my blog

SOP – Ti:Sapphire / Leica SP5 alignment

This SOP is published only for a social media discussion. The author does not take any responsibility for the utilization of this procedure. The system discussed here is a customized two-photon microscope, based on a Coherent Chameleon Vision 2 and Leica SP5. The optical path is fully enclosed and the SOP is written for maintenance. 

Basic rules

1.       Align laser with a lid room (smaller iris, smaller damage to the eye)

2.       Never align eyes with the height/direction of the laser beam

3.       Use the most appropriate personal protective equipment such as goggles and a white lab coat

4.       Perform laser alignments with the least number of people present in the room. Ideally, alignment is a 1 person job and a colleague is aware you are performing this task, within core hours

5.       Use devices such cards, cameras and viewers to visualize the laser beam

6.       Take short breaks every ~45 minutes of work. Do not continue alignment if too tired. Alignment of optics can be a stressful and lengthy procedure; try to identify the right moment to take a long break to relax

7.       Alignment is carried out only by authorized users

PPE for Ti:Sapphire laser

          VC5 IR card viewer from Thorlabs. WARNING: card viewers reflect part of the laser beam. Therefore, they must be used with caution, strictly using protective goggles, directing reflection away from the eyes

          Hand-held IR viewer from Newport. WARNING: hand-held IR viewers limit dexterity and must be used always with protective goggles.

          LG9 Amber lenses from Thorlabs. OD5+ on the 720-1090nm range; OD7+ on the 750-1064nm range. WARNING: goggles never fully protect from direct high power laser beam.

List of authorized users

Alessandro Esposito (MRC Cancer Cell Unit)

Coherent’s field engineer can align the laser under their own responsibility. Coherent’s field engineer can align the beam path until after the Pulse-Picker. The rest of the optical path must enclosed at any time or isolated with a beam stop.

Leica’s field engineer can align the complete beam path under their own responsibility with the exception of the Pulse-Picker. Alterations of the beam path have been discussed with Leica representatives.

Standard Operating Procedure

Room preparation

1.       Show warning at the door

2.       Lock the door

3.       Switch on the system as needed (shutters ON)

4.       Wear PPE as appropriate

5.       Open the beam path as needed (keep lens tube arriving to the scan-head until the last moment)

6.       If a large section of the beam path is opened, always block the laser beam with the beam stop after the optical element that is aligned in order to avoid the laser beam being reflected in dangerous directions (eye, skin, fire hazard) when misaligned

Beam alignment

7.       Always activate laser shutter when the beam is not undergoing alignment

8.       Apply #6 every time a section of the laser beam is aligned

9.       Start laser alignment, proceed with pairs of mirrors from the position closer to the laser up to the scan-head, trying to operate the laser beam within a central part of the mirrors

10.   Always ascertain that all optomechanics is stably connected to the optical table and that no optical device can fall, tilt, flip…

11.   Re-aligned section should be covered (at least temporarily) while progressing towards the scan-head

12.   When arrived at the EOM, remove the device (Leica’s shutter and half-plate may be removed as well). WARNING: the entrance window of the EOM is located within a brass cavity. Upwards reflections of the laser are possible.

13.   Using irises, make sure the laser beam is parallel to the table

14.   Reposition the EOM, coarsely aligned to the laser beam. WARNING: after the EOM there is a periscope. Use a beam-stop before the periscope, beam reflection towards undesired direction is otherwise possible.

15.   With a power meter, measure power of the laser before the EOM. Relocate the power meter after the EOM and iteratively maximize power through the power meter with the EOM in “high” state.

16.   Coarsely align the periscope if necessary, then reintroduce Leica’s shutter and half-plate if previously removed. BE SURE the periscope is locked to the optical table in a stable manner.

17.   Remove lens tube and MFP cover.

18.   Install Leica’s alignment tool on the scan-head

19.   Iteratively align the front iris of the alignment tool and the back aperture of the alignment tool.

20.   WARNING. During the iterative alignment of scan-head, PPE is usually hindering an already lengthy procedure. Avoid removing PPE. Check actions to be taken.

21.   When the two apertures are aligned, start scanning trying to see fluorescence from a bright sample on the screen. Keep adjusting alignment and MFP screws until alignment is completed.

