Although super-resolution light microscopy has broken new ground in our understanding of subcellular structures and the movement of individual molecules within cells, its use to investigate virus particles is in its infancy. However, the field could be about to explode following the publication of work by a Cambridge University team using a trio of fast and ultra-sensitive Andor iXon EMCCD cameras.
Led by Professor Clemens Kaminski and Dr Colin Crump, the team has successfully developed a methodology that allows dSTORM super-resolution microscopy to be used as a structural tool for the study of viruses. This has enabled the team to construct an ultra-high-resolution image of the Herpes simplex virus type-1 (HSV-1) and determine the position of individual protein layers within the virion with nanometre precision. They also determined the distance between the capsid protein shell and the centre of the HSV-1 virion. Additionally, multi-color dSTORM allowed the team to observe multiple layers simultaneously in individual virus particles.
"Pinpointing with nanometric precision the position of individual proteins within the HSV-1 structure is crucial in providing the research community with potential therapeutic targets," says Dr Romain Laine of the Department of Chemical Engineering and Biotechnology's Laser Analytics Group. "Understanding the structure gives us a chance to better understand the functions of the different proteins present in the virus, even to suggest new functionality, and to consider previously unforeseeable protein-protein interactions."
"When we looked for detectors capable of providing the speed required for dSTORM imaging while retaining excellent levels of sensitivity, we selected the Andor iXon3 897 electron multiplying CCD camera. The quality and sensitivity of these cameras is ideal for single-molecule fluorescence applications. Also, the bond of trust we have established with Andor is vital, having worked with the company's products and specialists over many years."
According to Orla Hanrahan, product specialist at Andor, the new methodology developed around dSTORM to elucidate the virus structure outflanks traditional EM and ET techniques in deciphering virus structure. "After using multi-color fluorescence super-resolution localization microscopy to visually distinguish virus proteins in individual virus particles, the Cambridge team designed a model-based analysis of the data, using particle averaging to reconstruct a high-resolution image and determine the position of individual protein layers within the virus particle. What's more, they investigated the effect of the fluorophore linker size and compared immuno-labeling strategies in the context of model-based analysis. The key to the positional accuracy attained by this powerful technique is the combination of speed and sensitivity delivered by the Andor iXon 897 EMCCD camera."