my blog

Coronavirus – getting ready in a lab

I am publishing here the recommendations I circulated to my colleagues, as this might help others to formulate their strategies or me to receive suggestions on how to improve. At the bottom of the post I also share my opinion about the situation, just to explain why we are taking action. Disclosure: I am no expert in this area, therefore I analysed data just to form my own opinion and to organize our work. Please check institutional guidance and reports.

Dear all,

      While we can still hope that no major disruption will occur, it is increasingly likely that the epidemic will not stop any longer. What is concerning, from a logistics point of view, is that this might last for several months as the responses of the public authorities will focus, rightly, in slowing down the epidemics. I would like to invite you to observe some basic rules, but also reflect on issues you might have not considered:

  1. The most important thing is to address the upcoming months with a scientific mind and no panic. Please follow the indications provided by the NHS, WHO, and the University.  While I do not doubt that all of us already wash their hands! Please do so also when you come in from outside, something we might usually not do.
  2. I am happy for you to work from home when you can. Most of you will have to carry out experiments, but I am happy for you to cluster reading and to write in specific days and to work from home. Would you need access to your computers from home, just organize this with IT but the Unit will provide appropriate IT arrangements soon.
  3. Some experiments could be rather expensive. Please let me know when this is happening so we could plan them properly. I would like not to delay important work though, so we might take some risks (on funds not on safety) but we could manage these risks proactively. For example ensuring that very expensive steps are executed in the shortest period of time and with sufficient people being aware of the experiment. For example, we have several commercial and in house developed cell lines that have not been archived yet.
  4. I can foresee two situations where we need to help each other. First, the case where a single individual will self isolate and they need help to store, throw materials, or shut down a microscope. I think Slack will suffice, but we should have also a ‘buddy system’. For example, if I started an experiment on a microscope that would last two days, I can inform someone else who would have the expertise to safely terminate the experiment.
  5. The second case is a bit more extreme but not really unlikely as other university campuses around the world have been closed. The Unit will soon provide specific guidance. Please think about which element would be critical, for example we will have to shut lasers down, air compressors to avoid them running out of oil etc.
  6. Do consider if you travel, even just within the UK, you might get stuck somewhere. Please check the University policies that are updated daily. I will not recommend specific actions related to personal trips, except to comply with public health guidelines and to think about the possible consequences to get stuck at home in one or another town.
  7. There is no indication – at the moment – that we will experience disruption to the supply chain. However, this might happen. Have a thought if we will run out of some consumables in a couple of weeks and perhaps order now.
  8. Also, very important. There are people coming from areas that are quarantined. Unless you are sure they are breaching rules, be supportive and do not make too many jokes. Some people are more sensitive than others.

To conclude, please do not allow the situation to make you anxious or too worried. For the general population, the main issue in not health but arranging life around likely restrictions of movement to permit the NHS to cope with the extra workload. For us, provided we will put first safety of ourselves, colleagues and family, we have opportunity to keep reasonably productive even in this situation simply organizing.

Feel free to propose ideas or to have a chat with me in private if you have any specific concern.

My opinion on the situation and on what is happening

I am growing of the opinion that the Italian situation is happening only earlier than in other European countries, not that is a special situation. Spain, France and Germany might be already on that path (10-14 days of delay compared to others). UK is probably an extra week late, meaning that by the beginning of April, or earlier, we will experience similar disruptions we are observing in Italy (hopefully not). Also, I had a look at mortality rates. Once taken into account the demographic and that in Italy we are experiencing a situation similar to Hubei (health systems overwhelmed) and not to the rest of China (managed containment of the disease), the stats of Italy do not seem odd to me any longer.

At this point, all other European countries will experience the same unless they enact strong preventive measures. To me it seems governments in Europe and USA have preferred to shield economy first rather than people, or they are simply incompetent, to then get caught off-guard and inflicting to the economy the same level of damage they would have got intervening earlier.
We can organize, minimize disruptions and deaths. Not eliminate them but we can do better we are doing. If only politicians would exert leadership, at local and national level, and – of course – people would comply with the indications…

Last word of caution. People might be complacent also thinking they have the best health systems. This is not the issue, the UK system will be as easily overwhelmed as the Italian one. In fact, there are fewer ICU beds in the UK than in most EU countries, including Italy. The point is to slow down the spread of the disease to keep our health system working within certain operational margins.

Bottom line. Am I writing to get your more worried or anxious? NO. The large majority of us will have minor health issues. However, the public health policies that will be necessary to minimize the negative impact on the NHS will cause major disruptions. Therefore, organize not by panic buying, but thinking ahead… how to work, look after family, etc, etc, when restrictions will be imposed.

Last thing. There is a tendency to minimize the situation as people dying is elderly affected by other pathologies. In Germany, it seems that they do not even consider those patients as CoV-related. The large majority of those people could have lived a much longer life, they are not (all) terminal patients. Moreover, with patients piling up in dedicated wards and ICU, everyone risks more because they will not receive adequate treatment, irrespective if they have been infected or not.

So… the apocalypse is not coming, but just the time to work together to get pass this.

Publishing: a business transaction

Until not so long time ago, desk-rejections (the editor decision not to proceed with peer-review of a submitted manuscript) or even rejections of a manuscript after peer-review with very little substance for that decision, could get me angry, at least in private. These emotions can motivate to do better, but most of the time – if we try too hard to get published in very selective journals – they can take a toll.

After speaking to several editors, I tried to focus on the fact that most of us (editors, authors, referees – sometimes the same people wearing different hats) are good and well-motivated people. That did not work. The sense of unfairness outranks that thought.

I tried to not care, and that did not work either. Until…

I believe that the large majority of scientists and editors do their job also for a clear vocation, to advance human knowledge for the benefit of society. For this reason, we often invest a lot more in our jobs that we should, emotionally and time-wise. This is why it might be difficult to have a detached view of what publishing is nowadays. Let’s make an effort together, watching the problem as a scientific one, analyze it, reducing its complexity to its components and mechanisms.

If you have a donkey and you want to sell the donkey, you go to the market. You might first go to a trader who pays very well as they have good contacts with wealthy farmers. However, they may not like your donkey even if you dropped the price. They do not like your donkey, why do you want to sell your donkey to them? Then you go to a different trader, they like your donkey and you settle for a fair price. But if your donkey is very old, you might come back to your farm with your old donkey. Perhaps you need the money and you get frustrated, maybe even angry, but which is the point? Business is business and the trader is simply doing their job.

Wait… what? D-D-D-Donkeys?

When you submit a paper to a journal, you try to initiate a business transaction. The editor is an expert trader, highly invested in their business and committed to maintaining their operations, legitimately, financially sustainable and profitable. The author trades-in two commodities, their manuscript and their reputation, and – additionally – pays a lump of money for the service. In return, the editor provides two commodities, their readership and their reputation, and – additionally – provides editorial services. I will perhaps elaborate in the future on the traded commodities and services, but for now, I keep this post to the bare essential.

