Fluorescence Excitation-Emission-Matrix Imaging.

Area of Science:

• Spectroscopy
• Microscopy
• Analytical Chemistry

Background:

• Traditional fluorescence imaging often faces limitations in spectral resolution and acquisition speed.
• Characterizing complex fluorescent samples requires detailed spectral information across spatial dimensions.

Purpose of the Study:

• To develop and validate a novel 4-dimensional (4D) fluorescence imaging system.
• To enhance data acquisition rates for excitation-emission-matrix (EEM) spectroscopy at each pixel.
• To demonstrate the system's capability in analyzing complex mixtures and dynamic fluorescence changes.

Main Methods:

• Implementation of a 4D fluorescence imaging system with 65,536 pixels, each containing an excitation-emission-matrix spectrum (31 excitation, 8 emission wavelengths).
• Utilized Hadamard-transform multiplexing with a 31-channel programmable light source for rapid data acquisition (8 seconds per image).
• Employed multivariate analysis, specifically parallel factor analysis, for data interpretation.

Main Results:

• Successfully acquired 4D EEM spectral data across spatial dimensions (x, y, excitation wavelength, emission wavelength).
• Demonstrated system performance with imaging of dye solutions in capillaries and layered solutions under varying temperatures.
• Generated spatial distribution images of fluorophores and their relative intensity changes over time.

Conclusions:

• The developed 4D fluorescence imaging system significantly increases data acquisition speed for EEM spectroscopy.
• The system effectively characterizes complex fluorescent mixtures and dynamic spectral changes.
• Multivariate analysis provides valuable insights into fluorophore distribution and behavior.