Observing and imaging biological structures is difficult due to the Abbe Diffraction Limit, formulated by Ernst Abbe in 1873. This limit ultimately arises due to the light being diffracted as it passes through the objective lens and is therefore defined by the wavelength of the light and the numerical aperture of the lens. The classical diffraction limit of a conventional light microscope is taken to be approximately 250 nm, this is the furthest distance two objects need to be apart to be visualised as two objects, or in the microscopy world - two proteins. Biological systems such as cells contain millions of components such as proteins and lipids, which we are often most interested in understanding. A single protein is only a few nanometres in size and when tagged with a fluorescent molecule the light emitted will give a signal through a fluorescence microscope of 250 nm. single point of light2If this protein was isolated within a 250 nm radius we would be able to image and centroid the light emitted, therefore calculating its position. Unfortunately for micropscopists this is hardly ever the case and instead there are hundreds inside such a small area, making them impossible to resolve using conventional microsscopy such as confocal or epifluorescence. This is where super-resolution comes in. We can use super-resolution techniques to reduce the size of this signal and be able to resolve structures which are 50-100 nm apart or go down further to isolate and localise the single proteins themselves.

Image 3b 1

 Super-Resolution is an ever-advancing set of tools for bypassing the diffraction limit using these conventional systems and adaptations of them. The most important part about using Super-Resolution, as any expert will tell you, is to think about your research question and what you are looking to find, find out which resolution you need and plan your experiment intimately from the sample preparation all the way to how you plan to analyse it at the end. We can assist with this process here at ESRIC to ensure you are using the right tools for your research question. You can find out about how each technique works in our Education Centre or if you need the specifications for our systems you can find this in Our Facilities.