The editor-trader first judges the quality of the product you want to trade-in. They are entitled to act discretionally applying their in-depth knowledge of their business to assess if they are about to initiate a potentially good deal. Can your donkey carry weight? Er, I mean, can your paper attract many citations and media coverage? If they do not want to do business with you, it is not a matter of fairness, even not of science, certainly nothing personal. It is the author’s responsibility to make their business pitch, and it is the editor’s responsibility to not lose good assets or not invest in bad ones.

If I read what I have just written ten years ago, I would have recoiled in disgust. Then I expect many scientists being horrified by what I have written and perhaps editors offended. I hope this is not the case, but if it happened, please let me clarify one point.

We (authors and editors) do what we do to advance human knowledge for the benefit of society. Boiling down everything to a mere business transaction feels perhaps bad. However, let’s keep in mind that scientific publishing is business. If it has to be or not, it is the subject of a different post and to the analysis of the nature of the commodities and services we trade.

For now, I just wished to share with you the trick I use to cope with the stress of rejections, particularly desk-rejections. That part of our job is just a business transaction. This thought helps me a bit more than anything else I tried before.

Lost in translation (dogma and science)

Once in a while I hear or read about dogmas as if they were models. I came to realize that some people might not be aware what a dogma is and before the (mis)use of this word spread even further, I hope you will agree to get it back into its original meaning.

The Oxford dictionaries define dogma as “A belief or set of beliefs held by a group or organization that others are expected to accept without argument”. Other dictionaries report similar definitions, but the Merriam-Webster also include the slightly softer “Something held as an established opinion, especially a definite authoritative tenet”. Many dictionaries also report to religious doctrines. Therefore, dogma can’t be used as synonym of model or hypothesis, particularly in science. Of course, most people are still using the word dogma correctly even in science, to refer to a model that has become established fact despite no, weak or even erroneously interpreted evidence for it.

I suspected that most of the damage has been caused by Francis Crick when he has introduced the “Central Dogma ” of molecular biology. Let’s be clear, I do not want to be pedantic and I care very little about semantics, but the correct use of the words dogma, hypothesis, model, theory, is rather important in science. There are instances when these four words might be interchanged but we should – I hope – all agree that dogma is to be used only with a negative connotation (in science).

I assume you know what the central dogma is but if you do not, the Wikipedia page is good enough to get an understanding. In lectures during the late 50s, Francis Crick stated that “Once information has got into a protein it can’t get out again” and named this statement “The Central Dogma”. Apparently the name was a bit of a joke, as it appears evident from the famous document stored by the Wellcome Library. The initial paragraph was entitled “The Doctrine of the Triad”, a clear reference to DNA, RNA and proteins with a rather obvious analogy to the Christian doctrine of the Trinity.

I must admit I did not read Crick’s autobiography, but it is well known that there, he writes that “I called this idea the central dogma, for two reasons, I suspect. I had already used the obvious word hypothesis in the sequence hypothesis, and in addition I wanted to suggest that this new assumption was more central and more powerful.” and “As it turned out, the use of the word dogma caused almost more trouble than it was worth. Many years later Jacques Monod pointed out to me that I did not appear to understand the correct use of the word dogma, which is a belief that cannot be doubted. I did apprehend this in a vague sort of way but since I thought that all religious beliefs were without foundation, I used the word the way I myself thought about it, not as most of the world does, and simply applied it to a grand hypothesis that, however plausible, had little direct experimental support.”

Then, I asked a friend who lived those times if perhaps the word dogma was used slightly differently in the past and I got this brilliant response: “A dogma in science is a fanatic intrusion into rational thought. When a big name in science makes a joke, accolades of small names taking it seriously are sure to follow… A model becoming a dogma is ready for the bin. Never had a dogma crossing my path.”

Well, nowadays I do see dogmas crossing my path but never mind, that is a different story. For the young students who might read the “central dogma” in text books and then adopt the term “dogma” as equivalent to model or hypothesis then, just two suggestions.

First, a scientists should be always skeptical and doubt about anything. It is unavoidable that sets of established facts, sometimes even wrong, become generally accepted in a discipline and crystallize into a real dogma that no one challenge. However, it is our duty to challenge any interpretation, any model, whenever is conflicting with evidence.

Second, let’s reserve the word dogma (in science) to critically identify established believes with insufficient or contradictory experimental evidence, or perhaps for jokes…

Ironically, the “Central Dogma” was a very good hypothesis.

Don’t fret about it, just get it (a FRET primer – Part I)

Why should you know FRET? Well, FRET is used when you do a real-time qPCR, or you might be using it in assays like HTRF, or to detect biochemical reactions in single living cells. You might measure protein-protein interactions, probe cell signalling, cell metabolism or nano-meter scale conformational changes. Or what about dimerization, protein – nucleic acids interactions, checking splicing variants by FISH, or detect fast conformational changes in structural studies? This is why some of us are very fond of FRET, and many others are using it without being fully aware of it. The usefulness of FRET arises from its capability to translate molecular properties occurring at a nanometer and nanosecond scales to optical signals that can be easily detected with a microscope or a spectrofluorimeter.

Figure 1. Energy flows from a donor ‘D’ to an acceptor fluorophore by FRET. The ration of fluorescence emitted by the donor and acceptors (IDD and IDA) can be used to estimate how much energy is transferred from donor to the acceptor, quantity that is proportional to the distance of the two molecules.

What is FRET? When a fluorescent molecule is in close proximity to another that might be, in principle, capable to absorb the light emitted by the first, FRET might occur. However, FRET is not the emission and re-absorption of light, but the non-radiative transfer of energy. This is important because the molecule that will donate energy and the one that will accept it become coupled and will inform us about the distance between the two molecules only if they are within a few nanometer ranges, with sub-nanometer precision. Most of us do not use this capability directly but to engineer probes that can sense specific biochemical reactions. Ok, now you are ready. What FRET stands for? RET is Resonance Energy Transfer and it says with three simple words what I have just described. For the “F”… you would think it is simple, but the community is a bit split on the meaning of that “F”. There are two camps. One that says “F” is for Foerster, from Theodor Foerster who developed the theoretical background to describe the phenomenon. Others say that “F” is for “Fluorescence” as it is detected by means of fluorescence emission. Who prefers Foerster-type energy transfer means to distinguish it from other possible mechanisms but, most importantly, to avoid misinterpretation of the acronym. Indeed, it is not fluorescence that is transferred from donor to acceptor and the acceptor does not need to be fluorescent. Those who use Fluorescence RET often say that Foerster did not discover FRET (correct, he did a mathematical description of a known phenomenon). Does it matter? Not really, but at least now we know what FRET means. Ah, I almost forgot… FRET for me is Foerster Resonance Energy Transfer… I heard you asking.

