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

Optical super-resolution microscopy techniques enable high molecular specificity with high spatial resolution and constitute a set of powerful tools in the investigation of the structure of supramolecular assemblies such as viruses. Here, we report on a new methodology which combines Structured Illumination Microscopy (SIM) with machine learning algorithms to image and classify the structure of large populations of biopharmaceutical viruses with high resolution. The method offers information on virus morphology that can ultimately be linked with functional performance. We demonstrate the approach on viruses produced for oncolytic viriotherapy (Newcastle Disease Virus) and vaccine development (Influenza). This unique tool enables the rapid assessment of the quality of viral production with high throughput obviating the need for traditional batch testing methods which are complex and time consuming. We show that our method also works on non-purified samples from pooled harvest fluids directly from the production line.

[1]  J. Rho,et al.  Handbook of pharmaceutical biotechnology , 2003 .

[2]  M. Heilemann,et al.  Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. , 2008, Angewandte Chemie.

[3]  Lynne Turnbull,et al.  Sub-viral imaging of vaccinia virus using super-resolution microscopy. , 2012, Journal of virological methods.

[4]  Geoffrey E. Hinton,et al.  ImageNet classification with deep convolutional neural networks , 2012, Commun. ACM.

[5]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[6]  Euan A. Ashley,et al.  Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments , 2016, PLoS Comput. Biol..

[7]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[8]  James D Manton,et al.  Ellipsoid Localization Microscopy Infers the Size and Order of Protein Layers in Bacillus Spore Coats , 2015, Biophysical journal.

[9]  Ian M. Dobbie,et al.  SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy , 2015, Scientific Reports.

[10]  W. Webb,et al.  Precise nanometer localization analysis for individual fluorescent probes. , 2002, Biophysical journal.

[11]  R. Horne,et al.  A negative staining method for high resolution electron microscopy of viruses. , 1959, Biochimica et biophysica acta.

[12]  M. Gustafsson Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[13]  M. Heilemann,et al.  Shedding new light on viruses: super-resolution microscopy for studying human immunodeficiency virus. , 2013, Trends in microbiology.

[14]  Rainer Heintzmann,et al.  Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating , 1999, European Conference on Biomedical Optics.

[15]  Luc Van Gool,et al.  Speeded-Up Robust Features (SURF) , 2008, Comput. Vis. Image Underst..

[16]  Clemens F. Kaminski,et al.  Structural analysis of herpes simplex virus by optical super-resolution imaging , 2015, Nature Communications.

[17]  Clemens F. Kaminski,et al.  A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors , 2016, Journal of visualized experiments : JoVE.

[18]  P. Collins,et al.  Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes , 2016, Cell.

[19]  M. Gustafsson,et al.  Super-resolution 3D microscopy of live whole cells using structured illumination , 2011, Nature Methods.

[20]  M. Sauer,et al.  rapidSTORM: accurate, fast open-source software for localization microscopy , 2012, Nature Methods.

[21]  Sara E. Miller,et al.  Modern Uses of Electron Microscopy for Detection of Viruses , 2009, Clinical Microbiology Reviews.

[22]  Bryant B. Chhun,et al.  Super-Resolution Video Microscopy of Live Cells by Structured Illumination , 2009, Nature Methods.

[23]  Ming-Kuei Hu,et al.  Visual pattern recognition by moment invariants , 1962, IRE Trans. Inf. Theory.

[24]  Clemens F Kaminski,et al.  Single Molecule Translation Imaging Visualizes the Dynamics of Local β-Actin Synthesis in Retinal Axons , 2017, Scientific Reports.

[25]  Pierre Mahou,et al.  Stimulated emission depletion microscopy to study amyloid fibril formation , 2015, Photonics West - Biomedical Optics.

[26]  Ricardo Henriques,et al.  VirusMapper: open-source nanoscale mapping of viral architecture through super-resolution microscopy , 2016, Scientific Reports.

[27]  Shayne C. Gad,et al.  Handbook of pharmaceutical biotechnology , 2007 .

[28]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[29]  Sachin Kumar,et al.  Newcastle disease virus: Current status and our understanding , 2014, Virus Research.

[30]  Leo Breiman,et al.  Random Forests , 2001, Machine Learning.

[31]  Clemens F. Kaminski,et al.  RNA Docking and Local Translation Regulate Site-Specific Axon Remodeling In Vivo , 2017, Neuron.

[32]  C. Breitbach,et al.  Going viral with cancer immunotherapy , 2014, Nature Reviews Cancer.

[33]  Clemens F Kaminski,et al.  HSV‐1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin‐Dependent Endocytosis , 2015, Traffic.

[34]  Ke Xu,et al.  Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy , 2015, Nature Methods.

[35]  Ullrich Köthe,et al.  Ilastik: Interactive learning and segmentation toolkit , 2011, 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro.