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Establishing a reference focal plane using beads for trypan-blue-based viability measurements.

Adele Peskin1, Steven P Lund2, Laura Pierce2

  • 1National Institute of Standards and Technology, Boulder, Colorado.

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Summary

Control beads ensure consistent image quality for trypan blue cell viability assays. This method improves accuracy and repeatability in automated cell analysis, overcoming challenges with cell-based autofocusing.

Keywords:
autofocuscell viabilityimage processing

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Area of Science:

  • Biomedical Imaging
  • Cell Biology
  • Image Analysis

Background:

  • Trypan blue dye exclusion assays for cell viability are sensitive to image quality and consistency.
  • Automated image analysis requires reliable image acquisition at a consistent focal plane and adequate signal-to-noise ratio.
  • Existing autofocusing methods struggle with cell sample heterogeneity, impacting accuracy and repeatability.

Purpose of the Study:

  • To develop a robust method for ensuring consistent image acquisition in trypan blue assays.
  • To improve the accuracy and repeatability of automated cell viability measurements.
  • To introduce a novel autofocusing strategy using control beads.

Main Methods:

  • Incorporation of control beads into cell samples for imaging.
  • Utilizing bead image features with stable profiles across varying focal and exposure levels.
  • Developing a predictive model based on bead image quality features to establish a reference focal plane.
  • Implementing an open-source Graphical User Interface (BB-FAIT) for semi-automated analysis.

Main Results:

  • A bead-based autofocusing method was developed, achieving over 95% accuracy.
  • The method reproducibly returns to a reference focal plane using minimal beads (less than 3 per image).
  • The approach demonstrates stability across a wide range of focal values and exposure levels.
  • The Bead Benchmarking-Focus And Intensity Tool (BB-FAIT) was created to implement the method.

Conclusions:

  • Control beads offer a reliable solution for maintaining consistent image quality in trypan blue assays.
  • This bead-based approach enhances the accuracy and repeatability of automated cell viability measurements.
  • The BB-FAIT tool provides a practical implementation for semi-automated cell analyzers.