A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

The overall goal of this procedure is to freeze muscle tissues, by plunging them directly into liquid nitrogen while eliminating freezing artifacts. Maozhang He, a student from my laboratory will demonstrate this procedure with Yizhong Huang. To begin, label the sample identity on each cryogenic vial.

Then, organize the following equipment:a tank of liquid nitrogen, safety glasses or a face shield, freezer gloves, 10 centimeter forceps, and 25 centimeter tweezers. Also, organize a styrofoam cooler, plastic film, a scalpel, and A4 PVC binding covers. To prepare muscle samples, begin by filling the cooler with liquid nitrogen.

Within five minutes of obtaining a specimen, gently place the sample on a piece of A4 PVC binding cover, and observe the direction of the muscle fibers. Next, use a scalpel to make two parallel incisions through the specimen in the direction of the muscle fibers, approximately 3 centimeters in length, and 0.6 centimeters apart. Trim the incised muscle into a rectangle approximately 0.6 centimeters wide, by 0.6 centimeters high, by 1.5 centimeters long.

Then, using ten centimeter forceps, gently put the sample on a piece of plastic film with the longitudinal axis of the incised muscle parallel to the long edge of the film. Place the muscle tissue into the lower middle of a labeled cryogenic vial, just between the inlet holes, and tightly cap the vial. To freeze the tissue sample, use 25 centimeter tweezers to quickly submerge the whole cryogenic vial in liquid nitrogen, in the pre-cooled styrofoam cooler, waiting until the liquid nitrogen no longer boils.

The liquid nitrogen should be adequate to immerse the whole cryovial, and the cryogenic vial should be entirely below the liquid level. Transfer the muscle samples to a liquid nitrogen storage tank, or a 80 degree celsius freezer for long-term storage. It is important to avoid accidental contact between the frozen muscles and the room temperature containers or instruments.

To prepare slides, pre-cool a cryostat to between 20 degrees celsius, and 22 degrees celsius. Remove and discard the old microtone blade, and wipe down the knife holder, and anti-roll plate in the cryostat. Then, install a new microtone blade in the cryostat, and set the cutting thickness to eight micrometers.

Remove the cryogenic vial with the muscle sample from the liquid nitrogen storage tank, or 80 degree celsius freezer, and place it in the liquid nitrogen, or on dry ice. Then, transfer the sample to the cryostat, and wait 30 minutes for the specimen to equilibrate with the temperature of the cryostat. After using OCT to embed the sample, mount the sample in the specimen holder.

Then, cut eight micrometer sections. Mount the sections towards the center of a room temperature micro slide. Immediately place the slide into a slide box.

Then, place the box of slides in a 80 degree celsius freezer. Do not allow the slide to dry at room temperature. As seen here, ice crystals formed when a sample of longissimus dorsi muscle from pigs was placed directly in a 80 degree celsius freezer, and caused a well known Swiss cheese effect on the muscle cryo-section.

Ice crystals also formed when the muscle tissue was immersed directly in liquid nitrogen. Using another method, many needle-like ice crystals formed in the cytoplasmic compartment of myofibers when the tissue was dipped in OCT mounting medium in the proper orientation, and then rapidly frozen in a slurry of isopentane. Freezing the tissue into the multi-hole cryovial achieves excellent specimen integrity, with a clearly visible cytoplasmic compartment and the tight apposition of myofibers to the surrounding tissue.

Using this method, ATPase activity was analyzed in fast-contracting, and slow-contracting muscle fibers. When given equivalent times, fast-contracting fibers appear light, with two degrees, and slow-contracting fibers appear black. In a standard freezing method shown here, muscle tissue was submerged in a slurry of isopentane without direct contact with OCT mounting medium, however a solid liquid slurry state of isopentane is difficult to maintain for more than one hour, and isopentane is not always available.