my blog

Our superpower is you (first draft)

This is a preliminary draft for a leaflet aimed at outreach events. Following the work we have done on Women in STEM at the last year Cambridge Science Festival, this year we’ll add a second leaflet to be more inclusive. This will be a word search puzzle that we will release into the public domain for your peruse. The idea is to have a list of people to include different genders, ethnicities, sexual orientations and disabilities. While my team is working on the graphics and the production of the leaflet, I prepared the first draft of the name list. As I believe it can be certainly improved  I wished to ask for suggestions and criticism.  

The message: “There is not much in common among these inspirational people, no colour, no gender, no physical ability. Hard working and smart, nothing else defines a scientist. A scientist is someone like you.”

Alan Turing (1912-1954),  Mathematician, a founder of Theory of Computation

Florence Nightingale (1820-1910), Social reformer and statistician, the founder of modern nursing. nurse

Neil Divine (1939-1994), Astrophysicist, a major contributor to the modern theory of
star formation.

Lynn Ann Conway (1938), Computer scientist and electrical engineer. Pioneer in electronics and computing.

George Washington Carver (1860-1943), Agricultural scientist and inventor. Promoted alternative crops to cotton and methods to prevent soil depletion.

Ernest Everett Just (1883–1941), Biologist, a pioneer in the studies of fertilization and early development,

Stephen Hawking (1942-2018), Theoretical physicist and cosmologist, a pioneer in the modern theory of cosmology and black holes.

Edwin Krebs (1918-2009), Biochemist. Pioneering work on post-translational modification of proteins and cell regulation.

Albert Einstein (1879-1955),  Theoretical physicist, founder of the theory of relativity, one of the two pillars of modern physics.

Charles Darwin (1809-1882), Naturalist, geologist and biologist, a pioneer in the science of evolution.

Rita Levi-Montalcini (1909-2012), Neurobiologist, pioneering discoveries in neurophysiology.

Chien-Shiung Wu (1912-1997), Experimental physicist who made significant contributions in the field of nuclear physics.

The design of the leaflet will be based on a diverse group of superheroes, with the message “Our superpower is you” meaning that science main resource is one: people.

 

 

The backstage story of a paper. Highs, lows, lessons to learn

Since a few months, the manuscript entitled “Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate“, and authored by Maximilian W Fries, Kalina T Haas, Suzan Ber, John Saganty, Emma K Richardson, Ashok R Venkitaraman, Alessandro Esposito, is available as pre-print at the bioRxiv repository. It has started its journey through the peer-review process, but here I wished to explain to students and young scientists what happened behind the scenes as, I believe, can be instructive.

The inception of the idea | I am unsure if it will be evident from the manuscript, but this is the culmination of a huge effort that started more than a decade ago. I was about to leave the Cell Biophysics Group led by Prof. Fred Wouters after I completed my PhD, on a train from Goettingen to Bonn where my partner used to work,  thinking: “What should I do next? … something that while capitalizing on my training can make my work distinct from my mentors and others? Where can I have the highest impact?” Moment that stuck in my memory.

I believe I read Santos et al. (2007) “Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate.” in that period, a paper that influenced me significantly. It made me thinking of cells as if they were computational machines, interpreting various inputs from the extra- and intra- cellular environment to trigger appropriate outputs, cell states or transition between cell states, i.e. cellular (fate) decisions. Everyone working with microscopy knows that cells treated equally often behave differently and, therefore,  I started to formulate ideas around the following question: “How does a network of biochemical reactions encodes for cellular decisions? Why do genetically identical cells take a different decision faced by a similar stimulus?” Basic principles, the science I love the most, but questions worth answering also to obtain mechanistic insights, questions also quite relevant to disease.

As a matter of fact, it is  of fundamental importance to understand how cells trigger pathological states or if differences in biochemical networks can be used as diagnostic markers for patient stratification or targeted for therapy, concepts that I started to work only later. Certainly, I thought back then, with my unique blend of physics, engineering, mathematics, molecular and cell biology I can do, in this area, what others might not be able to. Therefore, since 2007, my aim is to image not just a biochemical reaction, but biochemical networks within intact living cells, while they undertake decisions.

