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Updated: Jul 19, 2025

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Published on: October 28, 2018

Fluorescence Excitation-Emission-Matrix Imaging.

Oren Katz¹, Travis Ferguson², Emma Abbey¹

¹Department of Chemistry, University of Victoria, Victoria, British Columbia V8W 5C2, Canada.

Analytical Chemistry

|August 17, 2023

Summary

This summary is machine-generated.

This study introduces a fast 4D fluorescence imaging system capable of capturing excitation-emission-matrix spectra at each pixel. The novel system enables rapid data acquisition for complex fluorescent sample analysis.

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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.