It is yellow, the two proteins must interact!

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

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


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

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


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


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


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

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

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


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

Author: Alessandro

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