VirusMapper: open-source nanoscale mapping of viral architecture through super-resolution microscopy

Abstract

The nanoscale molecular assembly of mammalian viruses during their infectious life cycle remains poorly understood. Their small dimensions, generally bellow the 300nm diffraction limit of light microscopes, has limited most imaging studies to electron microscopy. The recent development of super-resolution (SR) light microscopy now allows the visualisation of viral structures at resolutions of tens of nanometers. In addition, these techniques provide the added benefit of molecular specific labelling and the capacity to investigate viral structural dynamics using live-cell microscopy. However, there is a lack of robust analytical tools that allow for precise mapping of viral structure within the setting of infection. Here we present an open-source analytical framework that combines super-resolution imaging and naïve single-particle analysis to generate unbiased molecular models. This tool, VirusMapper, is a high-throughput, user-friendly, ImageJ-based software package allowing for automatic statistical mapping of conserved multi-molecular structures, such as viral substructures or intact viruses. We demonstrate the usability of VirusMapper by applying it to SIM and STED images of vaccinia virus in isolation and when engaged with host cells. VirusMapper allows for the generation of accurate, high-content, molecular specific virion models and detection of nanoscale changes in viral architecture.

NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts

“…The SQUIRREL method (Superresolution Quantitative Image Rating and Reporting of Error Locations), implemented as the NanoJ-SQUIRREL plugin, is provided as part of the NanoJ high-performance superresolution data analysis package. It takes advantage of analytical features associated with NanoJ-SRRF (15) and NanoJ-VirusMapper (19). SQUIRREL operates on the assumption that all super-resolution images are representations of fluorescently labelled biological structures rendered at subdiffraction limit resolutions and signal intensities proportional to the local sample labelling density.…”

“…S3a). We recently described these structures in detail using SIM and STED, mapping a subset of molecular constituents of the virus (19). MVs provide an ideal test case for SQUIRREL as virion substructures cannot be discriminated by conventional fluorescence microscopy but are sufficiently large to be perceived as independent structures by most super-resolution methods.…”

Automating multimodal microscopy with NanoJ-Fluidics

“…Software and Hardware Availability. NanoJ-Fluidics follows open-source software and hardware standards, it is part of the NanoJ project (14,21,43). The steps to assemble a complete functioning system are described in https://github.com/HenriquesLab/NanoJ-Fluidics/wiki.…”

Structured illumination microscopy combined with machine learning enables the high throughput analysis and classification of virus structure

“…The potential of super-resolution microscopy (SRM) to unravel details of the structure and replication of viruses was explored early on 1,2 since the birth of SRM and has provided unprecedented insights into viral structure [3][4][5][6] . Most studies use the SRM techniques providing the highest resolutions such as Stimulated Emission Depletion (STED) 7 microscopy and Stochastic Optical Reconstruction Microscopy (STORM) 8 or dSTORM 9 , in order to extract highest level of details in the virus structure, but often at a cost of long acquisition times, thus limiting the imaging to static samples and low throughput.…”

“…Most studies use the SRM techniques providing the highest resolutions such as Stimulated Emission Depletion (STED) 7 microscopy and Stochastic Optical Reconstruction Microscopy (STORM) 8 or dSTORM 9 , in order to extract highest level of details in the virus structure, but often at a cost of long acquisition times, thus limiting the imaging to static samples and low throughput. Recently, the technique of Structured Illumination Microscopy (SIM) 10,11 was applied to study large viruses such as the prototypic poxvirus 5,12 with much higher imaging speeds. However, beyond understanding the fine structure of viruses, there is a need to identify and analyse classes of structures within large viral populations especially in the biotechnology industry where virus production is often hindered by the large heterogeneity obtained from batch production schemes.…”