Preparing the room to normal operation

22.   Close the optical path. Before securing all covers and panels, check that the alignment is still ok.

23.   Secure all safety panels

24.   With the laser ON, shutter OFF and during scanning, verify with the IR viewer that no beam is exciting the enclosed laser path.

25.   Remove safety warning on the door and operate equipment as normal.

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NyxBits and NyxSense? What?!

NyxSense&NyxBits paper here.

800px-Arte_romana,_statuetta_di_nyx_o_selene,_I_secolo_acI am not fond of new achronyms or ‘cool’ names, but then… guilty! you got me, I am contributing to the proliferation of four letters acronyms and fancy names like others! Lately, I have introduced a new one, HDIM as for Hyper-Dimensional Imaging Microscopy. But that is another story, and in a Supporting Note of that pre-print we explain our choice.

Earlier, we created the pHlameleons with the friend, my group leader back then, Fred Wouters. Well, first it was the Cameleon, the famous calcium reporter by the great Miyawaki and Tsien, brilliantly referred to as Camaleon because it is a protein that ‘changes colour’ upon binding calcium (Ca). Then it was the Clomeleon by Kuner and Augustine, as it senses cloride ions (Cl) rather then calcium. With all due respect for the authors, I must admit I did not love that name at first. Indeed, as we were deriving a family of pH sensors from yet another creation of Miyawaky (the CY11.5), we started to joke that we should have called this family of sensors the pHlameleons. Months after months, a joke ended up in a title of a paper, to be adopted as the name of these pH sensitive proteins. So, let’s not take ourselves too seriously too often. Sometimes we pick names for a bit of branding, other times to make our assays less heavy with too many technical terms, and other times, let’s just have fun with words (Clomeleon now for me is a great name, but I routinely joke about pHlameleons!).

Now that you know the little funny story about the pHlameleons, it is the turn of NyxSense and NyxBits. NyxSense is a software dedicated to multiplexing of FRET sensors. NyxBits are the components to create a multiplexing platform, a number of fluorescent proteins of distinct Stokes shift that can report, through their fluorescence lifetime, biochemical reactions probed via FRET with the use of dark/darker acceptor chromoproteins. A huge effort for us that took several years to bear fruit. Why Nyx?

During the revision of the drafts, colleagues found the manuscript a bit too technical and difficult to read. Thus I went back to pen and paper,  google and wikipedia, to find a name that could help us to refer to this sensing platform with a single word rather then a sentence. Greek mythology always provides great inspiration and eventually, I discovered Nyx the primordial goddess of the night (Nox in the Roman mythology). With Erebus (personification of darkness), Nyx gives birth to Aether (personification of the upper air and brightness), Moros (deadly fate), Moirai (destiny) and Thanatos (death). Then, I felt that this short name, Nyx, is intimately connected with our work for three reasons.

First, Nyx seems to link darkness and light, the day and night, a nice analogy with our bright donor fluorophores and dark acceptors. Second, Nyx is related to death and fate. We created the NyxBits and NyxSense to study cell fate, and our first application is cell death responses to an anti-cancer drug. Third, Nyx is a goddess and as I am really committed to gender equality at work (not just by picking names of fluorophores), it felt a little bit in tune with what I do, to honour a female deity.

But do not take these reflections too seriously – I do not – after all I needed just a simple name for a very complex sensing platform. As there is no way for me to tell the reasoning behind the names in the manuscripts, I thought to share with you why we picked NyxSense and NyxBit, light-heartedly.

 Now starting project Atlas… we’ll speak about this another time! 🙂

Embrace your public speaking anxiety

About a decade ago, I went to a PI during a retreat to ask a question. Nervously, but politely, he asked me to be left alone as he was rather anxious for a talk he was about to deliver.  A few hours later, a PhD student at the time, I was freaking out for my own talk, but it was comforting, in a way, to see that an established scientist I highly regarded and I had considered rather self-confident was in a similar state-of-mind.

Comforting? Why not scary? Would you never get rid of public speaking anxiety? I am no anxiety coach and, for that, browse around. However, I wished to share my own experience as it might be useful for students. I now noticed I am that ‘senior’ scientist at that retreat (or something similar) and that junior colleagues might misunderstand my confident speaking in public as evidence of no-stress, no-shyness, a gift from birth. So, even though your solution might be a different one, here I tell you which was mine.

