Unmixing of spectrally similar quantum dots using filter selection

This paper explores the possibilities for quantitative analysis of multiplexed Quantum Dot Immunohistochemical (QDIHC) staining using a 10-slot fluorescence microscope filter wheel. QDs are an ideal fluorophore for staining biomarkers due to their unique properties, including greater photostability and relatively narrower emission bandwidths compared to organic dyes. We imaged a slide containing 5 pure QD spots and 6 QD mixtures with a customized scanning fluorescence microscope. The QD mixtures contained either two or three QDs in equal amounts. Ten filter cubes were used to gather emission signal and then fast non-negative least squares regression (FNNLS) performed the unmixing process by assigning components of the 10-channel raw data to one of the five QDs used. the average error in the unmixing process was measured to be 7.60% when all filters were used and 7.80% when only 6 filters were used.