Next. How do we measure FRET? There are many ways to measure the occurrence of FRET but today I will focus only on ratiometric FRET and Fluorescence Lifetime Imaging Microscopy (FLIM). I am going to use an analogy that is very useful, that of buckets filled with water (Fig. 1). The tap is your light source, which is filling a donor bucket with water (energy). The bucket has one hole, from which water is dripping into a plate (a detector). That stream of water highlighted in green in Figs. 1-2 is the fluorescence signal that we measure, emitted by the donor. FRET is another hole punched into the donor-bucket. Water will flow into an acceptor-bucket from where it will drip (red flow) into a second plate (detector). The ratio of the water we collect in the blue and yellow plates will tell us the fraction of water that passed through the FRET “hole”. In a real FRET experiment, this fraction, called the ‘FRET efficiency’ is proportional to the inverse of the sixth power of the distance between the buckets, er… fluorophores.

Figure 2. Cross-talks between donor and acceptor excitation. DE: direct excitation of the acceptor. SBT: spectral bleed-through of the donor emission into the acceptor channel.

Unfortunately, the excitation and emission spectra of typical fluorophores are broad and spill-over of fluorescent signals (or water!) is usually unavoidable (Fig. 2). The buckets are large compared to their distance (the excitation spectra overlap) and part of the water we wish to put into the donor bucket will fill the acceptor bucket. This is called ‘direct excitation’ of the acceptor. The water we now collect in the yellow plate flows from one hole in the acceptor-bucket, but it originates from two different flows. Direct excitation (black flow) and FRET (red flow). The latter, FRET sensitised emission, is the signal that matters. At the same time, water flowing from the donor bucket spills-over into the yellow plate (the emission spectra overlap), adding a third (green) unwanted flow into the yellow plate.

So, how do we correct cross-talks? The good news is that sometimes you do not need to. If what you need to measure is a semiquantitative measure, the detection of changes, measuring the relative quantity of water that fell into the yellow plate compared to the blue plate will suffice. This, however, will require to ensure the stoichiometry of donor-acceptor fluorophores does not change, for instance when using typical FRET-based probes for kinase activity.

In other cases, you will need to correct for these cross-talks and techniques like ‘precision FRET’ and ‘three cube FRET’ comes to the rescue (see reference section).

Figure 3. FLIM measure the time the donor-bucket needs tobe emptied, thus inferring the size of the second (FRET) hole.

Another technique that can be used to measure FRET is Fluorescence Lifetime Imaging Microscopy or FLIM. FLIM does not need measuring the flow of water from the acceptor. FLIM requires to turn the tap on and off, and measuring the time that the donor buckets requires to be emptied. When a second hole (FRET) is punched into the donor-bucket, this will empty faster. We do not measure directly any signal from the acceptor and, therefore we avoid the need to correct for spill-overs.

This brings me back to the time I was a PhD student. A very smart master student entered my office and popped the question “how FLIM can detect the presence of FRET if the only photons we measure are those that do not experience energy transfer?”. Back then, I was taken aback from the question and I could not respond immediately in a satisfactory way. The bucket analogy should do the trick.

To conclude, this was just a brief overview of FRET and how we can measure it. There are plenty of great reviews out there to improve your understanding of FRET, but I hope that the analogy with buckets might provide a simple model for the non-specialist, albeit physically inaccurate for other aspects of FRET. Below, you can find a few references. Let me also refer to my new study published on Biomedical Optics Express entitled “How many photons are needed for FRET imaging?”. It is a theoretical study, but even the non-specialist might find some sections interesting and, plenty of more bucket figures there!


J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum Publishers, New York, 1999).

T. Förster, “Zwischenmolekulare Energiewanderung und Fluoreszenz,” Annalen der Physik 437, 55-75 (1948).

L. Stryer and R. P. Haugland, “Energy Transfer – A Spectroscopic Ruler,” Proceedings of the National Academy of Sciences of the United States of America 58, 719-& (1967).

G. Bunt and F. S. Wouters, “Visualization of molecular activities inside living cells with fluorescent labels,” International Review of Cytology 237, 205-277 (2004).

E. A. Jares-Erijman and T. M. Jovin, “FRET imaging,” Nat. Biotechnol. 21, 1387-1395 (2003).

J. Zhang and M. D. Allen, “FRET-based biosensors for protein kinases: illuminating the kinome,” Mol Biosyst 3, 759-765 (2007).

M. Y. Berezin and S. Achilefu, “Fluorescence lifetime measurements and biological imaging,” Chem Rev 110, 2641-2684 (2010).

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission,” Journal of the Royal Society, Interface/the Royal Society (2008).

A. Hoppe, K. Christensen, and J. A. Swanson, “Fluorescence resonance energy transfer-based stoichiometry in living cells,” Biophys J 83, 3652-3664 (2002).

M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy, “Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy,” Methods 29(2003).

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, “Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy,” Biophysical Journal 74, 2702-2713 (1998).

C. Berney and G. Danuser, “FRET or no FRET: A quantitative comparison,” Biophysical Journal 84, 3992-4010 (2003).

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophysical Journal 94, 986-1000 (2008).

A. Zeug, A. Woehler, E. Neher, and E. G. Ponimaskin, “Quantitative intensity-based FRET approaches–a comparative snapshot,” Biophys J 103, 1821-1827 (2012).

H. C. Gerritsen, A. V. Agronskaia, A. N. Bader, and A. Esposito, “Time Domain FLIM: theory, Instrumentation and data analysis,” in FRET & FLIM Imaging Techniques, T. W. Gadella, ed. (Elsevier, Amsterdam, The Netherlands, 2009).

R. A. Neher and E. Neher, “Applying spectral fingerprinting to the analysis of FRET images,” Microscopy Research and Technique 64, 185-195 (2004).

H. Wallrabe, Y. Chen, A. Periasamy, and M. Barroso, “Issues in confocal microscopy for quantitative FRET analysis,” Microscopy Research and Technique 69, 196-206 (2006).

S. Ganesan, S. M. Ameer beg, T. Ng, B. Vojnovic, and F. S. Wouters, “A YFP-based Resonance Energy Accepting Chromoprotein (REACh) for efficient FRET with GFP,” Proceedings of the National Academy of Sciences of the United States of America 103, 4089-4094 (2006).

J. Klarenbeek, J. Goedhart, A. van Batenburg, D. Groenewald, and K. Jalink, “Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity,” PLoS ONE 10, e0122513 (2015).

K. J. Martin, E. J. McGhee, J. P. Schwarz, M. Drysdale, S. M. Brachmann, V. Stucke, O. J. Sansom, and K. I. Anderson, “Accepting from the best donor; analysis of long-lifetime donor fluorescent protein pairings to optimise dynamic FLIM-based FRET experiments,” PLoS ONE 13, e0183585 (2018).

