Six months ago, FCS was a file type that the flow cytometer spits out, containing the complex, and often contradictory immunophenotyping data that I used to spend hours analysing during my PhD.
Fast forward to today, Fluorescence Correlation Spectroscopy sits firmly in my vocabulary, alongside a range of other biophysical terms and techniques that I now spend my days writing about.
I’m Charlotte, the Scientific Creative Writer at Exciting Instruments. I joined the team late in 2025, following a PhD in cancer immunology and a detour through the world of multiomics and spatial technologies.
The logical question, of course, is: ‘So, why are you working for a biophysics company?’
It is one that I asked myself, and caused a fair bit of imposter syndrome when I first started (and still does sometimes). Exciting Instruments’ mission is to democratise access to biophysical techniques, namely single-molecule spectroscopy in the form of smFRET, FCS and FCCS.
The fantastic scientists and engineers at EI have done just that – methods that once required dedicated dark rooms and career biophysicists can now be achieved using a benchtop instrument (and accompanying software) that is brilliantly described as having a ‘professor in a box’.
My role is very clearly part of this mission.
I was hired to work with our biophysicists to translate their expertise and incredible technology into something that not only resonates with biologists, but also allows them to actually make use of it. After all, biologists are tackling interesting and deeply challenging questions that literally underpin life and death! Such big questions deserve cutting-edge, powerful techniques that aren’t locked away in silos.
Still, the prospect was daunting. I didn’t know what the techniques were in any great detail, and importantly, how they could be used for anything other than complicated biophysical characterisation of proteins. Thus began a very intense December, where I aimed to get myself up to speed. Six months later, while I’m still learning every day, I feel like I can answer that dreaded question a bit better now.
Let me share with you some of the things that I’ve discovered – from one biologist to another.
The average is not a biologist’s friend
Let’s face it. Outliers are not fun. They skew our graphs, widen error bars and destroy any hope of seeing that magical p-value that gets everyone excited. As someone who used to analyse a lot of human blood samples, inter-donor variation was something I came to dread. Surprise, surprise…everyone’s an individual, and their blood cells are just as diverse.
The same is true for biomolecules – the same protein, formed from the same sequence, will exist in multiple different conformations within the same sample. Perhaps some have changed shape due to ligand binding or a newly phosphorylated site, while others might be wobbling between different states where the time spent in each of those configurations is influenced by salts or pH.
The point is, if that sample is analysed as a whole, this world of diversity is reduced to the average. Maybe only two conformations of the protein are captured instead of four, because the others are too transient and are lost to the more common states. If one of those transient states reveals a potentially druggable pocket, be it in the active site or at an allosteric one, that is information worth knowing.
If you have heard Tim Craggs, our CEO and Founder, give a talk for Exciting Instruments, you might have seen this picture – a rather large basketball player stood next to his coach who appears to be comically small in comparison. If their measured heights are analysed as an ensemble, the result is a number that doesn’t represent either of them.
We would never dream of reporting their heights as averages and expect it to be meaningful to them as individuals. And yet, this is so often how we analyse biological systems.
The ‘bio’ in biophysics is vast
So, I had the techniques and the concepts down. The next challenge was figuring out how this all applied to the world outside of biophysics. For the structural biologists, the complementarity of smFRET with techniques such as Cryo-EM, X-ray crystallography and NMR is evident. Biology exists in motion, so bringing exquisite atomic-resolution structures to life with dynamic data provides more clarity than either method could alone.
But how could someone like me, with a background in immunology and multiomics, really apply single-molecule spectroscopy?
My main concerns in the lab had been whether the change in brightness in my flow cytometry data was due to a true discovery or just lot-to-lot variation of antibodies. Around me, colleagues were trying to decipher how polydisperse their nanoparticle formulations were for drug delivery, with much frustration.
The turning point came when I realised single-molecule techniques could help with those issues, alongside a great deal more. And crucially, that these techniques do not require pristine, controlled conditions that are about as far away from physiological relevance as you can get – turns out, serum and cell lysates are fair game.
Antibody variability is a well-known problem. Biophysical QC is starting to take off (Abcam recently announced they are doing so), but there’s loads more that can be done to fix this. FCS can identify how many fluorescent dyes are attached to an antibody while determining target affinity and capturing aggregation tendencies and stability.
Definitely something I would have wanted to see performed on antibodies for my experiments, and something that could be applied to the fluorescent reagents used in NGS and spatial workflows too.
Common methods for analysing nanoparticles really struggle with complex biofluids and highly heterogeneous populations. FCS excels at this and is not completely disarmed by an overly large particle that skews the rest of the data. Add in a second fluorescent colour, and FCCS can directly confirm whether nanoparticles have been loaded with cargo (and if they’ve lost it somewhere down the line).
In the months that followed, I wrote about antibody-antigen binding, RNA folding, PROTACs, bispecific antibodies, ADCs, and so much more. The applications are vast, spanning academia, biopharma and, hopefully one day, the clinic.
At this point, I was starting to really get excited (apologies, we do lean into the puns).
But how democratised is it, really?
A good question.
The tricky thing about democratisation is that it can seem like technological rigour has been taken away in favour of simplicity.
I’m not here to tell you that single-molecule experiments are easy. Instead, I want to reassure you that they are accessible, irrespective of biophysical expertise, all without any sacrifice in quality.
The purpose of Exciting Instruments has been to take that complexity and distil it into something far more manageable across the lifecycle of an experiment. The benchtop instrument is testament to that, alongside extremely user-friendly acquisition and analysis software (replacing a slightly convoluted series of Python scripts and Jupyter Notebooks that make even seasoned users wince).
I have the privilege to work alongside world-class biophysicists who are supporting scientists through grant writing and experimental design to demos, proof of concepts and publications. They have supported me too, in teaching me how these techniques work and equipping me with detailed knowledge to share with the life sciences community. It is our job, collectively, to ensure that these techniques reach the people who can benefit from them and guide them through the process.
To quote Tim again, ‘You simply need curiosity, a question, and a place on your bench. Because when powerful technology becomes accessible, scientific progress accelerates.’
Final thoughts
I hope this has been insightful and has given you an idea of what is possible with single-molecule techniques. If you would like to find out more, we have a wealth of information on our website detailing the instruments, techniques and applications, alongside more in-depth technical notes.
Ultimately, my goal at Exciting Instruments is to translate the fantastic science that is happening here into something that resonates with people across sectors and specialisms. Everyone should have the opportunity to understand not just what is happening in their biological systems, but why, without having to spend three years learning how to build a microscope first.
At the six-month mark, I still have a lot to learn.
But I’m feeling a lot more confident when talking to biologists about single-molecule techniques, especially when they say: ‘But why is this relevant to me? I’m not a biophysicist.’
That’s the thing. Neither was I.