Be prepared! Be prepared? (take 1)

Trivial, isn’t it? I am not going to give practical suggestions here, except set yourself comfortable deadlines. With experience, you will be able to work on a talk until a few minutes before delivery, but earlier in carrier, you have to prepare all your material far in advance. However, even very experienced academics and businessmen when facing more unique scenarios work hard to prep a meeting and give this enough time and resources.

Be prepared! Be prepared? (take 2)

Perhaps, the most difficult thing you might find, it is to commit to a deadline, after which you have to be ready. But, here the challenging bit, even if you feel still unprepared (and some people may never be able to shred off that feeling) or if you are actually unprepared because you miscalculated something, you have anyway to commit to the next difficult bit, be mentally and physically prepared, something you might be completing neglecting. Deadlines are deadlines and the starting time of your talk is unmovable. Therefore, start to mature a process and to understand how long you need to be ready before a talk. Some people is a natural and need no or little preparation. Other people need time: never underestimate how long time you need. Most of my following comments are about this stage of preparation. The bottom line, when the deadline strikes, be sure you are ready and if you are not, do not allow doubts to undermine the next phase of preparation.

Commit physically: water and energy

During a stressful moment, your physiology will be heavily altered and you might lose control. So, think how not to. Personally, before a talk I try to drink lots of water to ensure I will be properly hydrated, and I also make sure I have water available during the talk. Once I didn’t, and I was not well. I coughed though all my talk and it was not a very ideal situation. Also, be sure you have energy, so a bar of chocolate or a juice, can help. Ah… ok, is this obvious?… pay attention – water in > water out. As basic as it seems, be sure you went to the toilet at the latest opportunity before the event. You do not want to be dehydrated, but even not to be distracted by your bladder while on stage.

Mind you that this is even more true when you have very long days, such as more articulated interviews or conference commitments.

Commit physically: oxygen

Breathing, for me, is the next most important issue. You might find yourself in need of oxygen after a few slides and attempting to do the world record in apnoea while speaking in public. You could pass through an entire 20 minutes presentation incapable to breath properly, increasing your level of anxiety at each slide. You are in front of an audience, it could be two people at an interview, or a thousand people in a theatre, if not a million in TV. However, giving a good breath permitting your lungs to be completely emptied and filled with fresh air takes a few seconds. This can be easily concealed in a transition between two slides, or during a question. And… if you cannot conceal it… do it anyway, 5 seconds spent silently breathing properly will be immediately forgotten by your audience, but a poorly delivered 20 minutes talk will be remembered.

Once again, get ready for it. First of all reflect on your breathing habits, far away from a talk. If you give enough thoughts about the issue, whenever you will struggle, a mental trigger will snap and make you aware of the occurring issue for you to take action. More importantly, if issues in breathing are recurrent for you, just do exercises in the 5 minutes preceding your talk. Breath in deeply and breath out slowly. This will decrease your anxiety and will prepare your breathing for the talk. You can do it while seating in the audience or even while speaking with others.

Commit physically: avoid distractions

Personally, I have a routine. Before a talk, I remove everything from my pockets, or even the badge, anything superfluous. After a few talks delivered with my pockets inside-out dangling from my trousers, I also double-check that I am generally presentable! So, on stage or seating in front of a panel, I have no distractions from the badge hitting the microphone, the phone vibrating, the keys stuck in my thigh. Well, the phone: switch it off well in advance of your talk and dump everything in you bag.

Commit mentally: have fun

Those were a few suggestions, and more or different tricks will work for you, to ensure your physical state will be ready to support the potential stress you might experience while speaking in public. Of course, your state of mind will play an equally important role. Perhaps, I should advise to not care, to convince yourself that the event you are preparing does not matter. This is probably key, more in general, to achieve the resilience necessary in the academic world. For me that does not work very well, as I tend to be heavily invested in everything I do. So, what it works for me is to repeat myself I need to have fun speaking about science, my work, or the work of others – otherwise is really not worth. A bit of self-couching targeted to focus your mood towards excitement, how great can be to speak or debate science.

