Antibodies have proven to be fantastically useful tools in life science research over the past 50 years. Antibody-based techniques like immunocytochemistry allow you to view the inner workings of the cell. For example, using antibodies, people have already imaged clathrin-coated pits on vesicles—things that were only being modeled when I was doing my PhD.
But technology is continually moving on, and sometimes even the best tools need to adjust to keep up with our ever-increasing ability to see things at higher resolution.
Traditional labeling: the problem
Whether you are a confocal superuser or a fluorescent microscope novice, the process of labeling your cells has remained the same for a long time: You fix the cells, incubate with your validated antibody of choice, add some fluorescence, and hey presto! you can locate your protein in the cell. Ok, so you may have graduated beyond this to live cell imaging, but the concept is still the same: label your protein with a glowing tag and have a peep to see its whereabouts.
Once you’ve located your protein, you can step it up to co-localization studies to investigate other proteins your pet protein interacts with, giving you more information on what it might be up to in there. An overlap of fluorescent signals from differently tagged proteins might suggest that your protein interacts with another. But what you are actually measuring is how close the fluorescent tags on the ends of the antibodies are to each other.
The size of a typical IgG antibody that you use in your ICC is 15 nm, so the end of the antibody is 15 nm away from your actual protein. Add on your secondary with a bright colorful tag and you’re 30 nm away. Put antibodies on your second target, and you could have two co-localized proteins that appear to be 60 nm away from each other according to your labelling. Alternatively, you could be recording co-localization for 2 molecules that are in fact 60 nm away from each other and getting on with their own business.
Still, this used to be good enough as the diffraction limit of a confocal microscope could only place proteins within ~250 nm of each other.
Science super powers: super resolution
Nowadays though, us scientists have super resolution powers that give us high-resolution imaging capabilities beyond the diffraction limit of the instruments. Techniques like SIMS and STED microscopy or single molecule localization techniques might now allow you to visualize whether things are actually co-localized.
This has forced a rethink on labelling in super resolution.
Scaling down to the cell’s level: aptamers for super resolution
Obviously, antibodies are now too big to use as labels for super resolution imaging. But scientists have already come up with a solution. They have taken the best features of antibodies—their target recognition and the ability to add nice glowing tags for visualization—and compacted this into still smaller molecules.
Aptamers are small affinity reagents generally with a molecular size of ~2 nm. They are generated in exactly the same way as recombinant antibodies, using phage display technology. They interact with their target in the same way as an antibody, but without the hefty backbone attached. As these smaller affinity reagents are designed for purpose, scientists can position the tag on your affinity molecule close to the binding site, giving you extra reassurance that you’re as close as you can be to your target.
The literature is starting to show that aptamers can be used in the same applications as antibodies, and they just might be better:
Aptamers have been used in live cell imaging, in which the use of nucleic acid aptamers allowed improved epitope recognition of the endosomal trafficking components transferrin receptor, epidermal growth factor receptor and prostate-specific membrane antigen.
If you increase the size of the labelling aptamers from 15 kDa to 30 kDa, there is a substantial decrease in the image quality obtained.
These smaller molecules gain access to smaller epitopes that are potentially obscured from the comparatively whopping antibodies.
Because aptamers lack disulphide bonds, they function perfectly within the reducing environment of the cell, whereas antibodies just fall to pieces.
While super resolution microscopy is still relatively new and expensive, the use of aptamers in place of antibodies extends the capability of high resolution imaging. Pretty soon we might even be able to see the molecular bonds that hold two proteins together!
If you have ever imaged biological samples, you have likely encountered autofluorescence. That pesky background coloration you see under the microscope, which can make it difficult to distinguish your actual signal from the noise.1 When you are trying to look for something as delicate as RNA, you don’t want to be hunting for your signal […]
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