Supported by CCR Office of Science and Technology Resources (OSTR)

Project Development

Image Analysis

A typical HCI screening project at HiTIF can be subdivided in 5 phases: Pre-Development, Subsidy Proposal, Assay Development, Primary Screening, and Secondary Screening.


The investigator contacts the Facility Head to inquire about the current availability of instrumentation time and manpower for the project to be run at the HiTIF. At this point the discussion will also involve an initial evaluation of the feasibility of the proposed project considering both biological and technological considerations. Typical questions to consider are: can the proposed imaging assay be carried out on tissue culture surfaces? Which is the most appropriate combination of fluorophores for the assay? Can the phenotype of interest be detected by confocal microscopy, and is it suitable for High-Throughput Imaging (HTI)? In case of screening projects, can the chosen cell line be efficiently transfected with oligo RNA? Are there appropriate biological and technical positive and negative controls for the assay? Is the assay window between positive and negative controls known? The availability of positive and negative controls and preliminary results in a low-throughput format, preferably including fluorescence microscopy data, will be essential to assess the feasibility of the project in a miniaturized, high-throughput format. During these initial discussions the HiTIF will provide guidance on how to design the HCI assay in exam.

Stars Subsidy for HiTIF Projects

To help offset project-based costs, HiTIF’s CCR Collaborators can apply for subsidies using the Supplemental Technology Award Review System (STARS), offered through the CCR Office of Science and Technology Resources (OSTR). Projects costing up to $20K are eligible for 50% subsidy and reviewed by OSTR for technical merit. Larger projects requiring more than $10K in subsidy will be directed to a more detailed STARS form. These STARS Applications require an experimental plan as well as scientific and budgetary justification and are reviewed by members of the CCR Science Board (CSB) for technical soundness, scientific merit, and potential impact. As with all STARS applications, technology subsidies should be requested before work is initiated.

For more information and/or questions about the STARS subsidies for HiTIF projects please contact Mariam Malik or Gianluca Pegoraro.

Assay Development

The HiTIF will train, advise and help in the use and troubleshooting of liquid handling and HTI instrumentation. Optimal cell growth conditions and confluence for the experimental conditions to be used during the screening phase will be optimized first in 384- well format, and if needed in 96-well format, in case the former format is not compatible with the assay. Image acquisition, siRNA or sgRNA transfection conditions will then be optimized. Based on the biological question addressed by the assay, HiTIF personnel will design custom image analysis pipelines to extract relevant quantitative phenotypic cellular features. Quality control descriptors (Z-score, S/N ratio, assay window) based on relevant positive and negative controls will be used to guide the optimization process. Multiple test plates with enough positive and negative controls will be run on independent days to determine the inter-plate and inter-day assay variability. Finally, a pilot screen with a library of ~ 300 reagents will be run to test, and possibly troubleshot, the cell seeding, cell culture reagent dispensing, automated image analysis and secondary data analysis workflow in a realistic screening scenario.

Primary Screening

Depending on the size of the libraries of reagents to be screened, primary screens will be run in multiple replicates on independent days. QC Filtering and hit-selection criteria based on biological potency and specificity will be determined by HiTIF personnel in concert with the investigators.

Secondary Screening

Based on the hit-list from the primary screen and on logistical considerations, several target genes or compounds will be selected for secondary screen to rule out off-target effects.