I did receive my dose of criticisms in my career, but let me tell you which is one of the best compliment I ever got. Do you remember the talk I was freaking out during my PhD? Well, after my talk, which might not have been even an excellent one, I overheard the head of a department advising two junior PIs to speak with the energy and enthusiasm I was speaking with. I guess you should remind yourself of how exciting the work you do is and if you disagree with this, change job or lie to yourself for a couple of hours.

Commit mentally: focus

You would not run the athletics world final 100m, physically unprepared and with no excitement. You would also not run it thinking about random stuff or worrying not to win it. Watch athletes on their blocks, the intensity of their eyes, the deep focus they concentrate on the start gun and those few seconds after. Focusing might take a fraction of a second if you were a natural or simply experienced. Also, keep your focus during the talk, try to nurture that unconscious little voice that can warn you everytime you are going off-track.

The top right-hand corner syndrome (TRiHCS) is a risky issue in our business. TRiHCS happen when your mind wonders off, but you keep speaking. TRiHCS happen when you zone out and speak for 2 minutes about an irrelevant detail being fixated on a corner of a room, while you are not engaging with the audience and perhaps even with the main topic of the talk. If you get TRiHCSed, your timing and narrative will derail. But, do not worry, if you notice it in time, you can easily recover.

OK, ok… TRiHCS? I just made this up, but I promise you, it is something that does happen!

Look after yourself…

Pay attention to yourself. It is easy to get anxiety compromise your health in the long term, or your performance in the short term. In an ideal world, you can sleep, eat, drink, meditate as a Yogi. In the real world, assaulted by too many things to do, it is likely you will experience periods of stress and long hours. However, you will have to know your limits and try to stay far from the edge and arrive to an event in good physical and mental conditions. Your institution and funders will offer you a provision of well-being courses, advice and activities. However, your institution and funders will implicitly ask you to neglect completely their own advice and deliver huge returns for them at any cost (for you). Like for any job, the day will come that you cannot run any longer over the edge. Then, manage anxiety, either it is just for public speaking, or for anything else… embrace it, as in ‘do not ignore it’, ‘do not fight it’ as it fights back, but manage it and if you can’t, ask for help.

Look after yourself… plan your cool-off stage

I did some crazy things aiming to present data still warm from the microscope (yes, it is a thing if you use high power lasers), consciously cutting sleeping times down (within reason) and working over the edge. Even if you do not, but public speaking really takes a toll on you, look after yourself after the main event. You need to consider two phases. One, which might be short or very long, depending on the event, is the immediate aftermath. I used to be a runner, and I used to give everything until the end of the race, which made it very likely for me to fall on the ground exhausted after the line… but you learn to immediately stand-up, walk, then do a run at slow space and hydrate.

Somehow, after a peak of stress you need to do something similar, often quietly and in public. This may have to happen in a few seconds before taking further questions. So, regain mental and physical composure, re-gather your focus and energy, again consider drinking water or a juice.  You will need this, particularly, in a day-long event full of meetings. It can really take just one minute, but if you do not do it, you might crash and underperform in the aftermath of a public speaking event. Do not underestimate the task you will have to follow after the main event and the energy you will need for them.

Then, at last, all is over. Really look after yourself because if the event you prepared took really a lot of energy from you, there might be consequences. You will discover what is best for you, if to completely relax and instruct yourself, or to simply take it easy for a few hours or a few days.

Conclusions

Keep in mind that what I have written here it is not an expert-opinion, but a personal experience. My suggestion to embrace your public speaking anxiety comes from trying to advise junior colleagues and realizing I did not wish to give the same suggestion a GP once gave to me: ‘you should avoid stress’. This is the wrong suggestion, in my opinion, as most of us, certainly in the ultra-competitive academic world, will have to manage plenty of stressful situation. Thus, the keyword is ‘manage’ not ‘avoid’, be the master or mistress of your stress-responses and, yes, avoid only those things that might push you too far beyond what you can manage. So, embrace your public anxiety speaking, mould your response to it in time and you will eventually grow out of it, or if not, at least you will manage.

Of course, whatever I described here is not something I usually think about, even during big talks. I made an effort to catalogue the various ‘tricks’ I – sometimes unconsciously -matured in 15 years of presenting scientific work in public. But recently, I had noticed that – either as a natural predisposition or by training – delivering a talk is more than just speaking in public. It is a process that requires physical and psychological strengths, like an actor preparing for a play or an athlete for a race. Scientists, noticing it or not, need to nurture these strengths, even not for their audience, but at least for looking after their health.