M. W. Fries, K. T. Haas, S. Ber, J. Saganty, E. K. Richardson, A. R. Venkitaraman, and A. Esposito, “Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate,” bioRxiv, 427237 (2018).

Signor Tenente (a song against mafia)

My holidays are spent with the nose into papers and the hands on the computer keyboard, working on quinquennial report. But I am back to my family in Italy, specifically in Sanremo, city of flowers, city of music, as it used to be the largest flower market and an important production center of flowers, and it hosts the most followed music festival in Italy. It is then not that surprising to walk in the streets and listen to music in the festive periods and in summer. Today, I got a break from work and went with my family to the main piazza of the town, where a group was singing various songs that contested the Sanremo Festival in the past.

The time came for “Signor Tenente” by Giorgio Faletti (1994), a song that was acclaimed by the critic and arrived second in the competition. A song that is musically flat, with a simple lyric, spoken rather than sung. A song that I had forgotten, but that is linked to an event I will never forget and changed me and many others in Italy, even very far from where it had happened.

In 1992, the prosecutor Giovanni Falcone was killed together with his wife Francesca Morvillo and three police officers in his security detail, Rocco Dicillo, Antonio Montinaro and Vito Schifani, when ‘Cosa Nostra’ blasted a segment of a motorway to kill his most feared enemy. Two months later, his friend and colleague Paolo Borsellino was killed with five police officers, Agostino Catalano, Walter Cosina, Emanuela Loi , Vincenzo Li Muli and Claudio Traina, by a car bomb while visiting his mother. Sanremo is a sea away from Sicily but in that tragic year we all felt Sicilians, raged against organized crime, close to the prosecutors, judges and the police forces – left alone by a political system that was about to be decimated by corruption scandals and that was in disarray.

“Signor Tenente” narrates that period from the point of view of the police (specifically Carabinieri) who, poorly paid and often in danger, do their duty while bombs kill.

These events might be difficult to understand outside Italy, or perhaps by the generation after mine. However, I wished to share with you, my friends, the feeling of pride I felt when, after a rendition of “Signor Tenente” finished, the square burst in a heart-felt applause, the warmest of the evening.

This is just a reminder that, in any country, most people are honest and good. There is time to criticize any authority, but there is also time to simply just thank, the police forces, the prosecutors, the justice system, and the people that in Italy and anywhere in the world fight injustice at great personal danger.

The coming year, talking about war

We have to be optimistic and hoping in a prosperous future for everybody, particularly in this period of the year, but optimism on its own makes very little to avoid sliding towards avoidable catastrophes. We can hope no storm will hit our towns in 2020 and live a happy life. At the same time, we can speak about the possibility of storms landing on our homes campaigning for strengthening river banks, coastal protections and flood barriers. Because our optimism should be well-spent in the hope that our actions will be successful rather than in the hope that our inaction will be rewarded by chance.

Lucio Dalla (1943-2012), a famous Italian singer, released a beautiful song in 1979, l’anno che verra’ (‘the year to come’). This song does not speak about war but of a troubled period of Italian history, when the country was shattered by political terrorism, when people’s worries were addressed by politicians by the constant renewals of promises of a prosperous future. This iconic Italian song is not just wonderfully and sadly contemporary, but also deeply meaningful outside Italy. It is thus a pity that, to my knowledge, no English rendition was ever attempted but scroll down to the bottom of this blog-post for a translation and the link to the song.

By many, l’anno che verra’ is considered an anti-war song. And being in Italy speaking with life-long friends, it came back to me. Yes, because during the last year, something has happened. Since the financial crisis, some of us has spoken about worries, at least privately, for an international political context similar to the period that preceded world-wide wars. Until recently, most people would respond to these concerns as if they were related to an abstract possibility, a distant scenario. Lately, I started to notice reactions that are more emotionally involved. Some people respond with an explicit wish for authoritarian figures that could bring back order and prosperity to people. Many others, quietly share their concerns, as to liberate themselves from an untold secret, something they never liked to speak about worried to be judged. Then I find myself speaking about the possibility of war with people from different countries and backgrounds, a discussion that is rarely met with skepticism by now. People does not appear pessimistic, desperately looking into the barrel of a gun, but realistically discussing about something that can happen and they wished to avoid.

Some politicians are promising us a prosperous future. At the same time, they are playing a complex chess game in an international scenario where the global geopolitical structure is in slow and constant flux, towards a new balance we cannot predict. Some politicians promise a wonderful year ahead, but advocate policies that lead to friction and conflict with other countries, a scenario that rarely leads to a peaceful and prosperous life. While I think that mainstream media could do a better job explaining to us what is happening, even just to reassure us, or to keep us alerted about the storms forming at the horizon, I feel I can do just one thing for now.

With a mild optimism that people will reject conflict and embrace international cooperation, with adequate scrutiny on the actions of their politicians, I dedicate to all of you Lucio Dalla’s song. Because there is still time to reinforce the river banks, the flood barriers and the coastal protections that defend our democracies, human and civil rights from the storms ahead.

Dear friend, …

The year to come by Lucio Dalla

Dear friend, I am writing to you so that I can distract myself a bit
And since you are very far away, I will write to you with that much more force.
Since you left, there’s been great change
The old year is over by now
But something still isn’t right here.
People don’t go out much at night, even when there are parties
And there are individuals who have put sacks of sand next to the window
And some are without words for entire weeks
And for others, they have nothing to say
Of the time that remains.
But the television said that the new year
Will bring a transformation
And we are all already in expectation
There will be 3 Christmases and people will celebrate all day
Every Christ will descend from the cross
Even the birds will make their return.
There will be enough to eat and it will be bright for the entire year
Even the mute will be able to speak
While the voiceless already do so.
And people will make love every day as they please
Even the priests will be able to marry
But only at a certain age
And without a lot of pain will someone pass away,
They will be perhaps the people who are too clever
And those who are too foolish in each era.
Consider, dear friend, what I write and say to you
And how content I am
To be here in this moment
Consider, consider, consider, consider,
Consider, dear friend, what one must make up
In order to be able to laugh through it all
In order to continue to hope.
And if this year were to pass then in an instant
Consider, dear friend
How important it becomes
That in this instant you be by near me again
The year that is coming will pass after another year
I am preparing myself and this is the news

