Fluorescence excitation emission matrix spectral imaging microscopy
Date
2026
Authors
Abbey, Emma X.
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Abstract
With this dissertation the capabilities of fluorescence microscopic imaging are greatly extended to allow for fast chemical fingerprinting in each pixel of an image. Spatial fluorescence imaging microscopy using multispectral and hyperspectral cameras with multiplexed excitation sources has been thus far not described in the literature. Three systems to acquire fluorescence excitation-emission matrix (F-EEM) spectra are demonstrated in this work, using Hadamard-multiplexed programmable excitation light sources. Computational approaches to bypass the limitations of multi- and hyperspectral camera hardware are implemented to increase the time resolution and emission spectral resolution.
The first chapter contains background and instrumentation used in F-EEM spectroscopy and multispectral imaging. Microscopy illumination methods are described as they relate to the methods used in this work, and a brief overview of parameters used for creating spatially distinct dye samples under a microscope are described. Raman spectroscopy and how it relates to multiplexed fluorescence spectroscopy is described and is followed by an overview of multivariate analysis methods used in F-EEM analysis.
F-EEM imaging requires a fully programmable excitation light source and an imaging detector. The work shown here develops and uses Hadamard-multiplexed excitation sources based on instruments built by our group in the past. The basis for multiplexed spectroscopy and the theory of the Hadamard transform are described in Chapter 2. The optical design and software integration of two programmable light sources—one white-light source based on a digital micromirror array and one using an array of discrete-wavelength laser diodes—are described in Chapter 3.
Acquisition of F-EEM images is done through one of two cameras—a snapshot multispectral camera using an eight-channel colour filter array, or an interferogram-based hyperspectral camera providing up to 141 spectral channels over a wide wavelength range. These commercial cameras and their integration into our systems are described in Chapter 4.
The processing and analysis for F EEM images acquired using Hadamard-modulated light sources and some unique challenges to these datasets are detailed in Chapter 5.
Four fluorescent components are spatially and spectrally separated in a macroscopic application of the multispectral camera and Hadamard-modulated white-light excitation source, described in Chapter 6. Here, an image of capillaries containing fluorophores and mixtures thereof is analyzed using multivariate analysis to demonstrate the spatial and spectral separation of four fluorescent components in an F EEM image.
The excitation light source is then modified for use in numerous microscopy illumination methods. F-EEM microscopic imaging using three distinct combinations of excitation source and imaging detectors is demonstrated in Chapter 7 using combinations of dye emulsions. Multivariate analysis of F EEM images taken with an 8-channel multispectral camera and using seven laser diodes can find ten fluorophores in a microscopy image when those spectral signatures are known. Without prior knowledge of the fluorophores, at least four fluorescent dyes in a microscope image are separated using multivariate analysis of an F EEM photomicrograph taken using the same multispectral camera and a white-light programmable light source.
The emission spectra of F EEM images acquired with the eight-channel multispectral camera are spectrally upscaled in Chapter 8 to increase the spectral resolution without hardware modifications. The upscaled spectra are demonstrated to provide a superior method of fluorophore identification and separation in an F EEM image—more fluorophores can be identified in F EEM images using upscaled spectra than raw spectra. A new computational method for increasing the time resolution of F EEM images acquired using a Hadamard-modulated excitation light source is also demonstrated. These two computational techniques allow us to obtain chemical identifiers and intensities for each of the 65,536 pixels per frame, when these frames are obtained at a rate between 3-10 per second.
Numerous avenues for experiments using the programmable light sources with the multispectral and hyperspectral cameras are described in Chapter 9, along with future work on a multi-wavelength multiplexed Raman and fluorescence spectroscopy experiment.
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Keywords
Fluorescence Imaging, Microscopy, Hadamard Multiplexing