[TALK] Goldilocks and the two ERKs; signalling in the ‘sweet spot’ underpins resistance to ERK pathway inhibitors

Friday 14/09 at 14.30 | Dr. Simon Cook (Signalling Laboratory, The Babraham Institute) will present the following talk, at the Clifford Allbutt Lecture Theatre, Clifford Allbutt Building (former LMB building). All welcome to attend.

 Goldilocks and the two ERKs; signalling in the ‘sweet spot’ underpins resistance to ERK pathway inhibitors

Simon Cook, Signalling Laboratory, The Babraham Institute

Tumour cells with BRAF or RAS mutations are ‘addicted’ to ERK1/2 signalling for proliferation and RAFi and/or MEKi are now approved for use in the clinic.  However, despite some striking clinical responses, resistance emerges within 9-12 months resulting in disease progression. Acquired resistance to MEKi often occurs through amplification of BRAFV600E or KRASG13D which act to reinstate ERK1/2 signalling.

Here we show that BRAFV600E amplification and MEKi resistance are fully reversible following drug withdrawal.  Resistant cells with BRAFV600E amplification become addicted to MEKi to clamp ERK1/2 signalling at a level optimal for cell survival and proliferation (2-3% of total ERK1/2 active, quantified by mass spectrometry).  This is seen in cell culture and in vivo where growth of resistant cells with BRAFV600E amplification as tumour xenografts is inhibited in mice that do not receive MEKi.  ERK1/2 hyperactivation (~20% active) following MEKi withdrawal drives expression of the cyclin-dependent kinase inhibitor (CDKI) p57KIP2, which promotes G1 cell cycle arrest and senescence, or expression of NOXA and cell death; these ‘terminal’ responses select against those cells with amplified BRAFV600E.  ERK1/2-dependent p57KIP2 expression is required for loss of BRAFV600E amplification and determines the rate of reversal of MEKi resistance.  Thus, BRAFV600E amplification confers a fitness deficit during drug withdrawal, providing a rationale for intermittent dosing (‘drug holidays’) to forestall resistance.

Remarkably, MEKi resistance driven by KRASG13D amplification is not reversible. ERK1/2 reactivation in the context of amplified KRASG13D does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to chemotherapy agents, arguing strongly against the use of ‘drug holidays’ in cases of resistance to MEKi driven by KRASG13D amplification.

 

 

1984 – A BREXIT tale

British people are lucky, as they have a wealth of literature to read from, a rich history to learn from and a great number of world-leading experts to listen to. They also have a lot of selfish, ideologically-biased and intellectually dishonest politicians to listen to. Not that all politicians are dishonest, but many – if not all – have the need to survive elections and when facts do not support their bid for a seat in the parliament, many politicians are simply keen to alter the facts. And in this era of non-accountability, this strategy works perfectly. So, why do many people decide to ignore facts and live in a fantasy world narrated by politicians? Spoiler alert: no answer to this question.

In the dystopian world beautifully described by Orwell in his novel Nineteen Eighty-Four,  the government alters recorded facts to make them fit with their policies and propaganda. For example, when the price of chocolate increases, the historical record of the price of chocolate is altered with higher prices; then people will have to take note that the price of chocolate has actually decreased in recent times. More importantly, in a society that respond with violence to those (‘enemies of the people’ and ‘saboteurs’) that do not conform with the ruler-engineered representation of reality, people will accept this contradiction between their own memory and what reality appears to be. But, importantly, they will eat less chocolate as the hard facts are… hard facts, even when they are ‘white, red and blue’.

Next year, the NHS will get an increased budget of £4 billions. Which is a great and greatly overdue news of course. The Tory MP and minister Andrea Leadsom commented “As we leave the European Union and stop paying significant annual subscriptions to Brussels, we will have more to spend on priorities such as the NHS”. Well, at least they spared us a new red bus at this round. Let’s be clear, NHS spending has nothing to do with the EU, it was a government decision to not increase budgets when needed, and it is a government decision to increase funding now. There is no money that was saved so far from the EU contributions and there will be no money for a while longer (facts). Even when funds will be released from EU commitments, people should be reminded that this will be just £8 billions a year, the net contribution of the UK to the EU budget, or ~1%  for the UK government budget. This saving will be partially reabsorbed by investments in administrating functions currently devolved to the EU, including a custom infrastructure. Who can do sums know that the ‘EU dividend’ will be real but down to very little money. Moreover, notwithstanding the fact that the UK economy might flourish outside the EU, even with bright and smart politicians steering us in the right direction, all predictions are for short-term economical pain in the near future.