Finding the resources, the initial success | Perhaps other students start less naïvely than me, but soon I would discover that having a good idea (let’s suppose it is a good idea) and having the right skills is only a tiny part of the job. First, aiming to coordinate my work with that of my partner (now wife), I accepted a job offer at the University of Cambridge to work with Prof. Clemens Kaminski and Dr. Virgilio Lew to study one exciting but quite unrelated project. While working on the homeostasis of P. falciparum infected red blood cells, I set up collaborations and wrote an EPSRC fellowship which was funded. Therefore, in 2009, two years after my first idea, I got the funding to work on biochemical multiplexing. With this fellowship, I was able to refine my expertise in biochemical multiplexing, permitting me to build advanced technologies for fluorescence sensing such as confocal  spectro-polarimetry and fast SPAD-based spectral FLIM. This EPSRC fellowship, together with my expertise and vision, and the benefit to have already established my name in the community thanks to the work I had done with and the support of Prof. Fred Wouters and Prof. Hans Gerritsen, were an excellent platform that permitted me to do the next jump and accepted a senior position at the MRC Cancer Unit.

Finding the resources, the struggle |  Rather than focusing just on technology, I then broaden my research to a research program that would require theoretical developments, engineering of new pairs of fluorescent proteins to achieve multiplexing, coding and, of course, biological applications. I recognize that expanding my research before seizing the appropriate resources was a significant mistake or at least a huge risk. Working within Prof. Ashok Venkitaraman group, I started to write ambitious EU grants. Some of them would receive excellent feedback (14 out of 15 points, first or second not funded…) but fall short of being funded. Hans once told me that “at this level of competition and quality, often it is just noise that decides the final outcome“. Probably true, even funny if you knew we worked together on photon-statistic (‘noise’). But great feedback does not replace funds, and thus I wrote an ERC grant.

I did not get ERC funding but, once again, ERC is very competitive and I was not sufficiently experienced, thus no drama. However, I started to notice one big issue. Physicists would judge my physics not great physics, biologists would judge my biology not great biology. Some colleagues would find my objectives impossible to reach. This is what I have then discovered to be the challenge of doing multi-disciplinary research (well, technically is called trans-disciplinary research, but this is the topic for another post). When your proposal is both trivial and impossible, you might have an issue that is not necessarily related only on your science. One referee commented that “A number of groups have being trying to improve the technologies for many years and although some of them have an enormous experience they are not anywhere close to where he intends to be in five years“. Around the same time, a renown scientist commented on the description of my work “It is impossible”, but then added in a wonderfully supportive and very appreciated manner “but if there is someone that could do it, it is Alessandro” – well, if funding-proposals could be judged with the human touch that people have when speaking in person knowing and respecting each others work…  I’ll cut an even longer story short, but with significantly less resources than I was asking and struggling to increase my funding, with the financial backing of Prof. Ashok Venkitaraman, we did everything we wanted to do in… five years!

The great technical success (NyxBits and NyxSense) | I wished to tell you a story of great success in a broader sense, but this has to be still written… if it will ever be. I did waste significant amount of time in looking for resources in what I found an amazingly inefficient system. However, from the end of my EPSRC fellowship since this year (~6 years), we have done a huge amount of work to realize what it was thought not to be possible:

