Autofluorescence prediction model for fluorescence unmixing and age determination

Background: Flow cytometry is a powerful tool for identifying and quantifying various cell markers, such as viability, vitality, and individual cell age, at single-cell stages. However, cell autofluorescence and marker fluorophore signals overlap at low fluorescence intensities. Thus, these signals must be unmixed before determining the age fraction. Methods and Results: A comparison was made between principal component regression (PCR) and random forest (RF) to predict autofluorescence signals of Saccharomyces pastorianus var. carlsbergensis in a flow cytometer. RF provided better prediction results than the PCR and was therefore determined to be better suited for unmixing signals. In the subsequent application for unmixing the autofluorescence signal from the marker fluorophore signal, the Gaussian mixture analysis based on RF was in better agreement with the microscopy-determined replicative age distribution than the PCR-based method. Conclusion: The proposed approach of single-laser spectral unmixing and subsequent Gaussian mixture analysis showed that the microscopy data was consistent with the unmixed fluorescence spectra. The demonstrated approach enables fast and reliable unmixing of flow cytometric spectral data using a single-laser spectral unmixing method. This analysis method enables age determination of cells in industrial processes. This age determination allows for quantifying the yeast cell's age fractions, providing a detailed view of age-related changes. Additionally, the bud scar labeling technique can be used to determine age-related changes in Pichia pastoris yeast for biotechnological applications or recombinant protein expression.