – 30 sections) the run can be paused to enable fine adjustment of the focus position.
- Check images for uneven ‘ripples ’
- When you are imaging a block, a tell-tale sign of softness is the appearance of uneven ‘ripples ’ across the image. This may not happen at the beginning of the sectioning since the top of the block is the least likely to be soft, but it will become much more evident as sectioning proceeds towards the base. If you see this with a sample, it is prudent to rebake the entire batch of samples.
Using HREM data
Image data on the DMDD website is captured at 12 bit resolution to maximise the information obtained. This enables us to apply subtle adjustments to grayscale mapping in order to give the best visualisation of different tissues. The precise adjustments vary between datasets and we try to achieve a consistent appearance of the final images. Data is scaled to obtain cubic voxels and the resulting images converted to 8 bit for display.
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