Förster resonance energy transfer (FRET)-based assemblies currently comprise a significant portion of intracellularly based sensors. Although extremely useful, the fluorescent protein pairs typically utilized in such sensors are still plagued by many photophysical issues including significant direct acceptor excitation, small changes in FRET efficiency, and limited photostability. Luminescent semiconductor nanocrystals or quantum dots (QDs) are characterized by many unique optical properties including size-tunable photoluminescence, broad excitation profiles coupled to narrow emission profiles, and resistance to photobleaching, which can cumulatively overcome many of the issues associated with use of fluorescent protein FRET donors. Utilizing QDs for intracellular FRET-based sensing still requires significant development in many areas including materials optimization, bioconjugation, cellular delivery and assay design and implementation. We are currently developing several QD-based FRET sensors for various intracellular applications. These include sensors targeting intracellular proteolytic activity along with those based on theranostic nanodevices for monitoring drug release. The protease sensor is based on a unique design where an intracellularly expressed fluorescent acceptor protein substrate assembles onto a QD donor following microinjection, forming an active complex that can be monitored in live cells over time. In the theranostic configuration, the QD is conjugated to a carrier protein-drug analogue complex to visualize real-time intracellular release of the drug from its carrier in response to an external stimulus. The focus of this talk will be on the design, properties, photophysical characterization and cellular application of these sensor constructs.
Efficient and specific delivery of particles and drugs intracellularly is a critical area of research which can allow for further understanding of cellular processes and increased efficacy of therapeutic treatments. Visualizing these delivery mechanisms occurs through use of florescent molecules, some of which are bulky and can change the processes being studied. Alternatively, semiconductor nanocrystals called quantum dots (QDs) are nanoscale particles that provide a scaffold for drugs or targeting moieties while providing superior optical properties that include size-tunable photoluminescence and resistance to photobleaching. Utilizing these platforms, delivery methods and intracellular assembly can be studied. Potential delivery mechanisms include either specified delivery through attenuation to targeting ligands or systemic delivery through microinjection or electroporation. Once within the cytosol, the QDs can assemble to express proteins, a process that can be visualized through the use of FRET. The effectiveness of various delivery methods and QD surface chemistry has been analyzed and is described here.
The controlled delivery of nanomaterials to the plasma membrane is critical for the development of nanoscale probes that can eventually enable cellular imaging and analysis of membrane processes. Chief among the requisite criteria are delivery/targeting modalities that result in the long-term residence (e.g., days) of the nanoparticles on the plasma membrane while simultaneously not interfering with regular cellular physiology and homeostasis. Our laboratory has developed a suite of peptidyl motifs that target semiconductor nanocrystals (quantum dots (QDs) to the plasma membrane where they remain resident for up to three days. Notably, only small a percentage of the QDs are endocytosed over this time course and cellular viability is maintained. This talk will highlight the utility of these peptide-QD constructs for cellular imaging and analysis.
The simultaneous visualization, identification and targeting of neurons during patch clamp-mediated
electrophysiological recordings is a basic technique in neuroscience, yet it is often complicated by the
inability to visualize the pipette tip, particularly in deep brain tissue. Here we demonstrate a novel
approach in which fluorescent quantum dot probes are used to coat pipettes prior to their use. The strong
two-photon absorption cross sections of the quantum dots afford robust contrast at significantly deeper
penetration depths than current methods allow. We demonstrate the utility of this technique in multiple
recording formats both in vitro and in vivo where imaging of the pipettes is achieved at remarkable depths
(up to 800 microns). Notably, minimal perturbation of cellular physiology is observed over the hours-long
time course of neuronal recordings. We discuss our results within the context of the role that quantum dot
nanoprobes may play in understanding neuronal cell physiology.
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