Resolution of complex fluorescence spectra recorded on single unpigmented living cells using a computerised method

Area of Science:

• Analytical Chemistry
• Biophysics
• Spectroscopy

Background:

• Analyzing complex fluorescence spectra is challenging, particularly with low signal-to-noise ratios.
• Accurate identification and quantification of fluorescent compounds are essential in various scientific fields.

Purpose of the Study:

• To develop a computational method for resolving low light level complex fluorescence spectra into their constituent components.
• To enable accurate identification and quantification of fluorescent compounds in challenging spectral environments.

Main Methods:

• A method based on the linear combination of characteristic fluorescence spectra and weighting functions was employed.
• Integration of fluorescence intensities across the entire spectra to generate a system of equations (n equations with N unknowns).
• Validation using complex intracellular fluorescence spectra from single living cells treated with benzo(a)pyrene.

Main Results:

• The method successfully resolved complex fluorescence spectra with low signal-to-noise ratios.
• Demonstrated reliability and sensitivity in identifying and quantifying fluorescent components.
• Successfully identified two, four, or five distinct fluorescent components in cellular samples, depending on cell type and treatment.

Conclusions:

• The proposed computational method effectively deconvolutes complex fluorescence spectra, even at low signal levels.
• This technique offers a sensitive and reliable approach for analyzing intricate fluorescence data in biological and chemical systems.
• The method has practical applications in studying intracellular fluorescence and compound analysis in living cells.