Re: Comments welcome on how super-resolution techniques have changed localization conclusions made with confocal microscopy

[Brandi Bickford]

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I would like to update this discussion. I am no longer using the confocal, or the TEM as we do so much digital imaging that I have left that to my Colleague who also forwarded this list serve to me. It seems that Digital Imaging can answer many questions and should be considered along with confocal. I agree about the analysis. Visiopharm had additional software available with their regular IAS which addresses this issue much more quickly and easily than all of this math people are proposing. I would recommend that you are using Olympus images, Olympus has far better resolution, which is really what we are talking about, than any other digital imaging system. BUT, I just would not do it since there is a better, easier, and very very clear way to determine colocalization. 3D Histech has a program that will create a 3D image of your slide, and believe me it is very very clear as to where precisely everything is. I have always advised, over the last 30 years or so, that FL microscopy is not a good tool for analyzing, it is a good tool for you see it or you do not. 3D provides this.  I also recommend very highly DJT solutions out of Miami, Domenic there has decades of experience with all microscopes and has the answer or knows who does, I love one stop shopping, he provides consultation services and I simply tell him what I need and he provides the answer.
Thank-you for your time and the opportunity to be of assistance.
Brandi Bickford
WFUBMC/Pathology/Digital Pathology

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Subject: Re: Comments welcome on how super-resolution techniques have changed colocalization conclusions made with confocal microscopy



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Hi Madison:

        Colocalization analysis is one of many microscopic techniques which appear easier than they really are. Red = green = yellow is not a valid way to determine coincidence of two labeled molecules in a digital micrograph-although it is surprising how many people still get away with publishing it! The only valid way to determine if the signals are colocalized is to analyse the digital image mathematically using a statistical analysis. Pearsons coefficient is the most frequently used method. It first determines the background if any and subtracts that from the analysis, then separates out pixels (or voxels) where both signals are present-the pixels where only one are there aren’t relevant. Then it analyses the pixels where both are present to determine if it is above what would be expected by random assortment alone and gives a value with 0 being random, 0 to 1 being probably colocalised and -1 to 0 being probably due to avoidance. The catch is that all these numbers are only valid within the resolution limit of the microscopic image, e.g. if your resolution is 200 nm in X and Y and say 450 in Z then the tagged molecules can only be colocalised within that space. If you take two “average” molecules, let’s say 15nm in size, then you can say they are colocalised only within that 200 x 200 x 450 nm space. Take the analogy of two people in a large room at a party. Colocalisation can tell you they are in the same room but are they standing next to each other or on opposite sides? Even if they are next to each other are they interacting? No way to tell really. To prove interaction you need another measure, biochemical or some kind of non-microscopic test. However a microscopic image is the only way to see where within a cell or tissue the possible areas of interaction are, and that is its value. Since super resolution techniques can increase the resolution of a digital micrograph they can narrow down the precise area of the colocalisation but there are also the issues of the super-res. techniques themselves to consider. You can’t see super resolution images with your eyes in a microscope, they all depend on mathematical processing of the digital data to determine the most probable sub-resolution area within the optical signal where the labeled molecule is located. So then you have to ask how good is the math that you use to apply the Pearson’s analysis too. The basic rule here should be don’t overanalyse your results. If you really want to show microscopically that two labeled molecules are next to each other a technique such as FRET analysis is much more accurate.

Good luck with your project

Chris Guerin
Senior Expert Scientist (retired)
VIB Bioimaging Core, Ghent