Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

This sample preparation technique's designed to combine the best quality of ultrastructure preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope. This protocol is especially useful for samples that require preparation by high pressure freezing for a reliable ultrastructure preservation but do not get enough contrast during the freeze substitution for volume imaging. For minimal resin embedding it is essential to remove as much resin as possible to allow proper targeting later on in the focused ion beam scanning electron microscope.

To begin, use the pipette to drip one drop of 20%PVP PBS on the cutting mat. Immerse the dissected nervus tibialis in the droplet. Use the scalpel to cut two millimeter length of the sample.

Use fine forceps to place the sample in a 0.2 millimeter deep type A metal sample carrier. Transfer the carrier into the middle plate of the cartridge. Place the hexadecane-coated type B carrier on the top as a lid.

Close the assembly by lowering the upper half of the cartridge. Close the three-piece cartridge in the loading station of the high-pressure freezer. Press the process button and proceed according to the manufacturer's operating instructions.

In a bath of liquid nitrogen, place sample carriers into cryo vials containing two milliliters of frozen 0.1%tannic acid and acetone and close the caps. Place the cryo vials into the freeze substitution unit set at minus 90 degrees Celsius. Start the program and keep the samples there for 100 hours.

In the freeze substitution unit, wash the samples with two milliliters of acetone three times for 30 minutes. Place 2%osmium tetroxide in 0.1%uranyl acetate into the freeze substitution unit for cooling down and add it to the samples for seven-hour continued staining at minus 90 degrees Celsius. When the program temperature in the freeze substitution unit reaches minus 20 degrees Celsius, keep the samples in there for another 16 hours.

When the temperature rises to 4 degrees Celsius, discard the liquid in the vials and fill in pure acetone. Remove the cryo vials from the freeze substitution unit. Sample handling before freezing and after osmofication is critical since the sample can be severely damaged.

In a fume hood, use fine forceps to remove the metal carriers out of the cryo vials and transfer the samples from the carriers to submerse them in a 150 millimeter deep watch glass dish filled with acetone. Use fine forceps to transfer the samples into two milliliter reaction tubes filled with acetone. Set the microwave at 250 watts for 40 seconds and start the microwave to wash the samples and repeat four times.

Pipette one milliliter of 1%thiocarbohydrazide solution into the reaction tube and put it in the microwave. Turn on the vacuum function with two minutes on and two minutes off, iterating for a total of 14 minutes and set it at 150 watts. Start the microwave.

Wash the sample four times in acetone as previously. Pipette one milliliter of 2%osmium tetroxide solution into the reaction tube. Place the sample in the microwave and use the same microwave settings as with the thiocarbohydrazide.

Wash the sample four times in acetone as previously. Pipette one milliliter of 25%resin in acetone into the reaction tube and put it in the microwave. Turn on the vacuum function for three minutes and set it at 250 watts.

Start the microwave. Use a toothpick to remove the nervus tibialis out of the reaction tube and place them on a piece of plastic film. Place a halogen lamp close to the samples to heat up the resin.

Using a toothpick, gently push the sample around on the plastic film until no remaining resin is detected. Put the plastic film with the sample on top in the oven and set the temperature at 60 degrees Celsius to polymerize the samples for at least 48 hours. Use a razor blade to cut out the polymerized samples together with the plastic film at a size fitting the SEM stub.

Use a toothpick to add conductive silver resin on the SEM stubs and attach the cut samples to it. And then add resin around the samples. Put the SEM stubs in the oven to polymerize at 60 degrees Celsius for at least 4 hours or overnight.

After polymerization, position the SEM stubs in a sputter coater. Set the sputter coater at 35 milliamperes and apply 10 nanometer thick gold coating or coat for one minute. After coating, place the SEM stubs inside the focused ion beam scanning electron microscope.

In this study, an enhanced protocol was performed for nervus tibialis of the mouse as well as C.elegans to illustrate the optimal preservation and contrast to perform 3D volume imaging with a focused ion beam scanning electron microscope. Standard protocols often suffer from a lack of membrane contrast where as the enhanced protocol provides a strong membrane contrast. The cross section images of C.elegans with enhanced protocol and standard protocol show a distinctive difference.

As well as for the cross section images of nervus tibialis, the enhanced protocol helps to show a stronger membrane contrast compared with standard protocol. After post-processing, the electron microscope data are visualized using IMOD, an image processing and modeling program. The blue-highlighted area shows segmented axons.

The red-highlighted area shows the Remak bundles. The yellow-and orange-highlighted areas show myelin sheaths. And the turquoise-highlighted area shows mitochondria.

Virtual reslicing and the three-dimensional model illustrates the fine-tissue architecture and helps to understand its function. Other volume scanning electron microscopy methods can be used as well such as serial block-face scanning electron microscopy and array tomography. This protocol can also be used for transmission electron microscopy and other sample types such as samples from the central nervous system.

Osmium tetroxide, thiocarbohydrozide, and uranyl acetate are toxic chemicals and should be used carefully. The resin is also harmful and should be used with care. Personal protective equipment such as gloves and lab coat should be worn for the full protocol.