Fluorescent Lifetime Imaging of Silica Nanoparticle Encapsulated Fluorescent Probes for Molecular Targeting
Fluorescence microscopy is a widely used technique that is capable of multiplexed detection of fluorescent probes that emit different colors. However, spectral fluorescence is limited by the number of probes that can be used at the same time due to limited spectral bandwidth. The focus of this thesis is the development of fluorescence lifetime as a second orthogonal property for multiplexing probes. Fluorescence lifetime is the amount of time in which a fluorescent material remains in its excited state before returning back to its ground state. The first part of thesis will focus on the development of a new fluorescence lifetime probe, in which different fluorescent dyes will be encapsulated into a silica nanoparticle at varying loading densities. Using Fluorescence lifetime imaging microscopy (FLIM) and the phasor analysis approach, lifetimes can readily be assessed and compared. We found that each dye exhibited a unique lifetime, which could be modulated to yield trajectory of positions as loading volume increased. Specifically, loading more dye decreased fluorescence emission efficiency as well as the lifetime location on the phasor diagram due to a dye-dye self-quenching mechanism. The second part of the thesis delves into other ways to tune lifetime of silica encapsulated dye in order to generate additional unique positions on the phasor plot. This was accomplished by doping in a quencher dye capable of absorbing light to accelerate lifetime or mixing two different dyes together to create a mixture which has combined lifetime between the individual probes. The different methods to modulate lifetime of individual fluorescent probes provided at least 20 unique lifetime positions on phasor plot which can be utilized to increase the number of detection channels.