  • Molecular Biology. I wished to develop two platforms, one based on spectrally multiplexed time-resolved anisotropy (open for collaborations here!) and one for spectral FLIM to manage the cross-talk between multiple FRET pairs and making biochemical multiplexing possible. With the limited resources I had, and initial help from Bryn Hardwick, Meredith Roberts-Thomson and David Perera in Ashok’s lab, we kick-started the project. The mole of work started to overwhelm me. Occupied with grant writing, training in a new field, engineering, software development and mathematics, I could not push this forward as fast as I wished. A great help then arrived from Max Fries who did 6 months with me as master student. Once he left, I was short of resources again, with the FRET pairs misbehaving and exhibiting aggregation or spurious signals, we abandoned one of the two sensing platforms.  Emma Richardson then joined me as a Research Assistant dedicated to cloning and testing FRET pairs and then Max came back to work with me for another four years as a PhD student. Committed and skilled, he tested tens and tens of FRET pairs. The work was a huge task, but a couple of paragraphs in the manuscript. We even have better pairs then we used in this work, all described in the Supporting Information. Indeed, under the pressure for publishing on high impact journals, I decided (probably anoher mistake of mine) to progress to applications, settling for what we recently baptized as NyxBits: mTagBFP, sREACh, mAmetrine, msCP576, mKeima and tdNirFP, so to focus on biological applications. NyxBits and NyxSense? Well, I have explained the choice of names elsewhere.
  • Mathematics and software. There is something I could not really write in the manuscript so explicitly and it is appreciated only by the experts in the field. There is something I also find impossible to communicate to review panels. As a testimony to this, I report here a comment I was once relayed to, something like: “Why do we need to offer him a carreer, once he has built the instruments we really need one person just clicking a button, no?” (I am sure I remember it much worst then it was. May be). The integration of technologies is so new and challenging, that we had to formulate new theoretical frameworks and write all new software, including how to acquire data, data format, and analysis. Also, some aspects of our work are difficult to appreciate. Let me tell you another small event that would push me in a particular direction. I really enjoy the conference Focus on Microscopy, even when criticized. Presenting new ideas, a colleague – respectfully – questioned the possibility for multiplexed imaging to be capable to measure several FRET pairs at the same time. This stimulated me to resume studying the Fisher information content in biochemical imaging. What is the biochemical resolution in microscopy? Can we enhance it? After years of thinking about this topic, in 2013 I cracked the problem, and published the mathematics in PLOS ONE where I formulate what I defined ‘the photon-partitioning theorem’. Then, with the increasing financial backing of my Director, Kalina Haas joined my growing team. Kalina implemented unmixing algorithms  and complex data analysis pipelines. Max and Kalina then became my dream-team to progress the project to the shape you can read today.
  • Technology. I mentioned some earlier technology platform that were designed for biochemical multiplexing. In my recent and first release of manuscripts on bioRxiv, we  also published a full implementation of Hyper-Dimensional Imaging Microscopy (HDIM)  with which we backed the photon-partitioning theorem with experimental evidence. We have done much more in that direction, but when we started biological applications, we realized the need for faster FLIM systems. Uncapable to wait for commercial solutions or to gain the benefits of other prototypes we had developed, I decided to build my own fast multiplexed electronics. This development was fostered by a negative criticism of a referee. During a PNAS submission of our spectral FLIM system, a referee mentioned we could do the same utilizing Hybrid PMTs. I disagreed, as achieving 64 channel spectral FLIM with the capability to run at hundreds of millions of photon-counts per second is all-together a very different application; however, there is merit in most referees’ criticisms, even the most negative ones. Only then I have realized PMT are now very fast and the bottleneck was just the electronics. Therefore, I got in touch with Surface Concept  who supported me wonderfully and  sold me one of their multi-hit TDC platforms. After several months of software development, we were then capable to run FLIM measurements with the quality of TCSPC and the speed of FD-FLIM. As usual, I presented this work at FoM where it was greatly received by colleagues and companies, but we did not publish the imaging platform as we were fully committed to pursue biological applications.
  • The biology. The bottleneck of our experiments was and still is data analysis and, with tens of experiments, thousands of biochemical traces to be painfully manually curated, we moved ahead very slowly, but working hardly. Mostly Max, Kalina and myself, suffered years of hard work, the occasional worry when something stopped working, and the excitement of seeing things that others could not see, for the first time. In this manuscript, we reveal the extent of non-genetic heterogeneity that  biochemical networks can exhibit and that eventually result into difference cellular decisions. Here, we focused on multiplexing simple biosensors for caspases as we aimed to de-risk and very ambitious project. We also decided to work with HeLa cells, again for the same reason. Despite the simplicity of the model system under study, we realized how complex and heterogeneous the response of biochemical pathways is, the cross-talk between enzymes, signaling pathways and cellular metabolism. All of this is, for me, fascinating and it shows that whenever we do ensemble measurements, we really see only the behavior of the average cell. It is then important to understand that the ‘average cell’, most of the times, does not really exist. If we are lucky, the bulk of the population responds with one phenotype and the measured ‘average cell’ will indeed represent the ‘most frequent cell’. However, in other instances when there are significant populations behaving in distinct ways, we would not just miss important information. The model inferred from the ‘average cell’ would be simply the wrong model of a non-existing cell. This is why it would be important to know, for any assay, if the sample behave synchronously with a stimulus and homogeneously. In this sense, single cell biochemistry, could bring not just an additional layer of information, but inform us if what the observations we obtain on a given model system with ensemble measurements can be reliable.

Enduring the struggle | I hope you did not mind I spoke so positvly about my own work. If you know me, you also know I am not so self-centered. However, I wished to let the younger scientists to know what there might be between a ‘good idea’ and its realization, passing through frequent failures and some success. Probably, one of the most precious quality of a scientist is resilience. We need thick skin to confront the constant failures that lead us to discoveries, the constant struggles in getting resources and eventually publishing good work in a highly competitive environment. Turning a negative event in something negative is part of this process. Understanding why one experiment did not work enables us to make troubleshooting, why an experiments falsified our hypothesis to build new and better models, why funding was not awarded or a manuscript was not published how we can improve our scientific proposals and reporting. Of course this is easier said than done.