While the political debate will now focus on an imaginary ‘EU dividend’, I wished to remind myself and those two people that read this blog a few simple facts. If we neglected for a moment the bright imagination of Boris Johnson about EU policies, and the hatred of Nigel Farage for everything European, we might still remember that the UK did very well within the EU, growing economically more than others. We will never know if the UK could have done better, but we certainly know that the UK had a prosperous time within the EU.

Then the financial crisis struck. Make a long breath and repeat – slowly: ‘the financial crisis struck in 2008’. It was a decade ago and very little was done to improve how our economies work, meaning that other financial crises like the 2008 are possible (or likely?). Private debt is again massive, very little was done to reform the financial sector, almost nothing was done to recoup the huge financial resources legally hidden away or simply moved around by wealthy people and organizations. All this money that is syphoned away from the UK, it is money that does not go into public services. It is the lack of funds that get your surgery rescheduled several times, get you wait six hours at the emergency, or four hours for an ambulance, get you pay a lot of money to travel on slow trains, get schools struggling or your prospect for a decent pension low. All these ‘little’ things we ‘normal’ people we actually experience.

So, now repeat – calmly: ‘the financial crisis struck one decade ago’. Also repeat with me, quietly: ‘wealthy hedge fund managers such as Nigel Farage and Jacob Rees-Mogg told me that the EU is evil and I forgot that the financial crisis even happened’. Repeat that a few times. If you do not get angry or feel fooled, it is ok. The Farages, the Rees-Moggs, the inept politicians and the smart financers we do not actually know publically, swept under the carpet, a blue and yellow carpet called the EU, all the damage they have done.

When the financial crisis was unfolding, I feared an increase in xenophobia, tensions between states, the formation of new blocks of countries and seeding new wars. I grew-up and I was educated in Italy, another country with a rich history and literature. And, although I was never the perfect student and I can barely remember what I ate yesterday, I do know what happens from an historical perspective. We are on that horrifying trajectory. Social and political tension is increasing, and if there will be hints to more significant financial stress, I am afraid politicians and those powerful people that have access to them will need to burn the carpet and its hidden secrets.

Oceania, Eurasia and Eastasia in a perpetual war to conquer the disputed regions in Africa and Asia (1984), a tool of propaganda to distract people and resources, that is what politicians need next to cover-up the major fuck-ups that are engineering around the world.

People have a choice. People can choose right, left or centre governments. People can choose to be pro or against Brexit. But people have also a choice to speak about war or pretend that it is not a possibility. Perhaps not tomorrow, perhaps not next year, but people’s political choices, carefully influenced by seemingly inept and bizarre characters with somehow huge resources, are directing several Western-countries towards discord and tension. People have a choice, the choice between peace and war, the choice between selecting their representatives or been lead like sheep. So, sometimes, let’s forget about Brexit and the little sit-com characters it delivered. Let’s just focus on the bigger picture.

Within the EU or outside the EU, people in the UK will be responsible for their own decisions. Our political system, trying to survive, created one extremely dangerous  situation. Soon or late, changing the record on the price of chocolate will work no longer. There will be two possibilities. The first is the one I would welcome, the one thought by an ‘optimist-me’ that prosperity for all of us is around the corner. That would be great. The second is that politicians will lose control, they will get us into another more powerful crisis, this time with no financial reserves, no political capital, no patience to leverage from people.

They will have only one way ahead, enflaming people souls, even more than now, and getting a big nice war to hide their pettiness. Now repeat calmly, after me: “I want no war, I want a life as a free man or woman”. Imagine yourself in the 1920s, in Germany, Italy, or any other European country of that period. Would you hail politicians on nationalist platforms to get your country first, to get back control, to reclaim sovereignty and prosperity for your people? Or would you warn people about the perils of populism and lack of cooperation among nations? Would you be an ‘innocent spectator’ not just of crimes ‘committed by a few’, but also of all the run-up to those more horrid years, that period during which everything got in motion? Then, repeat with me, with a bit of humility: ‘we need to fix the real problems, in our own country’ and shout this aloud to any politician that tells you that the price of chocolate has decreased, again.