Riprodotto da Muzikum

L’anno che verra’ by Lucio Dalla

Caro amico ti scrivo così mi distraggo un po’
e siccome sei molto lontano più forte ti scriverò
da quando sei partito c’è una grossa novità
l’anno vecchio è finito ormai
ma qualcosa ancora qui non va.
Si esce poco la sera compreso quando è festa
e c’è chi ha messo dei sacchi di sabbia
vicino alla finestra
e si sta senza parlare per intere settimane
e a quelli che hanno niente da dire
del tempo ne rimane.
Ma la televisione ha detto che il nuovo anno
porterà una trasformazione
e tutti quanti stiamo già aspettando
sarà tre volte Natale e festa tutto il giorno
ogni Cristo scenderà dalla croce
anche gli uccelli faranno ritorno.
Ci sarà da mangiare e luce tutto l’anno
anche i muti potranno parlare
mentre i sordi già lo fanno.
E si farà l’amore ognuno come gli va
anche i preti potranno sposarsi
ma soltanto a una certa età
e senza grandi disturbi qualcuno sparirà
saranno forse i troppo furbi
e i cretini di ogni età.
Vedi caro amico cosa ti scrivo e ti dico
e come sono contento
di essere qui in questo momento
vedi, vedi, vedi, vedi
vedi caro amico cosa si deve inventare
per poterci ridere sopra
per continuare a sperare.
E se quest’anno poi passasse in un istante
vedi amico mio
come diventa importante
che in questo istante ci sia anch’io.
L’anno che sta arrivando tra un anno passerà
io mi sto preparando è questa la novità

Riprodotto da Muzikum

Tackling cancer heterogeneity by live single-cell ‘systems biology’.

NOTE: This assay is the introduction to my research vision I wrote five years ago but that did not make into the programme grant we wrote. I think this is still current and, as it is unlikely I will publish this text, I am releasing it in the public domain with very little editing. I should note, particularly, that some paragraph remains unreferenced.

The genome, biochemical networks and phenotypes | Somatic mutations and gene copy number variations (CNVs) accumulate over time, stochastically altering the abundance and the functions of gene products. At first glance, efforts to identify and characterize somatic mutations provided a comparatively simple model: a few hundreds genes (proto-oncogenes and tumour suppressor genes) are often mutated contributing mechanistically to tumourigenesis (driver mutations). Some driver mutations are very frequent, but driver mutations that are less frequent in a cancer type overall dominate in number within an individual tumour, presumably conferring a more subtle growth advantage than others taken individually [1,2]. Also CNVs are very common in cancer. Sometimes, a clear role of CNVs in tumourigenesis can be established; most of the times, however, the effects of CNVs are difficult to predict or characterize because of the very different possible dependencies between phenotype and concentration of a gene products (e.g., haplo-insufficiency, quasi-sufficiency, triplo-sufficiency, etc.) [3,4]. Concentration effects and “subtle driver mutations” complicate the interpretation of genomic studies and may be best described by a continuum model for tumorogenesis where the all-or-none effects of individual genomic alterations are the frequent exception rather than the rule [4,5]. Notwithstanding the invaluable insights that genomics studies have provided and will continue to provide in our understanding of cancer, diagnostics and therapy, the role of these genomic alterations in tumorogenesis will be better understood in the context of the alteration of molecular networks underlying the respective cancer-associated phenotypes [1,6].

Few phenotypes are selected by mutation, those that enable cancer evolution [7] by increasing clonal heterogeneity (by genetic mutation, aneuploidy or epigenetic instability) and that permit growing in a hostile environment (avoidance of immunosurvaillance, metabolic deregulation and stromal hijacking). Moreover, cell survival, cell fate determination and, later in cancer evolution, cell migration are the key phenotypes that make of cancer the devastating diseases it is. Genomic alterations select for these phenotypes by influencing a comparatively small number of biochemical networks. Indeed, cancer-associated somatic mutations cluster in pathways controlling cell-cycle or cell-death, RAS/PI3K/MAPK, TGFβ, APC, STAT, NOTCH, WNT, HH and mTor [1,4,7]. Unsurprisingly, somatic evolution of cancer reshapes a comparatively small number of biochemical pathways that control cellular and tissue homeostasis to offset, often in a subtle manner, the net proliferative rate of cells. The study of these pathways is no less daunting than the understanding of complex genomic alterations. However, biochemical networks have evolved to exhibit robustness in the presence of intrinsic noise present in biological systems (e.g. stochastic variations in transcription or cytokines concentrations). Robustness of biochemical pathways permit to stably encode for cellular functions and cellular states. It is therefore conceivable that the myriads of possible genomic alterations and individual gene-products simply concur to generate a discrete set of biochemical states corresponding to cancer-associated phenotypes.

Other “big data disciplines” (e.g., transcriptomics, proteomics and metabolomics) have provided the opportunity to study the working mechanisms of biological systems alongside genomics. Some groups have suggested that integrative biology [8,9], the effort to integrate data from these various disciplines, may permit avoiding the biases and inherent flows of individual –omics techniques and, at the same time, may deliver a new approach to the study of human disease. This approach is summarized by the term “network medicine” highlighting that molecular networks altered in disease can be both the target for future therapeutic strategies and the possible source of novel biomarkers. A biochemical network, common to many different cell types or even species, exhibit a different “network utilization” in different physiological and pathological contexts. Mutations can therefore offset the utilization of molecular networks and their dynamics. On the one hand, better understanding of how networks encode functional states and cellular decisions under physiological conditions and how these are altered in disease will offer more and better targeted therapeutic opportunities. On the other hand, defining cancer-associated network utilizations and engineering tools (probes and instrumentation) to reveal them will provide fundamental insights to optimize patient stratification for improved theranostics and prognostics.

Heterogeneity, causality and phenotypes | Phenotypic heterogeneity, including genetic and epigenetic polymorphism, and polyphenism, is at the basis of both unicellular and complex lifeforms. These three levels of phenotypic heterogeneity are recapitulated in cancer and constitute often insurmountable obstacles to effective therapeutic intervention. Intra-tumour heterogeneity, either within the primary tumour, within a metastasis or between different metastatic foci is indeed the primary cause for the emergence of drug resistance and tumour relapse. The genetic basis for phenotypic heterogeneity within a tumour is rather established. However, other non-genetic factors can be regarded as equally important.

For instance, upon treatment, a fraction of tumour often exhibit drug resistance. In part, this can be caused by pre-existing tumour cell clones carrying mutations that, by chance, will confer resistance to any given drug. Alternatively, this may be caused by tumour initiating cancer cells, stem-like cells that are usually quiescent, less vulnerable to treatment and that can regenerate the tumour upon termination of the therapy. Moreover, non-Darwinian mechanisms for the emergence of drug resistance have been proposed as well, whereby cells trigger a transient drug-resistant phenotype that, in time, can be then converted to a stable inheritable state by subsequent somatic evolution.  Fractional killing may also be explained by non-genetic heterogeneity. For instance, Spencer et al. have shown that in a clonal population of cells, TRAIL elicits a heterogeneous phenotypic response with cells undergoing apoptosis at different times or surviving indefinitely. The authors elegantly demonstrate that this phenomenon is caused by stochastic variations in the abundances of the many proteins involved in the apoptotic molecular network.