The work we presented in bioRxiv is not the end of the story. The work, wonderfully-received in conferences, is still not peer-reviewed. Will colleagues appreciate and understand the vision of our work, its possible impact and the mole of work we had to do? Were we able to communicate properly? And even if we did it, we still have a long way in front of us. My dream is to establish a single cell systems biology of cell fate. A huge amount of work, from maths to biology, from biotechnology to physics, all still needed to be able to understand why cells do what they do, how physiological states are maintained and how pathological states emerge.

This is ATLAS.ONE (a high speed high resolution biochemical imaging platform)

Project ATLAS

In 2018, we decided to invest capital funds provided by the MRC and the MRC-DBT with the aim to make our technologies more accessible to the biomedical researcher laying down also the possibility to deliver advanced biophysical assays at high throughput [REF1] with a focus on 3D cultures. Why ATLAS? I often code-name internal projects, possibly with evocative names that might capture the pathos of the project. As these are capital investments to strengthen specific areas that will be essential for our long-term applications, I named this investment ATLAS, as the Titan that was condemned to hold up the heavens on its shoulders. In our case, I am building the base for two microscopes that will support my research projects, and support those of the others working at the MRC CU, in the longer period.

This is ATLAS.ONE

Here, I will briefly introduce ATLAS.ONE, the first of the two microscopes we have started to develop. The aim is to develop high spatiotemporal and biochemical resolution with a imaging platform that could be readily accessible by a non-expert user. We are testing the solid-state FLIM (Fluorescence Lifetime Imaging Microscopy) camera PCO.FLIM by PCO for video-rate biochemical read-out of genetically encoded probes or protein-protein interactions.  This is the commercial incarnation of part of my PhD work [REF2REF3] so brilliantly developed and delivered by PCO (no commercial conflict). After considering different possibilities to gain some resolution to better discriminate cellular compartments, we decided to integrate this platform with a simple SIM (structured illumination microscopy) setup based on LCoS spatial light modulators. Biochemical perturbations will be implemented with a CellASICS microfluidic platform. This is a capital investment and we will first focus on methodological advancements, however, we will deploy this platform to characterize genetic and non-genetic heterogeneity in cancer cell lines. While we will look for external funding, we’ll start working on KRAS-dependent signalling pathways and metabolic pathways.

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Who is involved?

You are welcome to get in touch if you wished to coordinate developments or to use this platform – once established – either with own resources or common grant applications. Currently, ATLAS.ONE is supported by the MRC and the MRC-DBT for capital funds and the following people for development and applications.

Andrew Trinh and Alessandro Esposito (MRC Cancer Unit), developing the system and applications together with Christian Frezza and Annie Howitt (MRC Cancer Unit), developing single-cell metabolic assays.

Guy Hagen, University of Colorado, to collaboratively develop SIM

Gerhard Holst, PCO to advise on camera integration.

 

[Open hardware] A safe laser by-pass

Well, I remember when I started this business, a beam stop was done with a recycled block of lead and reflections stopped with carton boxes 😉 Brown boxes, black carton catches fires, of course (tell this to my undergrad-self). Not any longer, of course!

About ten years ago, I started the procurement and development of my first two-photon microscope. For the first time, I was directly responsible of laser safety and I had to take decisions about how to build a system that was safe for a user facility in a biomedical research institute. As I was coupling commercially sourced systems (Leica SP5, Chameleon Vision 2 and Pulse Select) and I was not planning much customization for the excitation path of this instrument (I heavily develop assays and detection), I opted to fully enclose the laser in lens tubes. The resulting system is safe, stable, and no more difficult to align compared to other enclosures.

I think that enclosures around the complete table might make sense in many instances, particularly when compartmentalized in sub-sections, but this is the system that worked best for me at the time. One solution I wish to share, is a bypass for the Pulse Picker we had used to develop spectrally resolved FLIM utilizing smart SPAD arrays (detectors that integrate photon counting electronics with them).

20181112_184730As I start planning replacement of this systems, I wished to share this design, in case some of you might find it useful. In the image on the left, you can see the Ti:Sapphire on the top, the pulse-picker on the right and the first enclosure by Leica used to steer the beam to their in-coupling optics (bottom-right).

