Label-free viability assay using in-line holographic video microscopy.

Area of Science:

• Biotechnology
• Microscopy
• Cell Biology

Background:

• Cell viability assessment is crucial in biologics manufacturing.
• Traditional methods often rely on unreliable dyes or time-consuming manual cell counting.
• Label-free, automated techniques are needed for efficient cell viability analysis.

Purpose of the Study:

• To present Total Holographic Characterization (THC) as an efficient, automated, and label-free method for cell viability identification.
• To evaluate THC's accuracy in distinguishing live and dead yeast cells.
• To compare THC performance against traditional trypan blue staining and manual counting.

Main Methods:

• Utilized an in-line holographic microscope with a 40x objective lens and plane wave illumination.
• Applied Lorenz-Mie theory to analyze light scattering for single-particle characterization.
• Measured Saccharomyces cerevisiae yeast viability in the presence of varying isopropanol concentrations over time.
• Performed all measurements in the native sample environment without dilution or labels.

Main Results:

• THC accurately identified and distinguished between living and dead yeast cells.
• Cell viability was effectively determined by measuring the refractive index of individual cells.
• THC results correlated well with traditional trypan blue staining and manual cell counting.

Conclusions:

• Total Holographic Characterization (THC) is a viable, label-free technology for accurate cell viability assessment.
• THC offers an efficient and automated alternative to conventional cell viability testing methods.
• The refractive index of individual cells, as measured by THC, is a reliable indicator of cell viability.