Optical High Resolution Episcopic Microscope (HREM)
There is now an instrument available to realize the Optical HREM technique in your laboratory.
This technique achieves imaging for 3D reconstruction purposes. For whole samples/embryos by multiple sectioning (acurate cutting) samples of sizes 1mm to 20mm. High resolution imaging of each cut surface thus lends itself to 3D reconstruction of the whole sample and acurate measurement.
OHREM is an episcopic imaging procedure that uses plastic rather than wax as an embedding medium. Samples are embedded in a resin that has been made highly fluorescent by the addition of dyes.
Opaque and translucent tissue is imaged by its ability to suppress the fluorescence of the plastic. This results in extremely high resolution images of the block surface, irrespective of the tissue type or developmental stage. A standard section thickness of 1-5 micron is achievable and no re-alignment of the sections is required. OHREM is suitable for use over a broad range of magnifications.
Different tissue types are easily distinguished and as no realignment of the sections is necessary, the sections are simply imported directly into whatever 2D or 3D visualisation software you may be using.
Cut times and parameters:
Whilst it takes a few hours to cut a large block the resolution and section alignment achieved is excellent and the setup time is relatively small.
The speed of cut used and all camera and illumination parameters can be varied via the software to give the best section quality.
The Sample size is 20mm by 15mm approximately.
The Optical HREM is able to cut sections down to just below 1 micron but for most imaging 1.7 micron (and above) z section thickness produces good results.
Software:
The OHREM software controls all aspects of the system. Ensuring the fluorescence source is switched on at the start of the experiment and off at the end. The image capture parameters are set prior to the experiment commencing and then the system captures the 100’s or thousands of images without user intervention. The images are stored when captured and are in popular image formats for direct use in image processing and analysis packages as required.
Optical High Resolution Episcopic Microscope (HREM)
Resolution:
The resolution of the image captured depends on the camera used and there are a variety of cameras that are compatible with the software; Hamamatsu (4 megapixel- some earlier cameras are as high as 12megapixels) and Jenoptik Progress range (1.4 to 12 megapixels scanning) will be available in the coming release.
More cameras are being added as they are tested to be useful for this technique.
Sub Micron features can not only be viewed clearly in your 2D or 3D software (the images of sections taken can be JPG or TIF) by this system but measured accurately as the section thickness is measured at each cut and at the end of each experiment a measuring graticule can be imaged to ensure exact x y calibration from your camera when you import the sections into your imaging/measuring software.
