APPLICATION OF METAL-ENHANCED FLUORESCENCE FOR IMAGING OF BIOLOGICAL SYSTEMS

Candidate: Sumaiya Soha
Supervisors: Dr. Costin Antonescu; Dr. Roberto Botelho, Dr. Stefania Impellizzeri     

Abstract:

Fluorescence microscopy is a cornerstone of modern cell biology, yet its performance is constrained by low signal intensity, photobleaching, and phototoxicity. Metal-enhanced fluorescence, based on near-field coupling between fluorophores and metallic nanoparticles, offers a strategy to improve brightness and stability without increasing excitation intensity.
In this thesis I investigated silver nanoparticles (AgNPs) as platforms for controlled fluorescence enhancement and evaluated their potential applications in live-cell imaging. Three AgNP morphologies, small spherical (~3 nm), large spherical (~40 nm), and triangular (~80 nm), were synthesized and characterized for optical and plasmonic properties. Among these, small spherical AgNPs produced the most consistent enhancement of BODIPY fluorescence due to favorable spectral overlap. Imaging with total internal reflection and spinning-disk confocal microscopy confirmed that enhancement was spatially confined near the nanoparticle surface.
To translate this enhancement to biologically relevant systems, I introduced optimized small AgNPs were into macrophage cells. The nanoparticles localized within lysosomal compartments and amplified the fluorescence of green-emitting probes such as Alexa Fluor 488–dextran and DQ–BSA, without inducing cytotoxicity or disrupting lysosomal integrity. Comparative assays of lysosomal health investigation, such as pH, degradative capacity, and autophagy, amongst many more, confirmed that AgNP exposure at optimized concentrations maintained cellular homeostasis while improving fluorescence signal intensity.
In subsequent experiments I investigated strategies to coat coverslips with AgNP for single-particle tracking of cell surface receptors. I employed three different techniques to anchor the AgNP to the coverslip surface, namely: APTES functionalization, fibronectin overlays, and gelatin matrices. These approaches did not yield measurable fluorescence enhancement, highlighting the sensitivity of MEF to nanoparticle distribution, fluorophore–metal spacing, and surface immobilization parameters.
Finally, silver nanoparticle–dextran–FITC conjugates were synthesized to integrate plasmonic and fluorophore components within a single biocompatible construct. Despite nominally identical synthesis protocols, these conjugates exhibited substantial batch-to-batch variability and limited reproducibility, precluding definitive conclusions regarding their MEF performance.
Collectively, this work demonstrates both the potential and the practical constraints of AgNP-mediated MEF in biological imaging. The findings underscore the importance of precise control over nanoparticle morphology, spatial distribution, and surface functionalization to achieve reproducible fluorescence enhancement in complex biological environments.