P.S. These are just a few thoughts, clearly not a technical analysis. Of course, my opinion is largely shaped by the British political landscape and events, where I live for longer than a decade. However, I am equally critical of most of the events and politicians I follow, from Italy to USA passing through Germany and Turkey. All opinions expressed on this website are my own, even those more confined to my academic activities. I do not and I will not use this platform to share my political opinions too often. However, even though not known to the masses, because of my work I am somehow a public figure and – with no expectation that my words will make any difference – I could not refrain to declare publicly my worries about our society and our future. If we do not speak out against war, violence of any type, if we do not speak in favour of civil and human right, or against threats to our freedom, perhaps it is not worth speaking at all.

 

Volume rendering: is this localization-based super-resolution?

Project outcome published in Biophysical Journal in 2010.

  • Esposito A*, Choimet JB, Skepper JN, Mauritz JMA, Lew VL, Kaminski CF, Tiffert T, “Quantitative imaging of human red blood cells infected with Plasmodium falciparum“, Biophys. J., 99(3):953-960

Most papers have an untold backstory that we cannot reveal in it so to focus on a main message and the most relevant discoveries. This one has a little one I wish to share. Volumetric imaging of red blood cells is not the most difficult thing I have ever done. However, accurate morphological and volumetric imaging of red blood cells infected by Plasmodium falciparum, the causative pathogen of malaria, caused me a few headaches. Let’s forget the time spent waiting for the cultures growing at the right speed to deliver bugs at the right stage of development, undecided if to sleep before or after the experiment, and always getting the decision wrong. Let’s not speak for now about the optimization of the sample preparation that that by trying and failing lead to other interesting observations. And here we focus on the very simple concept of accurate volume rendering.

In one way or another, volume rendering and estimation will require some sort of thresholding on the data so to discriminate the object from the background. As imaging conditions change even slightly from experiment to experiment, setting this threshold might confound the final outcomes. When you deal also with a sample that undergoes major morphological transitions, a simple problem soon became one for which I spent a lot of time to identify a solution for. As it happens, one perhaps does not find the best, most elegant or even the simplest solution, but the solution that they can find with their skills and tools. Mine was a brute-force solution of isosurface volume rendering, iteratively deformed by local refitting of a random sample of vertices in order to respect a specific model set for the transition of object to background. This was a method that permitted us to preserve high resolution morphological descriptions, at high accuracy and reproducibility for volume rendering.

This work was carried out while many of my colleagues were focusing on super-resolution, e.g. maximizing the spatial resolution in optical microscopy. Then, it was simple to notice that fitting a surface onto volumetric data delivers volume estimates at higher precisions than what the optical resolution of a microscope should permit. Indeed, whenever you have a model for an object, in my case the boundary of a red blood cell, in single-molecule super-resolution methods the point-spread-function of an emitter, it is possible to fit this model with a precision that is not (fully) constrained by diffraction, but – in the right conditions – only by the signal-to-noise ratio, the analytical tools and the adequacy of the model for the object.

In this Biophysical Journal paper, we focused on the biological application and, together with other published work, on the modelling of homeostasis of infected red blood cells. Also to avoid criticisms from referees, probably legitimate ones, I decided not to mention the concept of super-resolution. As my research focus is on biochemical resolution and its utilization to understand cellular decisions in cancer, I will not pursue this work any further, but I thought to write this little story.

While writing this brief story, I recalled my friend Alberto Diaspro often citing Toraldo di Francia on resolving power and information. I believe that my work was far from being breakthrough from an optical standpoint, but I wished to use it as a reminder of a fundamental issue that, often in biomedical applications, get forgotten. The resolution at which we can observe a phenomenon, irrespective of the tools used, depends both on the qualities of the instrument used and the quality of prior information we can utilize to interpret the data. Once technology permitted to image single emitters in fluorescence microscopy, the prior of point-like sources could be use to analyse images so to reveal the fullness of the information content of an image that is carried by photons.

