We describe High Content Analysis (HCA) to be the implementation of automated imaging for high throughput assays that include image acquisition with cellular or subcellular resolution. HCA is most commonly implemented using wide-field or confocal intensity imaging, often with multispectral detection, for which there is a range of available commercial instrumentation.

Our work applying advanced microscopy techniques, particularly FLIM/FRET, to biomedical studies highlighted the challenges to achieve reproducibility and statistical robustness when undertaking manual microscopy experiments, noting that the sample preparation could change between repeats of experiments and the acquisition and image analysis could be subjective and therefore open to operator-bias. Accordingly, we were motivated to implement our microscopy techniques in an automated multiwell plate imaging format. The consequent near-uniform sample preparation in each well and the opportunity to image 100’s of fields of view under similar conditions enables averaging over large sample numbers – typically with many replicates of the same experimental conditions and wells with control or calibration samples. This, combined with the high consistency of sample preparation across a multiwell plate, significantly reduces the impact of random noise in both measurement and biological systems (for example, expression of fluorescent proteins). The low random noise, consistent sample preparation and replicates of experimental conditions then enable any systematic errors in the measurement to be easily identified. 

We are developing a modular open source HCA platform: openHCA, for MicroManager that will be extendable to multiple imaging modalities. This will eventually incorporate our existing HCA modalities:

High content analysis

High Content analysis references

  1. High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis
    C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, M. Katan, C. Dunsby, M. A. A. Neil and P.M. W. French
    J. Biophoton. 1 (2008) 514–521
  2. Rapid global fitting of large fluorescence lifetime imaging microscopy datasets,
    S.C. Warren, A. Margineanu, D. Alibhai, D.J. Kelly, C. Talbot, Y. Alexandrov, I. Munro, M. Katan, C. Dunsby and P.M.W. French, PLoS ONE 8 (2013) e70687
  3. Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
    F. Görlitz*, D. J. Kelly*, S. C. Warren, D. Alibhai, L. West, S. Kumar, Y. Alexandrov, I. Munro, J. McGinty, C. Talbot, R. A. Serwa, E. Thinon, V. da Paola, E. J. Murray, F. Stuhmeier, M. A. A. Neil, E. W. Tate, C. Dunsby and P. M. W. French
    J. Vis. Exp. 119, (2017) e55119
  4. Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation
    D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineanu, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A.A. Neil, C. Dunsby and P. M.W. French,
    J. Biophotonics 6 (2012) 398-408
  5. Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)
    Anca Margineanu, Jia Jia Chan**, Douglas J. Kelly**, Sean C. Warren**, Delphine Flatters, Sunil Kumar, Matilda Katan, Christopher W. Dunsby, Paul M.W. French
    Sci Rep. 6 (2016) 28186
  6. Automated FLIM HCA of protein-protein interactions between endogenously-labelled kinetochore proteins in live budding yeast cells
    Wenjun Guo, Sunil Kumar, Frederik Görlitz, Edwin Garcia, Yuriy Alexandrov, Ian Munro, Douglas J. Kelly, Sean Warren, Peter Thorpe, Christopher Dunsby*, Paul French*
    SLAS Technology, 24 (2019) 308-320
  7. easySTORM: a robust, lower-cost approach to localisation and TIRF microscopy
    K. Kwakwa, A. Savell, T. Davies, I. Munro1 S. Parrinello, M.A. Purbhoo, C. Dunsby, M.A.A. Neil and P.M.W. French
    J. Biophotonics 9 (2016) 948–957
  8. Accelerating single molecule localisation microscopy through parallel processing on a high-performance computing cluster HPC STORM, I. Munro*, E. García1*, M. Yan*, S. Guldbrand, S. Kumar, K. Kwakwa, C. Dunsby, M.A.A. Neil# and P.M.W. French#, J Micros. 273 (2019) 148-160
  9. Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.
    Vincent Maioli, George Chennell, Hugh Sparks, Tobia Lana, Sunil Kumar, David Carling, Alessandro Sardini & Chris Dunsby
    Sci Rep 6, (2016) 37777