High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

The overall goal of this procedure is to generate digital volume data of organic materials. Examples are embryos and embryo tissues of biomedical model organisms, tissue blocks harvested from adult animals and humans, including skin or muscle biopsies and material like uncoated paper and skin substitutes. HREM helps answering fundamental questions in the fields of medicine and biomedical research.

It permits three-dimensional analysis and visualization of organs and tissues of for example, genetically-modified mouse embryos or manipulated chick embryos. It also permits analysis of cancer models as well as biopsies taken from human or animal tissues. The main advantage of HREM is that it provides a simple way to generate stacks of images which are of near histological quality.

The implication of this technique extend towards researching wound healing by using animal models, as well as spinning and mending tissue pathologies in humans. Demonstrating the procedure will be BMR Marlene Rodler and Johannes Gunther. Both from the Medical University of Vienna, Division of Anatomy.

Begin with fixed embryos or embryonic tissue of no larger than 5 x 5 x 5 cubic milliliters. Remove the fixative. And add PBS.

Wash at four degree Celsius with constant rocking for 24 hours. Add 70%ethanol to the tubes containing the tissue and place the samples on a rotator at four degree Celsius. After two to three hours continue to dehydrate the samples in ethanol solutions of increasing concentration for two to three hours each time.

While the samples dehydrate, prepare infiltration solution by adding 0.3125 grams of benzoyl peroxide and 0.1 grams of ESN to 25 milliliters of solution A in a beaker. Stir on a magnetic stirring plate at four degree Celsius for two to three hours until the ESN and catalyst are fully dissolved. Place the samples in infiltration solution.

And rock or rotate the samples for 12 to 24 hours at four degree Celsius. Begin by preparing embedding solution. Add one milliliter or solution B to 25 milliliters of fresh infiltration solution.

After preparing the molding cup trays, fill the deep cavity of the molds with embedding solution. Transfer the samples into the molds using a spoon. Ensure that the sample is fully covered with embedding solution to avoid trapping air.

Orient the sample inside the mold using needles or forceps. It is critical to optimize the orientation of the sample during embedding and to mark its precise position on the block surface. This way the region of interest can be kept small and the objective with the highest possible magnification can be used.

As soon as the embedding solution starts to become viscous, place a block holder on top of the mold and add embedding solution through the central hole of the block holder until the embedding solution raises to one to two millimeters above the base of the block holder. Seal the molding cup tray by covering it with liquid paraffin wax and wait until it is hardened before moving. Allow the blocks to finish polymerization by storing the sealed molding cup trays for one to two days at room temperature.

For post polymerization processing, place the molding cup trays with the polymerized blocks in a standard laboratory oven and bake at 70 to 80 degree Celsius for a minimum of one to two days. Remove the blocks from the molds. Then identify the field of view by directing white light obliquely to the block surface.

Trace the shadow the sample with the black marker on the block surface. Mount the resin block with indicated field of view on the microtome and to move the block holder to its stopping position. Start the digital camera and data generation software and acquire a live image.

Choose an objective with appropriate magnification to cover the region of interest. Move the optic up and down and the microtome laterally until the region of interest matches the field of view displayed on the computer screen. Section the block until the first structures of the sample become visible.

Move the microtome to arrange the block surface in the focal plane of the optic. Choose a section thickness between 0.5 to five microns. Then after setting up the software as per the manufacturer's instructions, start the software and begin data capture.

This HREM section image shows a sagittal section through a mouse embryo harvested at embryonic day 9.5. This volume rendered 3D model shows the surface of this embryo. This image shows a virtual sagittal section through a volume rendered model of the neck of the mouse embryo harvested at embryonic day 15.5.

The surface model highlights the lumina of the cardiovascular system in a volume rendering of all tissues of a chick embryo, a developmental Hamburger Hamilton Stage 18. This image shows an HREM section through a human nerve. The inlay enlarges the section of this image.

This image shows part of an HREM section image through porcine liver. This image shows a volume rendered 3D model of a biopsy taken from a human thumb pad. This image shows the surface rendered models of dermal arteries, veins, and nerves in front of a virtual resection through HREM data.

This animation shows a volume rendered model of thick skin of a human thumb pad. Different thresholds and hence different dermal structures, such as blood vessels, nerve fibers, sweat glands and the ducts of sweat glands. HREM allows for rapid visualization of the architecture of fibrous material.

This image shows a volume rendered model of native dermal substitute material. This animation shows the volume rendered model of dermal substitute material. Note the different shape and the caliber of the fibers.

Samples have to be dehydrated, infiltrated, and embedded in resin so the whole procedure can take up to several days but includes only two to three hours of operational works for preparing solutions, changing solutions and so on. The data generation itself is fully automated on the HREM apparatus and 1000 images can be produced in two to three hours. After its development, HREM paved the way for researchers in the field of developmental biology to precisely score the phenotype of mutant mouse embryos.

One example is the deciphering mechanisms of developmental disorders or DMDD project where HREM is used phenotyping, embryonically lethal mouse embryos in the yet unknown detail. It turned out that HREM is also an excellent alternative to positional light microscopy for examining blood vessels, nerve, or fiber architecture in human tissue samples.