In 1873, the German physicist Ernst Abbe realized that the resolution of optical imaging instruments, including telescopes and microscopes, is fundamentally limited by the diffraction of light. For many years even using the best lenses and optical systems the 2D resolution within the x-y plane was limited to 300nm. Cell biologists were unable to differentiate between two structures unless the lateral distance between them was greater than this, meaning that if two molecules were within 300nm of one another they appeared to co-localise.
Super resolution microscopy has transformed light microscopy by overcoming the physical diffraction limit. The Nobel Prize in Chemistry last year was awarded to Eric Betzig, Stefan W. Hell and William E. Moerner for the development of super-resolved fluorescent microscopy and the ability of this technique to surpass the limits of the light microscope. But now that we have resolved the limit of diffraction in microscopy, the limits of what we can visualise are being imposed by labelling.
The initial challenge in labelling samples for super resolution microscopy was to find fluorescent dyes that were excited by the excitation laser and de-excited by the stimulated-emission laser. Chemists took this on and produced a number of suitable compounds, so that we can effectively consider this problem solved. But now a further unexpected challenge remains- the size of the labelling reagents.
With a molecular weight of 150kDa and length of 10-15nm for each antibody, labelling a cell or tissue sample with both primary and secondary antibodies results in a combined probe length of up to 30nm. As super resolution microscopy offers resolutions of 20-40nm probes of this size pose obvious problems. Firstly, the fluorophores may be up to 30nm away from their targets, resulting in potentially misleading localisation information and secondly, the antibodies are unable to bind every target within the cell due to spatial constraints. New tools were required to make use of the advances in microscopy.
As Affimer molecules are smaller and lighter than antibodies with a molecular weight of only 12-14kDa and a length of 2-3nm, they pose no problems at resolutions down to 20nm or less in super-resolution microscopy. Their small size also makes it possible to label almost all antigens present in a sample, even when there aren’t many. For example, only 10 antigens may be present within a synaptic vesicle, and while 10 antibodies couldn’t possibly fit into the available space inside a vesicle, no such problem would exist for 10 Affimer proteins.
With the use of smaller labelling probes in fluorescent microscopy researchers can expect improved accuracy and sensitivity, which may be of use in key applications such as live cell imaging. Smaller probes can help to visualise smaller structures, be taken up by cells more easily and the increased number of epitopes recognised may result in brighter staining, thus reducing the requirement for high laser powers to detect the target and allowing time-lapse super resolution imaging.
Until recently the available tools have limited our ability to see inside the cell, but with the forward leaps that have occurred in both microscopy and fluorophore chemistry, Affimer technology now offers the essential protein detection tools to connect these technological innovations and realise the full potential of super resolution microscopy.