Comparison of Near-Infrared Fluorophore Pairs for Lifetime-Based FRET Imaging

Abstract

Near-Infrared fluorophore pairs were comparatively studied to perform quantitative lifetime-based FRET imaging. The best pair, as defined as the brightest and leading to accurate estimation of FRET component, is suitable for pre-clinical application. OCIS codes: (170.3880) Medical and biological imaging; (170.6920) Time-resolved imaging IntroductionTargeted drug delivery has been studied for decades to improve therapeutic index of drugs, particularly cancer drugs. Transferrin (Tfn) is one of the most common molecule used as a receptor-mediated drug and gene delivery vehicle for diagnosis treatment of medical conditions [1]. Tfn can be used for deliver drugs for treatment of cancers as malignant cells express more transferrin receptor (TfR) [2]. The homo-dimerization of transferrin receptors during endocytosis provides a few nanometers distance between two transferrin molecules. At this distance, Förster resonance energy transfer (FRET) occurs. Hence, FRET imaging may be used to sense and quantify TfR-Tfn uptake into tumor cells.FRET is a phenomenon where two fluorescent molecules, a donor and an acceptor, are close to each other within 2-10 nanometers distance. During FRET state, the donor transfers the photon energy to the acceptor; the donor fluorescent intensity is reduced. The lifetime of the donor, another characteristic that can be observed, is also significantly reduced. The efficiency of FRET largely depends on the distance between donor and acceptor. Specifically, the efficiency is inversely proportional to the sixth power of the distance. The Förster distance (R0) is the distance where the efficiency is 50%. The larger R0 provides more chance of FRET phenomenon. The donor emission spectrum must overlap the acceptor excitation spectrum for a certain degree to provide sufficient energy transfer. The spectral overlap is the characteristic of the donor-acceptor pair and it affects R0 as well. Hence, FRET imaging performances are highly dependent on the fluorophore pair employed.Near-infrared (NIR) fluorophores are attractive for imaging as they are less affected by the interaction of photons with biological specimens. Most components of cells and tissue absorb the photons at visible spectrum. Proteins are the main component absorbing the light at UV to visible spectrum. Absorption of photons in vivo is primarily influenced by hemoglobin, melanin, fat and water. The absorption coefficients of these components are minimal in the NIR spectrum [3]. Furthermore, in vivo fluorescence imaging is affected by autofluoresence of endogenous fluorophores from extracellular matrix proteins. Autofluorescence mainly occurs from the UV to the visible spectrum and is relatively weak in the NIR. Because of these biological interactions, near-infrared fluorophores are the most suitable for in vitro and in vivo biological imaging applications. However, these fluorophores usually have shorter lifetime than visible fluorophores. These shorter lifetimes are challenging to resolve in time-resolved imaging as the lifetime may be cl...