Size and fluorescence calibrated imaging flow cytometry: From arbitrary to standard units

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Cell Biology

Background:

• Imaging flow cytometry (IFCM) enables high-throughput detection, sizing, and phenotyping of extracellular vesicles (EVs) in complex biological fluids without prior isolation.
• Current IFCM data interpretation is hindered by arbitrary units in side scatter (SSC) signals, complicating inter-instrument and inter-institute comparisons.
• Standardized calibration of fluorescence signals is achievable, but SSC signal calibration remains a significant challenge for IFCM.

Purpose of the Study:

• To develop and present an approach for relating SSC signals to particle size in IFCM.
• To conduct a comparability study of three different IFCM instruments using a standardized plasma EV test sample (PEVTES).
• To improve the standardization and reproducibility of EV measurements using IFCM.

Main Methods:

• Developed a method to calibrate IFCM SSC signals to particle size using polystyrene (PS) and hollow organosilica beads (HOBs) with a 405 nm laser.
• Applied Mie theory to correlate scatter signals with particle size, achieving a coefficient of determination >0.99.
• Performed fluorescence calibration using MESF beads and size/fluorescence calibration across three IFCMs in two laboratories with stained PEVTES.

Main Results:

• Successfully related IFCM SSC signals to particle size, with 81 nm PS beads discernible from background.
• Demonstrated the detection of 216 nm HOBs, confirming SSC sensitivity for EV detection, with a lower size limit of ~100 nm.
• Size and fluorescence calibration reduced the coefficient of variation in EV concentration comparisons from 33% to 21%, significantly improving inter-instrument comparability.

Conclusions:

• Presented the first scatter calibration for IFCM using a 405 nm laser, yielding a scatter-to-diameter relationship comparable to sensitive commercial flow cytometers.
• The developed calibration method enhances the reliability of IFCM for EV research by providing robust standardization and reproducibility.
• This standardization is crucial for understanding the biological significance of EVs and facilitating multi-center research.