Genomics, transcriptomics, proteomics and metabolomics allow the characterization of tens of thousands of biomolecules at the same time. Furthermore, the increasing sensitivity of these techniques provides – or may provide in the future –  single cell “–omics” characterization. However, the invasiveness of these techniques will limit their applications to the study of individual time points. Thus, causality can be established only by inference. Techniques capable to provide low invasiveness and biochemical information on living cells are thus extremely useful to complement models derived by ‑omics techniques and to provide a tool for testing hypothesis derived from analysis of big data.

It is thus evident that time-lapse imaging of individual living cells with biochemical information is strategic for the understanding of the heterogeneous response of biological systems and to establish causality between biochemical events and cellular decisions. At the same time, genetic heterogeneity within a tumour and between tumours induces differences in network utilizations with significant consequences for prognosis and treatment. Also in this context, biochemical imaging techniques are necessary to understand the phenotypic heterogeneity of a tumour and, at the same time, may be useful to define network-based biomarkers.

The next generation of Systems Biology | Several groups have identified the need to integrate fluorescence microscopy in the systems level study of the cell and organisms [10-15]. The term “Systems Microscopy” has been suggested for the description of microscopy tools applied to this field [14]. In order to strategically complement other approaches, Systems Microscopy has to deliver single cell resolution, temporal characterization of living cells and high quality quantitative data and has to be applied to the most appropriate biological context (e.g., for epithelial cancers, adherent 2D, 3D, organotypic cultures or in vivo rather than in suspension or cellular homogenates) [10]. Whereas –omic techniques can sample the biological space over the fullness of biochemical moieties (genes, RNAs, proteins, metabolites) albeit with poor sampling of individual cellular behaviours and spatio-temporal organization, Systems Microscopy samples the fullness of the spatio-temporal organization of molecular networks but reports about a limited number of gene products or biochemical events [10]. Therefore, Systems Microscopy elegantly complements big data studies.   

We envisage two (not mutually exclusive) approaches to Systems Microscopy: high throughput screening platforms and single cell biochemical multiplexing. High Content Screening (HCS, also known as imaging cytometry or high throughput imaging) is the current tool of choice for Systems Microscopy. Relying on robotics, automation and unsupervised or semi-supervised data analysis, HCS enables the screening of large numbers of cellular perturbations (e.g., siRNA or compound libraries) with commercial instrumentation making the correlation of these perturbations with morphological estimators and fluorescent markers possible. Several groups have also highlighted the importance of integrating quantitative biophysical imaging techniques such as Fluorescence Correlation Spectroscopy (FCS) and Foerster Resonance Energy Transfer (FRET) in Systems Microscopy in order to deliver data of high quality. Despite this, HCS has been integrated with these techniques only in a few academic-based efforts [16-19]. HCS expands the sampling of biological space of imaging technologies to deliver another set of “big data” but with single cell resolution.

We are pursuing a different approach to Systems Microscopy that maps in space and time an increasing number of fluorescent markers within the living cell. Fluorescence is not amenable to the simultaneous detection of many fluorescent molecules because of the broad excitation and emission spectra of common fluorophores. Therefore, we are determined to develop new techniques (bioprobes and instruments) that exploit all properties of light (photon arrival times, colour and polarization) efficiently to maximize the biochemical resolving power of microscopy. We aim to monitor nodes of molecular networks (e.g., quantifying the dynamic phosphorylation of several substrates) in living cells in response to stimuli, discerning between physiological and pathological (oncogene-driven) network behaviour (topology). The integration of Optogenetics tools (e.g., light-inducible oncogenic signalling) enables perturbational analysis of biochemical networks and facilitates the execution of complex biochemical imaging assays fully automated with no requirement for sample manipulation other than by light. Therefore, these techniques will be strategic for the study of biological networks at low throughput with high quality data; thanks to this all-optical approach, they may also be integrated with HCS increasing the quality and quantity of information and decreasing steps in chemical manipulations of the samples (e.g., addition of doxycycline to stimulate the expression of a gene)

[1] Vogelstein et al. (2013) “Cancer Genome Landscapes” Science
[2] Wood et al. (2007) “The genomic landscapes of human breast and colorectal cancers” Science
[3] Solimini et al. (2012) “Recurrent hemizygous deletions in cancers may optimize proliferative potential.” Science
[4] Davoli et al. (2013) “Cumulative Haploinsufficiency and Triplosensitivity Drive Aneuploidy Patterns to Shape the Cancer Genome” Cell
[5] Berger et al. (2011) “A continuum model for tumour suppression.” Nature
[6] Jorgensen&Linding (2010) “Simplistic pathways or complex networks?” Current Opinions in Genetics and Development
[7] Hanahan&Weinberg (2010) “Hallmarks of Cancer: The Next Generation” Cell
[8] Erler&Linding (2010) “Network-based drug and biomarkers” J Pathology
[9] Barbasi et al (2011) “Network Medicine: A Network-based Approach to Human Disease” Nat Rev Genet
[10] Megason et al. (2007) “Imaging in Systems Biology” Cell
[11] Verveer&Bastiaens (2008) “Quantitative microscopy and systems biology” Histochem Cell Biol
[12] Ankers et al. (2008) “Spatio-temporal protein dynamics in single living cells” Current Opinion in Biotechnology
[13] Pepperkok&Ellenberg (2006) “High-throughput fluorescence microscopy for systems biology” NAt Rev Mol Cell Biol
[14] Lock&Stromblad (2010) “Systems microscopy: An emerging strategy for the life sciences” Exp Cell Res
[15] Conrad&Gerlich (2009) “Automated microscopy for high-content
RNAi screening” J Cell Biol
[16] Esposito et al. (2007) “Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening” Mol Cell Proteomics
[17] Mathews et al. (2008) “A high-content screening platform utilizing polarization anisotropy and FLIM microscopy” SPIE BIOS
[18] Talbot et al. (2008) “High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis” J Biophotonics
[19] Barber et al. (2013) “The Gray Institute ‘open’ high‐content, fluorescence lifetime microscopes” J Microsc

Be kind to people

“I have worked hard for three years and now that I believe I understand the mechanism, the funding is over”. “I am at the third referee round in five different submissions and I am always getting different requests”. “My grant was not funded because of insufficient preliminary results”. “I do not understand why they got a promotion and I am struggling to keep my job with a similar track record”. “I worked days and nights and the panel dismissed me with meaningless questions”. “My friend never recovered from a mental breakdown”. “I have written the proposal for a month and it was rejected with one sentence, on subjective grounds”. “The referees were very positive but the panel was unimpressed”. “I did not get funding but those in the panels did”. “I got bullied but a committee found that nothing was wrong”.