In an experiment, information content is the most precious thing. Irrespective of the methodologies used, our protocols are designed to maximize signal-to-noise ratios and, thus, maximize information content, precision and resolution. However, as trivial as these statements are, in the biomedical sciences we often do not follow through the process of maximizing information content. Significant information can be provided by our a priori constrains and models. Moreover, a thorough understanding of information theory related to a specific assay can provide levels of precision and resolution that go beyond what we assume, at first, possible. However, priors and information theory are far too often neglected. This happens out of necessity as most people do not have the training and understanding of both biological and physical processes, and even those that might, have to invest their limited resources carefully. I wish that in the future there will be more collaborative work between the life sciences, physicists and mathematicians, aimed to better understand how to extract maximum information from experiments in the biomedical areas.

So… was our volumetric imaging super-resolution? I am not sure I care to really answer, but I wished to provoke some thoughts and make you think a little bit about the relevance of information theory in biomedical research.

Photon partitioning theorem and biochemical resolving power

Project outcome published in PLoS ONE in 2013.

  • Esposito A*, Popleteeva M, Venkitaraman AR, “Maximizing the biochemical resolving power in fluorescence microscopy”, PLOS ONE, 8(10):e77392

After my 2007 theoretical work on photon-economy and acquisition throughput, I occasionally worked on a more general framework attempting to falsify my hypothesis that multi-channel or multi-parametric imaging techniques can deliver better results than other simpler techniques.

My proposal to develop instrumentation to achieve spectrally and polarization resolved lifetime imaging (later defined as HDIM) was met with scepticism by many. The recurrent question was: if you struggle to do a double exponential fit with the small photon budget we have available in biological applications, how could you possibly dilute these photons over several channels and analyse them with more complex algorithms?

Here, there are a few fundamental misunderstandings. First, the analysis should not be carried out on each “detection channel” independently, but the entire dataset should be used to exploit all information at once. Second, the use of dispersive optics rather than filters permits to acquire a higher number of useful photons. Third, limitations in current technologies (e.g., speed or photon-collection efficiency) should not be an obstacle to the development of these techniques because these are not conceptual flaws, but simply technology obstacles that can be removed.

Although I have a lot of (unpublished) work I used to describe performances of multi-channel systems, I achieved a breakthrough only when I understood I had to focus my efforts on the description of the general properties of the Fisher information content in fluorescence detection rather than the Fisher information in a specific experiment. Fisher information is the information content that an experiment provides about an unknown we wish to estimate. Its inverse is the smallest variance ever attainable within an experiment, or what is called the Rao-Cramer limit. In other words, by maximizing Fisher information, we maximize the precision of our experiments.

Photon-partitioning theorem

The second breakthrough was the understanding that the best description of precision in biophysical imaging techniques was possible only defining the concept of biochemical resolving power that is a generalization of the resolving power of a spectrograph to any measured photophysical parameter and then to its application to biochemistry. The biochemical resolving power is proportional to the square root of the photon-efficiency of a microscopy technique and the number of detected photons. Maximization of Fisher information leads to the maximization of photon-efficiency and, therefore, net improvements in biochemical resolving power. This definition complements the definition of spatial resolution in microscopy and allows to define when two objects are spatially and/or biochemically distinct. It is worth to mention that this is equivalent to stating that two objects are spatially and photo-physically distinct, but we use the photophysics of fluorophores to do biochemistry, hence my nomenclature. I see possible implications for other techniques, including super-resolution and, perhaps, this will be the subject of a future work.

The third breakthrough was the utilization of numerical computation of Fisher information rather than the analytical solutions of equations that are not always available. This process is very common in engineering but not in our field. Therefore, we can now optimize the properties of any detection scheme in order to attain the highest performance.

This work is a very specialist one and I assume there will be not many people interested in it, although the implications of this piece of theory for everyone’s experiment are significant. I believe that this is my most elegant theoretical work, but I guess it is a matter of opinion. The paper in itself had to be expanded well beyond what I wished to publish during the refereeing process and it is now including examples, software, etc. I think the theoretical introduction and the mathematical demonstrations are the best part and the description of the numerical optimization of Fisher information the most useful.

NOTE: there are two typographical errors in the published manuscript within the definitions of photon economy and separability. These are described in a comment on PLOS ONE