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

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.