Quantifying Fluorochrome Detection Thresholds

The detection threshold of fluorochromes using the imaging system and chambers was determined by using DNA origami (GATTAquant, Graefeling, Germany). We obtained 17- and 30-fluorochrome standards (GATTA-Brightness RGB17 and GATTA-Brightness RGB30) to construct an RFU-per-fluorochrome instrument response function for the conditions used in the study (optics, filter configurations, image exposure times). The probe set corresponds to the dyes used for MASEV profiling, including AF488 and AF555 (exact matches), as well as ATTO647N, which has excellent quantitative spectral overlap with AF647, requiring minimal correction for normalized brightness as a function of microscope optics, extinction coefficient, and quantum yield. The standards containing either 17 ± 3 or 30 ± 5 fluorochromes were affixed to a #1 thickness (0.12–0.17 mm) coverglass of equivalent thickness to the MASEV flow cell. Samples were imaged within the same day using identical settings as during a typical MASEV experiment. We used ImageJ to measure the signal intensity above the local background for hundreds of GATTAquant tiles in each fluorophore channel across three fields of view. The mean intensity of the tile population was then estimated by fitting a Gaussian curve to a histogram of these values in MATLAB; error bars are calculated/plotted as the standard deviation of the mean intensity from three distinct fields of view. Signal intensity per fluorochrome was calculated by performing a linear fit between results for 17- and 30-fluorochrome constructs, enabling extrapolation of the detection threshold.