Today I had the pleasure of learning the ropes for freezing fibres! This doesnt maybe sound like the most exciting thing…unless you get to do it with liquid nitrogen! All of this said the freezing process used liquid nitrogen to cool Isopentane to liquid nitrogen temperatures to freeze fibres in. So I wasn’t just adhoc throwing fibres into liquid nitrogen and seeing what happened. The reason for this is liquid nitrogen is highly volatile. So if I want to rapid freeze a fibre the heat of the cooled fibre would mean the liquid nitrogen would boil off slowing the freezing of the fibre. For this reason Isopentane is used. It is less volatile and will allow instantaneous freezing of the fibre bundles.
I had an induction given to me where I was shown how to collect the liquid nitrogen and how I was to dispose of the liquid nitrogen and Isopentane once I had finished my freezing. Apparently there is nothing worse than freezing one of your own toes off. So sandles/flip flops should not be worn whillst using liquid nitrogen.
After my induction I was set loose with a container for my liquid nitrogen and a set of fibres for freezing. Once the nitrogen was collected I needed to lower a metal container holding the Isopentane into the liquid nitrogen. I then waited afew minutes to allow the isopentane to cool down (It’s easy to tell when its cold, it begins to freeze over). I was taught the proficiency of leaving a piece of cork floating in the Isopentane so that I could remove it once it had frozen over. A technique very similar to fishing through ice!
The objective was to take a bundle of fibres prepared in a solution of 0.5M Trehalose and flash freeze it. Hopefully allowing vitrification (i.e. no ice crystal formation) to reduce damage to the sarcomere. To do this I grabbed the longest tongs I could find and extracted my fibre bundle from its 0.5M Trehalose solution and dunked it into the isopentane. At this stage the bundles look much like a frozen popsicle.
A pictoral representation showing the structure of the sarcomere and how it is arranged into muscle fibres
After this was achieved the day was done. The test of the success of this method will come in the next few days. The diffraction pattern of the fibres wil be assessed to gain a subjective analysis of how intact the sarcromere still is. If the fibres look ok in this respect they will be contracted to see if the contractile force of the fibres has been altered by the cryopreservation method.
I will update you again in a couple of days with hopefully some results to my experiment!