09 March, 2022
The Babraham Institute is a world-leading organisation and an amazing place to work. It always feels very welcoming to walk into the building each morning passing familiar faces, trading smiles and greetings, and continuing conversations with colleagues that started weeks ago perpetuated by these brief but always friendly interactions. My work includes the use of a technology known as Flow Cytometry which analyses single cells on both a physical and chemical level but perhaps most importantly, it can allow you to break down a sample of cells into individual components. A nice analogy here would be the concept of a fruit smoothie, and you would like to break it down into all the bits that are banana, apples, and grapes and separate them into individual cups; which is the main function of a cell sorter and I will delve deeper into this below. I work as a Flow Cytometry Specialist and my responsibilities include but are not limited to: operating the cell sorters, helping users of the lab with data acquisition and analysis, offering training for anyone wishing to use our instrumentation (currently a complement of 17 machines!), and below I hope to highlight a deeper insight into my core duties.
I start my day by battling the traffic on the A1307 and making my way into the lab to start the first task of the day; turning the analysers on and performing quality control checks which help standardise the machines and reduce variability (this can be really helpful when running experiments with different timepoints). By routinely running these checks, I am able to track machine performance and with any luck – everything checks out fine! In the rare situation that something seems amiss, I can then troubleshoot the issue further and perform any maintenance that may be needed. A combination of these aforementioned steps means I can spot trends forming in any generated data suggesting an issue requiring further attention to avoid any unnecessary downtime and, in the worst case, I always have an engineer on speed dial!
I may need to run some training on the in-house analysers: the Fortessa, Aurora, Yeti, and ImageStream. In a nutshell, the Fortessa and Yeti systems are equipped with multiple lasers and detectors allowing analysis by detecting the emission of light within a specified wavelength of light and can be used for basic and intermediary applications; the Aurora is a newer addition to the lab and builds upon the aforementioned systems but upgrades the capacity of detection across the wave spectrum of light which increases the flexibility, sensitivity, and applicational range of the technology; the ImageStream marries conventional flow cytometry with high resolution microscopy using cameras to collect large numbers of digital images. I invite anyone who requests an introduction to these systems to the lab and encourage them to bring their own cells so get a sense of what to expect when analysing their samples. This is a great networking scenario as well, as I can engage with the person on the training and figure out how I can best help them with their future work.
The cell sorters require more manual intervention to get ready for the day. I start by pressuring these systems up to 100PSI and equip the correct nozzle size which is dependent upon cell type and size (for example, a T cell measures 5-7μm so can fit a smaller nozzle whereas a keratinocyte measures up to 50μm and would require our largest nozzle and choosing the wrong nozzle could cause the machine to block!). Older and more conventional sorters, such as the Jazz and Influx, need their lasers aligned to the sample stream manually, which I do using 3μm single peak fluorescent beads through a series of gimbal adjustments on a XYZ linear translation stage. I then calculate the drop delay; the time between the point of interrogation and the point where the droplet breaks off, so we can accurately encapsulate a cell of interest in a droplet of PBS and sort that droplet accurately into a collection vessel - the “droplet” is created using a vibrating crystal that can resonate up to a frequency of 96,000MHz which, when applied to a stream of liquid, generates a tiny series of droplets big enough to contain a cell!
Example of what flow cytometry data can look like
This interrogation point is the position at which the lasers intercept the stream and visualise the phenotype of the cell by an antibody conjugated to a fluorophore (a fluorescent chemical compound that can re-emit light upon light excitation) or by the integration of a fluorophore into the DNA of a cell. This allows me to measure specific quantities of proteins found in, or on, a cell/particle (in the plot below, cells have been stained with fluorophores conjugated to CD8 and CD4 to visualise T helper and cytotoxic T cells). When a fluorophore is exposed to the energy of a laser become “excited”, however, as energy cannot be created nor destroyed but rather transferred, as the fluorophore leaves this excited phase back to a resting state, it releases a photon of light that a series of detectors and electronics within the machines can visualise and display as an event on a plot. The above may be an oversimplification of the process but gives you a basic idea of what’s happening, there is a lot of other things going on too!
In the rare moments I am not working in the lab, you can find me in the office. This is a space you can sneak a few snacks, have a quick drink, and where I engage in perhaps something that doesn’t seem as “science-y”; responding to emails. However, emails tend to be where the majority of the science starts as this is the platform I used to collaborate and communicate with all the core’s internal and external users. In one email, I may bridge the gap in someone’s knowledge which could be the final component to them getting their experiment started or finishing some data analysis; in another I may start a series of conversations slowly but surely designing an experiment from the ground up, helping optimise fluorophore panels (a combination of fluorescently tagged antibodies used commonly for analysing all the cells in an immune system) and develop their methodology. These experiments can perhaps last weeks, if not months, so it always feels very rewarding to have provided some help and guidance in the initial foundation work.
The end of the day is nice and consistent; cleaning and shutting down all of the instrumentation. Taking the time to do this now is something that the tomorrow morning version of myself is always thankful for when I’m still half-asleep. Routine cleaning prevents contamination spreading throughout the machine but also ensures good data quality is generated so it’s a win/win for everyone. Some days are more complex than others and some can be more relaxed but it’s always a mixed bag and that’s half the challenge!
The Flow Cytometry Core Facility itself is a massively collaborative space and atmosphere full of a mix of both internal and external users from varying groups who are always excited to talk to you about their science. It’s also home to cutting-edge technology as the facility is an early adopter of all things new in flow cytometry, which is one of my favourite things about working here. I have worked in this field for nearly six years after a bit of dabbling with flow cytometry during my dissertation at university and was able to join the Sanger Institute as a Research Assistant where I stayed for over four years before joining the Babraham. It’s something I would recommend to anyone who wants to combine both biology with technology and enjoys working collaboratively on an assortment of projects encompassing all types of science.
09 March 2022
By Sam Thompson