Yasaman Shahrestani

Topic

Enhanced Fluorescence Excitation–Emission–Matrix Imaging Using Different Illumination Techniques

Department of Chemistry

Date & location

• Wednesday, October 28, 2026
• 10:00 A.M.
• Elliott Building, Room 230

Examining Committee

Supervisory Committee

• Dr. Peter Loock, Department of Chemistry, University of Victoria (Supervisor)
• Dr. Alexandre Brolo, Department of Chemistry, UVic (Member)

External Examiner

• Dr. Mohsen Akbari, Department of Mechanical Engineering, UVic

Chair of Oral Examination

• Dr. Tao Lu, Department of Electrical and Computer Engineering, UVic

Abstract

This thesis describes the design, construction, and characterization of a custom fluorescence microscopy platform for precise fluorescence detection and background noise minimization. Two separate illumination approaches are presented: total internal reflection fluorescence (TIRF) microscopy and fiber-coupled dark-field illumination. Both techniques share the same goal; selectively exciting fluorophores while suppressing unwanted background contributions from scattering. In both setups, a multispectral camera (MSC) serves as the detector, and a programmable multi-wavelength excitation source is used to acquire fluorescence excitation-emission matrices (EEMs) at each pixel of the image. This per-pixel EEM approach provides a two-dimensional spectral fingerprint at every spatial location, enabling the identification and separation of multiple fluorescent dyes even when their emission spectra overlap.

Conventional fluorescence microscopy uses one excitation wavelength and filters to detect emission, which makes it difficult to study complex samples that contain multiple fluorophores with overlapping spectra. This work addresses that limitation by recording full EEM spectra at every pixel of an image, generating a four-dimensional dataset (𝑥, 𝑦, 𝜆_ex, 𝜆_em. Multivariate analysis such as Parallel Factor Analysis extracts unique spectral fingerprint for each fluorophore and then assigns concentration values (“scores”) for each fluorophore to each of the pixels.

The programmable light source, controlled via a MATLAB™ interface, allows multiple excitation wavelengths to be delivered in rapid succession using Hadamard-multiplexed patterns that enhance signal-to-noise ratio without increasing total exposure time. Emission detection is performed by a multispectral camera with 8 spectral channels, providing the information needed to resolve overlapping fluorophores.

Two illumination experiments are reported. First, the Total Internal Reflection Fluorescence (TIRF) illumination setup was employed in which fiber-coupled laser diodes are clamped to the side of a microscope slide, delivering excitation light into this planar waveguide at angles that exceed the critical angle and generate an evanescent field confined to within ~100–200 nm of the glass surface. This geometry selectively excites fluorophores close to the microscope slide’s surface while strongly suppressing background fluorescence from the bulk sample and eliminating the inner-filter effect that arises in conventional top-illumination configurations.

We attempted to further enhance the signal from the TIRF illumination by adding reflective material (aluminum foil) to the edges of the microscope slide, and thereby enhance the evanescent wave excitation and fluorescence signal. Six microscope slides with different aluminum-foil coverage patterns were prepared for this purpose. A cavity-enhancement model was used to extract reflection coefficients. The results showed that aluminum-coated back edges achieved 85–98% reflectivity, compared to only 6–10% for uncoated glass, while the side and top/bottom surfaces already undergo total internal reflection and are minimally affected by the aluminum coating.

Second, a custom fiber-coupled dark-field illumination setup was designed and built. Twenty-five multimode fibers (NA ≈ 0.39) are arranged in a ring formation within a disk-shaped fiber holder. A second thin disk, attached just below the fiber holder, positions a ball lens at the tip of each fiber to partially collimate the diverging light emerging from the multimode fiber ends. Beneath both disks, a Fresnel lens mounted in a separate adjustable carrier focuses the combined output of all 25 fibers to a sharp spot on the microscope slide’s surface. This optical configuration delivers oblique, annular illumination to the sample, reproducing the dark-field condition without the need for a traditional condenser. A linear fiber bundle fabricated in-house transfers the spectral output of the digital micromirror array (DMA)-based programmable light source into the annular ring geometry, providing digital spectral control of the illumination.

Both platforms successfully achieve precise fluorescence detection with minimized background noise, and PARAFAC analysis demonstrates effective separation of two and three fluorescent dyes with overlapping emission spectra.

MATLAB™ software integrates the programmable light source, the camera, and Hadamard multiplexing to acquire full EEM images. Parallel factor analysis (PARAFAC) is applied to decompose the resulting EEM data cubes into individual fluorophore contributions. Experiments on samples containing two and three fluorescent dyes demonstrate successful spectral separation of species whose emission spectra substantially overlap.