Multicolor whole-cell bacterial sensing using a synchronous fluorescence spectroscopy-based approach.

Area of Science:

• Microbiology
• Biotechnology
• Spectroscopy

Background:

• Fluorescent proteins (FPs) are valuable for bacterial reporter gene studies.
• Spectral overlap between multiple FPs limits simultaneous detection.
• Existing methods struggle with precise separation of complex fluorescent signals.

Purpose of the Study:

• To develop a novel method for detecting and separating overlapping fluorescent signals from multiple bioreporter strains.
• To overcome the limitations of spectral bleed-through in multicolor bacterial studies.
• To enhance the selectivity and sensitivity of fluorescent signal analysis.

Main Methods:

• Coupling synchronous fluorescent spectroscopy (SFS) with Canonical Polyadic (CP) decomposition.
• Utilizing CP decomposition (also known as Candecomp/Parafac) for blind spectral decomposition of 3D data arrays.
• Applying the method to mixtures of labeled E. coli strains and monitoring gene expression in P. aeruginosa.

Main Results:

• The SFS/CP approach successfully extracted contributions from at least four overlapping FPs in E. coli.
• The method demonstrated reduced spectral overlap and improved selectivity.
• Simultaneous monitoring of three iron-responsive genes and pyoverdine production in P. aeruginosa was achieved.
• The technique was implemented in a microplate format for convenience.

Conclusions:

• The SFS/CP method offers a powerful tool for multiplex fluorescent reporter studies in bacterial systems.
• This approach enhances the ability to study complex biological processes with multiple variables.
• The developed technique improves the resolution and accuracy of multicolor fluorescent analyses in microbiology.