It is yellow, the two proteins must interact!

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

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

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

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

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

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

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

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

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

 

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

The Space Race | STEM outreach activities for primary schools

Well, we are not rocket scientists but we could not miss the opportunity to speak about the space race at the Science Day of our local Primary School so close to the 50th anniversary of the moon landing. The inspiration came from the book “Space Race” by Deborah Cadbury. After reading it, a summary of the space race became one of the bedtime stories we tell our daughter. When the time came to pick a story to tell at the Science Day, after discussing work-related topics ranging from DNA extraction to optics, we opted for the space race and the moon landing. We are no experts in outreach but after a few years of volunteering, we can tell you that a well-done job is a hard job and a rewarding one. Also, like for any other communication-based activity, the three main tricks to reach impact are i) tell a compelling story ii) think about your audience and iii) be prepared.

The space race and the moon landing can be still very inspirational story to tell. It is a story of exploration, science and technology, it is a race but also a monumental teamwork. It has its roots in the cold war and the manufacturing of weapons of mass destruction… a story that ended up with a blast-off to the moon to inspire generations instead.

The backstage

The first step in the organization for us was to see which are the basic experiments people do in the classrooms around the world. We clocked several hours over a few weeks trying to understand what is possible and what might excite pupils. Google and YouTube were the most obvious starting point. This activity was fun (well, particularly if you are a bit geeky!) but also stressful when we noticed we were not converging to a particular set of experiments we wished to demonstrate. Everything changed when we decided which story we would tell, as we were able to rethink all the material we explored from a different perspective.

The second step was gathering materials and more information. We studied facts about the moon, rockets and the space race. Most of it was general information that could have been useful to answer questions, some of it ended up in an introduction supported by a few slides. At the same time, we went shopping both targetting specific items but also browsing toy shops randomly trying to identify anything that could be useful. We kept brainstorming about a possible story-line and experiments to demonstrate, finally converging to a plan.

The third step was to prepare the day. We prepared a few slides and selected a few fun facts to share. While unnecessary strictly speaking, in private we discussed sensitive topics, the drive of science and technology during the cold war to prepare weapons of mass destruction, how this turned to a different type of race to reach the moon, with elements of competition and team working. While, of course, we did not discuss these topics in the classroom, eventually we were able to emphasize concepts that are important to us, the use of science and technology for good purposes (exploration and discovery) rather than bad ones (war), racing as a fun activity but highlight how teamwork is essential to reach very high goals.

Before the day came, we just needed to be sure that the day at school was organized properly, and we were lucky that Emily Boyce from the Babraham Institute had organized an excellent schedule for the entire day, logistics and liaised with teachers, so we could spend all the time we could just on the activities. Finally, risk assessments. Yes, they are boring and sometimes they seem superfluous but if done properly they help you think about what could go wrong and avoid accidents to happen. As they are anyway a legal requirement, make best use of them to help you planning the event logistics.

On the day

We had prepared a few slides with full screen images from the Apollo mission (a fired-up Saturn V, the moon lander, Armstrong’s footprint, a map of the solar system) and we ad a passionate and engaging chat with the students (see ‘Let’s talk about the Moon’ section). While the students were engaged, one of us set up all the contraptions needed for the latter part of the session.

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Prepare a short introduction with fun facts, engaging students and setting up the narrative for the following experiments

Next, we wanted to introduce the concept of propulsion and Newton’s third law of motion. We started with this toy we found in a store:

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build up the story starting from simple concepts and visual demonstrations

We just showed how air pushed to the ‘rocket’ can lift it up, just small jumps catching the rocket with the hands. With the reception class, we let some children playing with it, while with year 3, we did some jokes (e.g., ‘do you see a big man or woman pushing a large pedal under the rocket?’ while pointing to the image of a fired-up Saturn V ready for lift-off) and we asked to explain to us what was happening.

Next, we told that this is not how rockets work and release rocket balloons in the room that we had inflated before entering the room and clipped. When thrown (not just released them speedless), these balloons are propelled around the room.

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We inflated rocket balloons (we had an electrical air pump we previously bought for parties to help) and clipped them ready to be released in the classroom

We engaged the students asking what they thought it was happening and clarified that air is getting out of the balloon and pushing the ballon ahead. The uncoordinated movement of the rocket balloons let us introduce the next contraption. We had placed a mock-up moon in the corner of the classroom. Because of the limited time available we prepared it at home with recycled materials within a plastic bag forced into a spherical shape with cello tape then covered with aluminium foil. We left that knotted handles of the bag out of the aluminium foil to anchor two fishing lines. The fishing lines were several meters long to cover the length of a classroom. There are plenty of instructions over the internet on how to build a rocket balloon guided by a string. I would recommend a more visible line than the one I found in the local shops but here the materials we used.

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Materials to build racing rocket-balloons. Rehearse at home to understand the logistics, and which material could work best. The rocket balloons we originally picked for this purpose did not work on the fishing line and with drinking straws we had at home. However, they were fun to release in the class. Also, we used the straws included in their package for their inflation to guide our rocket-balloons, standard medium-sized party balloons.

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We inflated the balloon with an air pump, pasted the straw on the top of the balloon with two long pieces of cello tape and we drew a fun face on the balloon with a permanent marker. We then took one of the prepared fishing lines and demonstrated how the rocket balloon could reach the moon, asking the children to do a countdown after which we released the clip. This was just an introduction to the main activity of the session where we split the class into groups and gave materials to prepare and decorate their own balloons. As we pre-made two fishing lines, we let them race in pairs of groups to the moon.