“Yes, I understand you. It is unfair but this is how academia works”

No… it is not me moaning but a collection of whispers, complaints and shouts you can hear in the corridors of Academia. Along with comforting words, the response to a colleague in a temporary moment of discomfort or a prolonged stage of distress are often two. One might be an explanation of what a colleague might have done objectively wrong or how to avoid typical traps in the various stages of academic assessment. The other is just the acknowledgement that at least in many, if not all, cases… well… this is how Academia works and we have to be resilient and keep going*. However, this post is not about complaining but more about the human factor often lost in Academia.

In the last few days, twice I heard or read appeals of ‘being king’ to people in the academic context. Once, in a speech by our Director, Prof. Ashok Venkitaraman, opening our retreat on Friday. His speech did mention academic excellence but it was particularly focused on people as described by our colleague Dr Ben Hall.

His words resonated with most of us as kindness is far too often forgotten in Academia, probably because in very competitive environments, people are supposed to be all so full of themselves and thick-skinned that everything goes. In truth, like in any work environment, the large majority of people treat each other with respect and just a few then spoil it for everyone else.

Just a day later on Saturday, in a private conversation completely unrelated, a friend pointed out that the Teichmann laboratory at the Wellcome Sanger Institute, adopted as a lab motto the words “Be bold. Be brilliant. Be kind.

These two almost trivial observations (from our Director and another successful Academic) made me think. Why do we need to make such appeals for kindness? After two decades of living a life within Universities, my experience of the Academic environment is of a very tolerant, liberal and progressive environment. Of course, there are plenty of issues to be fixed, common to other sections of society, but the general attitude and ethos – in my experience** – was mostly positive. Then why do we eventually feel the need to appeal to kindness?

My opinion is that the obsession for ‘independent’ academic assessment and competition is in part selecting for certain characters. Being ruthless and selfish helps in any competitive environment, as it increases the likelihood to seize resources. However, I do not think this is just the issue. Most academic assessment is either performed anonymously or by panels that often have no knowledge of the person they have to judge. Various forms of peer-review (either for publishing or funding) are designed to be objective and independent. While peer-review is the best system I can also think of, its issue is that – eventually – it is not objective and it is not independent but in trying to be, it loses any human touch. Even when interviews are at the core of assessment, these are brief (5-20mins) and very focused, in any case preceded by anonymous reviews. The lack of human connection and two-way personal dialogue, I think, dehumanize the process of assessment and triggers ‘unkind’ behaviours. The problem, perhaps, we focus too much on projects and not enough on people.

I might be still naive, but in my opinion, the most important resource in any work environment, and also in Academia, is people. Recently, we prepared a leaflet for outreach with the motto “Our superpower is you”, meaning that science main resource is one: people. Unfortunately, the structure of academic assessment and a highly tapered career pyramid with huge turn-overs at its base, create rent-seeking behaviours and an environment that can be harsh in general, or at least in key moments of one’s career. We should think about people investing in people for the benefit of people, not just in projects.

I know that this is perhaps a tiny bit too idealistic and any type of assessment has flows. Probably, we cannot really solve this problem, maybe it is not a problem in itself. But I would like to leave you, my friend, with a provocation. I dare you not just being kind (if you read until here you might agree with the general concept) but challenge everyone that is not, be kind when you review a paper or a grant, particularly when you have strong criticisms to share. If you are an Editor, the head of a panel, academic or not, I dare you challenging unkind behaviour and disqualifying any critique that is not delivered with respect. I dare you all speaking publicly about the need to be excellent in science, but also in our humanity. Because if we wait longer for a top-down change, even though many at the top are wonderful people agreeing with the ‘be kind’ concept, we will keep losing our human capital. I dare you last, to use this or any other badge of your choice in your website or public communication. The large majority of people is good people, in any environment, we just need to remind everyone that it is not acceptable to be otherwise:

* to avoid misunderstandings, I should clarify that I might also respond in this way, it is not a criticism on trying to be helpful explaining how the system might work.
** VERY IMPORTANT TO ME, this is my own experience. I am fully aware of other very different experiences, and structural problems. Here I am speaking about a general attitude and – as I am committed in Equality Diversity and Inclusiveness in Academia, I am fully aware that there are plenty of problems to be solved. I do not want that this specific statement about Academia being generally a liberal and progressive environment (which is what I think) will be misunderstood as if Academia is perfect, indeed my post would suggest otherwise.

My remembrance day, a pledge for peace in support of soldiers

As an immigrant in the UK, it took some time to understand the deeper meaning of the remembrance day. In fact, remembrance day is lived by different British people in different ways, and to truly embrace this event, one has to stare a red poppy and feel what it means for them. You should have an intimate meaning for the red poppy to relate to the remembrance day. If you do, the wearing of the red poppy becomes not only a charitable gesture but a deeply meaningful action. As an immigrant from a country where the red poppy is not a tradition, therefore, it is only after one decade in the UK that I can finally embrace this day full-heartedly.


Remembrance day is approaching. I hope most people will reflect on what this day actually represents. It is the day where Commonwealth nations remember the soldiers fallen during the first world war and by extension, it is the day many intend to pay tribute to those who died in wars. In these weeks, many people will use war rhetoric and will revive patriotic emotions. Many people will proudly wear red poppies, to support veterans, as a statement of national pride, to remember the fallen soldiers, or for social pressure. Like every year, the news will invite comments, there will be vast support, but also critical opinions, and critical rebuttals of those critiques. Eventually, remembrance day ends up to be all those things. This year, however, I will have my first true remembrance day when I will not care about what this means for others, but I will care only about what it means for me. The reasons are two. One is that after many years I relate to British traditions as my own. The second is that my daughter, a British citizen who self-define as English, is in year 1 at school and I have to dialogue with her about the red poppy.


The red poppy is not a symbol for blood nor a symbol for death and, in the UK, it is adopted by the Royal British Legion as a charitable act, and to manifest support for Armed forces, veterans and their families. The red poppy should be worn as a personal choice and there is no ‘correct’ way on how to wear it.

However, the colour of this flower and the origin of this symbol  – the devastated fields where soldiers died during the first war and were then covered by red poppies – are so evocative that many people cannot refrain to associate the colour of the scarlet red poppy to the blood of soldiers who died in the war.


So, what is for me the red poppy? It is the blood of the soldiers shed during wars, but it is also the blood of the civilians crashed between opposing fronts. The bloody tears of those who survived, the broken families, the broken hearts, the children, the mothers and fathers, the elderly who died in battle or were visited by death at home. To me, the red poppy and remembrance day are reminders that we should always do anything possible to avoid conflict and war.

As war rhetoric came back fashionable also in democratic countries, when authoritarian movements are gaining the consensus of the public, and when too many people are proud to divide nations rather than to unite, we should not escape from the deeper meaning of this day.


To me, the red poppy is the blood that should never be spilt again but that will, and does. 

