Fluorescence Multiplexing with Spectral Imaging and Combinatorics.

Area of Science:

• Biophotonics
• Analytical Chemistry
• Molecular Imaging

Background:

• Fluorescence assays are widely used but challenging to multiplex due to overlapping emission spectra.
• Current multiplexing methods are limited by spectral overlap, hindering simultaneous detection of multiple targets.

Purpose of the Study:

• To introduce a novel fluorescence multiplexing approach called multiplexing using spectral imaging and combinatorics (MuSIC).
• To overcome the limitations of spectral overlap in fluorescence assays and increase multiplexing capacity.

Main Methods:

• MuSIC involves creating new, independent probes by covalently linking combinations of individual fluorophores.
• Utilizes spectral emission scanning and combinatorial mathematics to infer probe levels in complex mixtures.
• Leverages existing fluorophores and measurement tools for enhanced multiplexing.

Main Results:

• Theoretical analysis and simulations predict a 4-5 fold increase in fluorescence multiplexing capability.
• Experimental validation demonstrated robust demultiplexing of nine solution-based probes.
• Successful demultiplexing was achieved using a limited excitation wavelength window (380-480 nm).

Conclusions:

• MuSIC offers a powerful strategy to significantly enhance fluorescence multiplexing using readily available probes and technology.
• The approach is compatible with emerging technologies like white lasers and advanced spectral scanning systems.
• MuSIC provides a viable solution for increasing the complexity and information content of fluorescence-based assays.