Digital holographic microscopy for rapid bacteria segmentation and counting in microfluidic cartridges: basic...

Area of Science:

• Microscopy and Imaging Technologies
• Biotechnology and Bioengineering
• Microfluidics and Lab-on-a-Chip Systems

Background:

• Digital holographic microscopy (DHM) is valuable for particle segmentation and monitoring bacterial growth in microfluidic devices.
• Optimizing DHM-based assays for applications like antimicrobial susceptibility testing requires understanding influencing factors.
• Limited comprehensive studies exist on how instrument, consumable, and sample parameters affect DHM phase image quality.

Purpose of the Study:

• To systematically investigate the impact of microfluidic parameters on the signal-to-noise ratio (SNR) of DHM-reconstructed phase images.
• To identify key factors affecting image quality for improved DHM assay development.
• To establish a predictive framework for DHM system performance.

Main Methods:

• Constructed an off-axis digital holographic microscope with a robust numerical processing pipeline.
• Analyzed particle counting and SNR using silica microspheres and bacteria (Staphylococcus warneri, Escherichia coli) in microfluidic chips of varying heights.
• Quantified system performance across different suspension densities and particle concentrations.

Main Results:

• The DHM system accurately reflected particle dilution steps over 2-3 orders of magnitude.
• Microfluidic chamber height and suspension density were identified as primary contributors to background noise, impacting SNR.
• Particles themselves had a negligible effect on SNR, allowing for the derivation of an analytical SNR prediction function.

Conclusions:

• A DHM system capable of counting suspended particles across wide concentration ranges and microfluidic heights was successfully developed.
• An analytical framework was derived to predict and optimize DHM system performance based on key parameters.
• This work provides a foundation for developing more robust and reliable DHM-based microfluidic assays.