We had planned to stop here if we ran out of time but prepared also a different ending. Our sessions were 45 minutes long and we discovered there was enough time for it. We pointed out there is no one inflating rockets and we introduced the concept of rocket fuel.

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Malt vinegar and bicarbonate of soda react to generate CO2 that bubbling through the protein-rich malt vinegar will generate a lot of froth.

Before the beginning of the session, we poured two shots of malt vinegar in a tall glass. When the time came, we uncovered the glass and chatted about liquid and solid fuels, introducing the concept of chemical reactions used to propel a rocket. We then added a teaspoon of bicarbonate of soda to show the formation of large amounts of froth. During testing at home with the materials we could find in the local shops, we accidentally realize that malt vinegar would generate a lot of froth and that we could use this as a trick for comparing the froth to the vapours and flames coming out of a rocket engine.

Finally, we showed how this could be used to propel a rocked by inflating a balloon.  We tested a few materials and opted to use a small plastic bottle with white vinegar. Keep in mind we used what we could find at the local shop and other combinations could work better. We added four shots of vinegar into the empty juice bottles. The labels were removed and we wrote the content with a marker. We also always had the bottle under control, but the obvious shape of the bottle attracted attention from younger children and we probably would use a different bottle or covered it with paper if we were to redo it, just to avoid a child grabbing it and trying to drink from during the confusion of some of the activities.

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Vinegar and bicarbonate of soda react forming CO2 that can inflate a balloon illustrating the mixing of chemicals used in a rocket engine

To make things simple on the day, we prepared balloons filled with two teaspoons of bicarbonate of soda, gently clipped, with excess powder blown away from the opening of the balloon.

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Prepare balloons with bicarbonate of soda. We pre-inflated them to write on them ‘to the moon’ a few times and filled with two teaspoons of bicarbonate of soda.

At the right moment, we removed the clip and attached the balloon to the neck of the bottle paying attention to not let any powder drop into the bottle. Then we raised the balloon permitting the powder to mix with vinegar while holding the neck of the bottle firmly with the hands to avoid the balloon shooting in the class and spraying vinegar. We kept the vinegar a bit warmer than room temperature by pouring some hot water in a cup and keeping the bottle of vinegar in it. This was done in a staff room for safety. The lukewarm vinegar reacts faster with baking soda resulting in very fast inflation of the ballon.

This is how we prepared our Science Day activities. Each of the experiments is rather common and we got inspired by a lot of materials we read and watched. However, it is important to test every single experiment at home, identify the most appropriate materials and doses in order to ensure the timely and safe execution of each of them. Together, we probably invested about 50 hours of work in this activity in addition to the day spent at the school, spending evenings and spare time to plan the activities.

Let’s talk about the Moon

Presentation HERE.

1) Who can tell what the Moon is? It is a space rock we call a satellite that turns around (orbits) the Earth. It was formed about 4.5 billion years ago when a large space object hit the Earth, and the debris from this crash formed the Moon. The Moon completes its turn around the Earth in 27.3 days.

2) What colour is the Moon and what it is made of? It’s made of mostly dust and rocks, there is no atmosphere, no water and no life. Just mountains and large craters. The Moon itself does not produce any light; we see it shining because the Moon reflects light from the Sun.

3) We see only one side of the Moon (also called near side), why is that? While orbiting around the Earth, Moon also rotates around its axis, and this rotation takes the same amount of time as it does to complete the turn around Earth. That’s why we can only see only one side of the Moon (about 60% of its surface).

4) What is the temperature at the Moon? Hot or cold? Well, both actually. During the day when the Sun hits the surface of the Moon temperatures can reach 127°C. You can fry an egg without a stove. During the night, the temperature can go down to freezing -173°C.

5) Did you know that you weigh six times less on Moon? That’s because the gravity (the force that pulls us down to the ground) on the Moon is weaker than the gravity on Earth. You can jump really high on the Moon. In fact, astronauts have to wear their heavy boots to keep them down on the ground.

6) How far is the Moon from us? It is really really far, about 384.000 km. If you are to drive this distance by car it would take you about 150 days. However, thanks to the rockets built by very talented teams of engineers and scientists we can reach the Moon in just 3 days, and we have exciting opportunities to explore the space!

We humans have always been curious about the world around us. The Moon was always one of our biggest curiosity. Using his telescope, Galileo have documented many observations about the Moon in 1600s. We have come a long way since then and thanks to the space rockets we have built we can explore places “Where No Man Has Gone Before”

Rockets were initially developed for wars, unfortunately. Luckily, later on, we realised we could use and develop rockets for much better goals – to explore the deep space. The Space race began. The first country to send an astronaut into Space was the Soviet Union, with Yuri Gagarin and his Vostok 1 capsule. The Soviet Union, sent also the first satellite in orbit (Sputnik) and the first rocket to the Moon, with the spacecraft Luna 1 passing very near to the Moon and Luna 2 crash-landing on our Satellite in 1959. It was then in 1969, that an incredible adventure lead by USA brought the first people on the Moon. Engineers and scientist in USA built a massive rocket, Saturn V and brought three brave astronauts up to the Moon with their Apollo 11 mission. The team was led by Neil Armstrong who made the first step on the Moon. As Neil Armstrong said, this was “ one small step for [a] man, one giant leap for mankind!”

Armstrong’s footstep will be a long lasting one as well. It will last in our culture, as the most exciting moment of a long adventure. It will last a long time on the Moon, where there is no wind to wipe it off.

Suzan Ber and Alessandro Esposito

#outreach #spacerace #moonlanding #science #technology #rockets