The development of super-resolution microscopy (SRM) has led to renewed interest in the use of optical microscopy in a number of areas of life sciences research, especially neuroscience and molecular cell biology. First proposed in 1978, this technique overcomes the so-called Abbe diffraction limit – described by German physicist Ernst Abbe over 100 years earlier in 1873 – allowing structures of less than 200 nanometers to be distinguished with fluorescence-based light microscopy.
Super-Resolution microscopy remained a niche technique until around 15 to 20 years ago, when researchers began combining a number of separate super-resolution technologies – including light sheet, 4Pi, STED (stimulated emission depletion), PALM (photoactivation localization microscopy), and STORM (stochastic optical reconstruction microscopy) – to create systems capable of achieving spatial resolutions down to around 20 nanometers. This development led to Eric Betzig, Stefan W. Hell, and William E. Moerner being awarded the 2014 Nobel Prize in Chemistry, and has greatly enhanced our understanding of many intracellular processes and molecular interactions.
SRM allows cell biologists to non-destructively investigate cellular structures in previously unattainable detail, and its use has grown rapidly, particularly since the arrival of commercially-available super-resolution systems.
One of the main benefits of using these optical microscopy systems is that, unlike other methods offering comparable resolution – such as scanning electron microscopy (SEM) – they can be used to investigate metabolic interactions in living cells. The rise of SRM has therefore prompted many laboratories to revisit imaging-based research in areas such as cell division and intracellular transport – which had previously been hindered by diffraction-limited image resolution – by combining selective labelling with the ability to distinguish co-localised molecules of interest using spectrally-separated fluorescent dyes.
As with many new research tools, the development of SRM technologies has been primarily driven by academia with a majority of the systems in use today originating either directly from research labs or via university spin-off companies. The first generation of these instruments was, by necessity, built in-house by physicists using standard parts from other microscope platforms.
Given the 10-fold improvement in resolution achieved by SRM techniques, the ability to position samples and objectives accurately was obviously a key requirement to be able to create such systems. Fortunately, piezo-based positioning systems – with nanometer resolution – were already commercially available when SRM development began, and were widely used by other high resolution imaging techniques, such as atomic force microscopy (AFM) and SEM. As a result, piezo-based nanopositioners became a key enabler for the development of SRM techniques.