Quantum dots are fluorescent nanoparticles with unique optical properties that have the potential to revolution- ize cellular microscopy and bioimaging. These fluorophores have emerged simultaneously with an ongoing revolution in fluorescence microscopy called 'super-resolution'imaging whereby molecules, cells, and tissues can now be optically imaged at resolutions approaching that of individual proteins, with molecular specificity. For example, cellular structures as small as the neuronal synaptic cleft (~30 nm) can now be resolved dynami- cally in live cells, which previously required static, fixed cell imaging through electron microscopy. Quantum dots have a unique niche here: unlike fluorescent proteins and organic dyes, their emission intensity is so bright that individual molecules can be readily observed, and their emission does not photobleach. Theoretical- ly, these particles can be used to image and track single proteins involved in neuron-neuron communication within the tiny synapse to reveal the heterogeneous, dynamically changing processes involved in neural signal- ing and how intercellular communication is disrupted in diseases such as Alzheimer's, Parkinson's and in strokes. However the implementation of quantum dots for advanced microscopy of live cells has been hindered by their bulky size (~20 nm) and non-specific labeling, which greatly restricts specific access to the crowded neuronal synapse. Our preliminary data show that small-sized quantum dots (~7 nm) have much greater ac- cess to the neuronal synapse, substantially greater than previous bulky dots. This allows tracking of individual neurotransmitter receptors for long durations (~1 hour). However the production of particles in this size range remains a major problem primarily due to their coating, which serves to stabilize the particles colloidally in solu- tion. There is simply a fundamental tradeoff between size, stability, and nonspecificity that has yet to be over- come with current coatings. Here we propose to generate a new series of coatings based on our previous work and the best literature results to date. These polymers and ligands are ultra-compact and stabilized by strong multidentate binding;quantum dots coated with these new materials will be tested for optical stability, colloidal stability, and nonspecific interactions using a battery of quantitative assays. We will assess the capacity of these new particles to bind specifically to the AMPA neurotransmitter receptor on living neurons and the capac- ity to preserve native receptor behavior through direct comparisons with compact (but unstable) dyes and fluo- rescent proteins. This proposal is a collaborative effort between Prof. Paul Selvin, an expert in microscopy, op- tics, and biophysics, and Prof. Andrew Smith, an expert in quantum dot development and colloidal synthesis. Success will open the door to super-resolution observation of a multitude of cellular and molecular processes underlying disease that have resisted understanding using classical molecular and cellular biology approaches. These include tumor cell chemotaxis and metastasis, motor protein dysfunction, and neuronal dysfunction in diseases.
Basic bioscience and applied clinical medicine are broadly in need of fluorescent tools that have minimal perturbation, are bright enough to be seen at the single molecule level, and have unlimited stability to report events from milliseconds to hours. We propose to develop compact quantum dots, <10 nm, to meet this need, as current quantum dots are simply too bulky for the most pressing biomedical applications. As a key application, we will use compact quantum dots to examine the neuronal synapse, the small (~ 30 nm) region where nerves communicate, and employ super-resolution microscopy to understand neurotransmitter receptors that control memory, learning, and many neuronal diseases.
