Inter-Instrument Quantification of Fluorescence Read-out Signal Using DNA Origami Calibrator Beads

Comparing experimental results across different instruments and laboratories is challenging due to variations in instrument sensitivity and resolution, particularly for analyzing nano-sized and dimly fluorescing particles like viruses and extracellular vesicles. To address this challenge, we conducted a study using fluorescent DNA calibrator beads to test, calibrate, and compare the sensitivity of five different flow cytometers. Our approach involves DNA beads produced through a bottom-up DNA origami method. These beads contain a defined number of fluorophores (0-220) and exhibit no autofluorescence. Fluorophores are precisely conjugated onto the beads in a controlled array, minimizing dye interactions. This allows triggering on one fluorophore and calibration based on another, facilitating precise and accurate calibration in absolute fluorophore numbers. We tested five flow cytometers--FACSAria III, NovoCyte 3000YBG, NovoCyte Quanteon 4500, LSRFortessa, and CytoFlex S--using Cy5 and FAM DNA calibrator beads. We investigated various instrument parameters, including count rate, trigger and calibration gain, gain amplification linearity, and fluorescence trigger sensitivity. Using fluorescent triggering, we demonstrated the capability to detect dimly fluorescent particles, enabling instrument calibration and fluorescence sensitivity comparison using DNA calibrators. In conclusion, our study offers a robust method for addressing instrument fluorescence channel resolution in absolute fluorophore numbers in the analysis of nano-sized and dimly fluorescent particles.