In the middle, the laser bypass we utilize to direct the laser through the pulse-picker or around it.

In the image below, you see a close-up photo of the by-pass. The black box with the rectangular aluminum cover is the Leica spectral flattener used to reduce power of the Chameleon Vision at the peak wavelength. One of the few customization I needed here was simply to have a hole on a Thorlabs SM2 lens tube to accommodate this filter. This is screwed in a C4W-CC cube that can host a movable turning mirror with high reproducibility. The alignment of the microscope without the pulse-picker is done with the pair of mirrors provided by Leica. The alignment of the Pulse Picker is done with the kinematic mirrors visible on the left (M1 and M2). I placed a light-block behind them just in case one would become lose or to block the small amount of light transmitted through them. A kinematic cube is used to host ultrafast beam sampler by Newport to direct a small fraction of light to the Thorlabs PIN diode I use to feed the electronics of the pulse picker. In front of the PIN diode I have an xy-translating cage element. An empty four-way cube is used to allow the laser beam to pass from top to bottom (bypassed) or from left to right (coupled pulse picker). The aluminum block tagged as L1 is just a cover for the C4W-CC when empty.

20181112_184735

At the output of the pulse-picker, you see the mirror image of this bypass (on the right) and the two steering mirrors by Leica (the cylindrical towers). On the far right of the picture there is the in-coupling optics by Leica, preceded by two diagnostics ports.

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Below, you can see a close-up picture of this part of the coupling. Because of the layout, I needed to add one extra mirror (top left) and aiming to isolate users (placed on the top of the image) from accidental damages of the in-coupling optics, I added a light barrier.

Both diagnostics ports are based on a 4-way kinematic cube from Thorlabs hosting Newport beam samplers. The first port is used to sample the pulses after the pulse-picker and to feed our FLIM electronics. The second has two scopes. First, for course alignment of the system. I have two irises in the system that are aligned when the laser is aligned (roughly) to the in-coupling optics of Leica.

I usually remove a cover at the exit of this diagnostic port and use a fluorescent card to verify alignment, but in this picture you see the fiber coupling a spectrograph we occasionally use to diagnose faults of the laser.

 

20181112_184714

The alignment is simpler that it seems. First we start with a microscope that is fully aligned without pulse-picker as per normal operations. Then, when we need the pulse picker, we insert the two turning mirrors (L1 and R1). We do this with the laser off and with the pulse-picker crystal retracted (coarse alignment) or protected by an alignment card (fine alignment). M1 and M2 are then used to align the beam with the crystal. Then we align the PIN diode and proceed with the fine alignment of the pulse-picker cavity.  Once this is done, we align the cavity with the microscope utilizing M4 and M5. For course alignment, the signals from the two diagnostics ports is very useful until some signal is picked on the microscope monitor, after which the final fine tuning of all the optics can proceed.

Be aware, alignment of Class 4 lasers can be dangerous. Therefore, do your own risk assessments and think carefully about the logistics of your system. Now that I am starting to consider the redevelopment of the system, I thought to share these notes with you, hoping that it could be of some use.

Sharing is caring: an open access FLIM trial

Are you interested in cell biochemistry, but in single living cells, organoids or tissues? Is there a Western blot or IP you wished to do on a living sample? Or did you wish to see where in a cell a protein-protein interaction occurs.

Well, if you are interested in quantifying a ligand concentration, a post-translational modification, a protein-protein interaction, chromatin states, oligomerization of proteins, you might be interested in FLIM or FRET, but you might not be in your comfort zone to set-up or execute such assays. 

The specialist expertise and instrumentation required to perform fluorescence lifetime imaging microscopy (FLIM) is often a barrier to adoption of quantitative biochemical imaging techniques. The same can be true, although at a lesser extent, for intensity-based measurements of FRET.

Well, we have the expertise and we have the instrumentation. Not just this, but today, instrumentation and data analysis is becoming simpler and simpler. During 2019, we are going to trial a system by which we can support you for setting-up and test FLIM/FRET experiments. We have limited resources and, therefore, we will open only a few experimental sessions to start with, but there will be no strings attached. No fees, no authorship to include in that paper you really care.

Although we still have to setup the “Sharing is caring” trial, feel free to inform us about your interest. Initially, projects will be selected at our discretion, with priority given (but not confined) to cancer-related work and work with a potential to impact public health in the short or long period.

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.

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.