Improving contrast in biological imaging: time-resolved microscopy and protein-targeted dendrimers
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This dissertation presents the results of two projects that shared the common goal of improving contrast in cellular imaging; one, instrumental and the other, probe-based. The instrumental approach is focused on the development, optimization and characterization of a Time resolved luminescence (TRL) microscope for lanthanide imaging that employs a light-emitting diode (LED, λem = 365 nm) for pulsed epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection. Instrument performance was evaluated in terms of short-lifetime fluorescence background rejection, photon collection efficiency, image contrast, and signal-to-noise ratio (SNR). The performance analyses show that TRL microscopy is sufficiently sensitive and precise to allow high-resolution, quantitative imaging of lanthanide luminescence in living cells under physiologically relevant experimental conditions. The probe-based approach entails the synthesis and characterization of monovalent, ligand-functionalized, dendrimer-encapsulated, gold nanoparticles that were designed to label specific proteins for correlative luminescence and electron microscopy (CLEM). Specifically, the synthetic strategies for functionalizing cystamine core G4 and G5 PAMAM dendrimers monovalently with a protein targeting ligand (TMP and biotin) and with multiple fluorophores as well as templating gold nanoparticle growth are presented.