And therefore, I will embrace this remembrance day as my own. With gratitude for brave soldiers that defended our freedoms but with shame because we have asked and we will ask them again to kill and to die instead of just being vigil, watching with pride our democracies working peacefully together.

It is yellow, the two proteins must interact!

In fluorescence microscopy, colocalization is the spatial correlation between two different fluorescent labels. Often, we tag two proteins in a cell with distinct fluorescent labels,  and we look if and where the staining localizes. When there is a “significant overlap” between the two signals we say that the two molecules “colocalize” and we might use this observation as possible evidence for a “functional association”. We might argue that measuring colocalization in microscopy is one of the simplest quantitation we can do. Yet, many horror stories surround colocalization measurements.  This post is not a review of how to do colocalization, but a brief casual discussion about a few common controversies that is – as often I do – aimed to junior scientists.

This is a slide I often use in a presentation to introduce FRET but useful to understand colocalization. You can see the average size of a globular protein, fused to a fluorescent protein compared to the typical resolution of diffraction-limited and super-resolving fluorescence microscopy. When the signals from two molecules are within the same pixel, these two molecules can be really far apart from each other. However, the spatial correlation of distinct labelling can inform us about possible functional associations.


I am imaging GFP, but the image is blue, can you help me?”. Well, this is not a question related to colocalization but it illustrates a fundamental issue. In truth, cell biology is such an inherent multidisciplinary science that – in most cases – a researcher might require the use of tens of different techniques on a weekly basis. It is thus not surprising that many researchers (I dare say most) will be an expert on some of the techniques they use but not all. Microscopy is particularly tricky. To be a true expert, you need to handle a feast of physical, engineering and mathematical knowledge alongside experimental techniques that might span chemistry, cell culture and genetic engineering. However, the wonderful commercial systems we have available permit us to get a pretty picture of a cell with just a click of a button. Here the tricky bit, you want to study a cell, you get a picture of a cell. One is lead to confusing the quantity that intends to measure with the information that is actually gathering and with its representation. This is true for any analytical technique but as ‘seeing is believing’, imaging might misrepresent scientific truth in very convincing ways. Hence, with no doubts that upon reflection the non-expert user would have understood why the picture on the screen was ‘blue’, the initial temptation was to believe the picture.

Question what you set out to measure, what the assay you have setup is actually measuring and what the representation is showing. Trivial? Not really. It is an exercise we explicitly do in my lab when we have difficulties to interpret data.


It is yellow, they colocalize, right?”. Weeeeeeeeellll… may be, may be not. Most of you will be familiar with this case. Often researchers acquire two images of the same sample, the pictures of two fluorescent labels, one then is represented in green and the other in red. With an overlay of the red and green channels, pixels that are bright in both colours will appear yellow. I would not say that this approach is inherently flawed but we can certainly state that it is misused most of the times and, therefore, I try to discourage its use. One issue is that colour-blindness, not as rare as people think, renders this representation impractical for many colleagues (so my colour highlights!), but even people with perfect vision will see colours with lower contrast than grey-scale representations, and green more than red. Eventually, to ‘see yellow’ is almost unavoidable to boost the brightness of the underlying two colours to make the colocalization signal visible. This can be done either during the acquisition of the image often saturating the signal (bad, saturated pixels carry very little and often misleading information) or during post-processing (not necessarily bad, if declared and properly done). Either way, at the point you are doing this, your goal to be quantitative has been probably missed. The truth is that a lot of biological work is non-quantitative but faux-quantitative representations or statistics are demanded by the broader community even when unnecessary. Let’s consider one example with one of the stains being tubulin and the other a protein of interest (PoI). Let’s assume the PoI is localizing at nicely distinguishable microtubules in a few independent experiments. Once the specificity of the stain is confirmed, the PoI can be considered localized at the microtubules (within the limitations of the assay performed) without the need for statistics or overlays. Unfortunately, it is not very rare to see papers, also after peer-review, to show diffuse stainings of at least one of the PoI and perhaps a more localised stain of the second PoI and a ‘yellow’ signal emerging from an overlay is considered colocalization, instead of what it is: just noise. Another common issue is localization in vesicles. Again, any cytoplasmic PoI would appear to colocalize with most organelles and structures within the cytoplasm with diffraction-limited techniques. Sometimes punctuated stainings might partially overlap with known properly marked vesicles, let’s say lysosomes, but not all. Then the issue is to prove that, at least, the overlap is not random and, therefore, statistics in the form of correlation coefficients are necessary.


The two proteins do not colocalise, two molecules cannot occupy the same volume” Really!? Well, from a quantum mechanics standpoint…. No, do not worry, I am not going there. I have received that criticism during peer-review in the past and until recently I thought this was a one-off case. However, I have recently realised that I was not the only person reading that statement. I am really uncertain why a colleague would feel the need to make such an obvious statement except for that condescending one-third of the community. I should clarify that to my knowledge no one implies physical impossibilities with the term colocalization. That statement is perfectly ok in a casual discussion or to make a point to teach beginners the basics. Some of us also might enjoy discussing definitions,  philosophical aspects related to science, controversial (real or perceived) aspects of techniques, but better at a conference or in front of a beer, rather than during peer-review.  The issue here is that while it is reasonable to criticise certain sloppy and not too uncommon colocalization studies, in general colocalization can be informative when properly done. 


So, is measuring colocalization useful?” Homework. Replace ‘colocalization’ with your preferred technique. Done? Now try to make the same positive effort for colocalization. Every technique is useful when used properly.

You might have noticed I marked some words in my introduction: colocalize, significant overlap and functional association. It is important we understand what we mean with those words. Colocalization means co-occurrence at the same structure, a non-trivial correlation between the localization of two molecules of interest, within the limits defined by the resolution of the instrumentation. The “significant overlap” should be really replaced by “non-trivial correlation”. Non-trivial, as diffuse stainings, unspecific stainings, saturated images can very easily result in meaningless colocalization of the signals but not of the molecules of interest. Correlation, as the concept of overlap might be improper in certain assays, for instance in some studies based on super-resolution microscopy. After we did everything properly, we still cannot say that if protein A and protein B colocalize they interact (see slide). However, we can use colocalization to disprove the direct interaction of two proteins (if they are not in the same place, they do not interact) and we can use high-quality colocalization data to suggest a possible functional association that might be not a direct interaction, and that should be then proven with additional functional assays.

Then, my friends, do make good use of colocalization as one of the many tools you have in your laboratory toolbox but beware that just because it is simple to acquire two colourful pretty pictures, there are many common errors that people do when acquire, analyse and interpret colocalization data.


P.S.: if I cited your question or statement, please do not take it personally. As I have written, not everyone can be an expert of everything and the discussion between experts and non-experts is very useful, so making real-life anonymous examples.