My final day in the lab has now come and gone. University has started again and my time in the lab already feels like such a long time ago.

I have really enjoyed myself over the past 8 weeks. Learning many new techniques that will hold me in great stead for the coming year. I now feel very at home in a lab environment thanks to the warm welcome I got from everyone. I got many helping hands along the way from many different people. All of whom have been very supportive.

I have cultured many friendships through this experience, and even the odd game of squash! Getting to know many people in this environment has been very helpful as they are all great for advice on furthering your academic career and pointing you in the right direction.

This placement has helped me cement my choice to apply for postgraduate studies with an aim to go on to an eventual PhD.

I would like to thank everyone from the lab that has helped me along my way. I have learnt so much and had such fun doing it. I would recommend a studentship like this to anyone thinking about furthering their careers in academia, or even if they are unsure of what to do in the future.

I would also like to thank the Biochemical society for funding my studentship. Which may not have been possible otherwise. They are providing an amazing opportunity for undergraduate students like myself to venture into the lab environment for the first time. – CT

It has come to that stage in my 8 week project with one week left to go. Its all go and I am now starting to have enough data to perform some significant statistical analysis. It’s incredibly exciting as I can now finally see wether the work I have been doing has been fruitful or not.

It hasnt become clear yet wether my Trehalose infusion before deep freezing has prevented any damage to the contractile function of the muscle fibres. But I have been taught how to statistically analyse the data that I have amassed over the past several weeks.

This is an invaluable skill to learn and I’m sure it will put me in good stead for my  further studies. What I have found the most useful about this placement is how much more invigorating learning these techniques is when you can see them applied in a practical context. It really helps to enforce what yo have learnt and makes it far more memorable.

At the moment there is plenty to do to make sure I make the most of my remaining time. I am already starting to feel sad that my time is running out. The project has given me a great insight into what real experimental science is. – CT

The day had gone well I had intended to stay late to get a few experiments done. Unfortunately on the last contraction of the day I as always took my measurements but absent mindedly twisted the wrong dial. I thought this was no problem I would have just overstretched my fibre as the dial would have pulled the pins further apart. I looked down the microscope to re-adjust and saw that the force transducer arm had come lose. Oh no! It was borken.

I told my supervisor Tim much to his amusement at the time! He wasn’t too worried and said he couldn’t even remember how many he had broken over the years. They are incredibly delicate a slight touch in the wrong direction and they break. This is because they need to measure small forces of a single muscle fibre contracting. Luckily it was no big deal to fix, although it would take a day.

So I set about learning how to fix the transducer. This entailed a bit of electronics, which I have very little expertise in. I got plenty of help and encouraging words one I remember more then others, one of the post-doctoral researchers joking with me about how on this day I had become a true man and that you couldn’t have been one without breaking a force transducer.

It was a day that I learnt one of the last techniques I hadn’t yet learnt meaning I am now almost 100% independent to do the work I need to do. This is a great feeling! Complete independence in a lab that lets me go about my work and achieve what I want to achieve it was brilliant. – CT

Diagramatic representation of a sarcomere showing the thick and thin filaments as labeled.

To measure the sarcomere length a program can be used. This allows you to measure the average size of each sarcomere. To do this I have to take a digital image of the muscle fibre mounted under light microscope with a camera attached.

With the picture I can then select an area with the programme and it will calculate the average sarcomere length. This will allow me to standardise my results for sarcomere length. – CT

This sounded relatively straight forward, unfortunately it wasn’t as easy as it had previously seemed. In the wise words of one of the research fellows in the lab ‘On paper and in theory everything looks straight forward, in practice not so much!’. I think this is a statement that can be applied to many experimental procedures.

After having spent a few hours mounting fibres I tried to move them out of my dish filled with relaxing solution to fix the fibres between two pins on the stage. This took me quite a long time and much muttering, much to the amusement of many of my colleagues (there is always a friendly atmosphere in the lab and quite a few jokes!). After some while I had correctly mounted the fibre and was ready to contract it! This was the moment I had been practising for, for the last two weeks. I clicked the acquire button on my programme to move the fibre along the solutions to contract it………… after two weeks of training it was over in 8 seconds. I checked to see the force that had been recorded….. nothing. After much scratching of my head and one of the research fellows trying to figure out what had gone wrong we tried again.

This time with our fingers crossed and breath held it worked! Heres the result:

As an explanation to this graph the greatest horizontal line between 3000-5000 ms (x-axis) is the max force of contraction of the fibre as a voltage (y-axis). Later I will convert this voltage to a force using the calibration setting of the force transducer. But this means I have contracted my first fibre and it feels great. All of the work in the lab preparing for this has finally paid off. It is a very rewarding feeling! Now for the repeats! – CT

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.

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!

Over the last week I have been keeping myself very busy with learning how to manipulate the single fibres under the microscope to mount them to test their force of contraction. I thaw the frozen fibres and move them into a dish containing relaxing solution. This allows me to manipulate the bundle of fibres and tease a single fibre out. Once this is done I have to make sure the fibre is straight and not damaged along its length. Then I have to trim it to an adequate size usually for my rig this will be anywhere from 2.5 – 4 mm long. Once the fibres are the required length I  attach minute T-clips to either end used to mount the fibres between the two hooks of my fibre contracting setup.

Photo of muscle fibre attached at both ends with T-clips(bottom) . T-clip unfolded shown (top).

This takes a bit of practice and is very delicate work. You need patience and perseverance to learn the techniques but they are so rewarding once mastered. In the next few days I get to look forward to freezing some of my fibre bundles in liquid nitrogen and then contracting them!

So far the work has been very fulfilling and there is never a dull moment. There is also much to observe and learn in a lab from many of the people working in it, who have also helped me along my way and taught me tricks of the trade to make life easier.