Quantum Dot Fluorescent Imaging: Using Atomic Structure Correlation Studies to Improve Photophysical Properties.

Efforts to study intricate, higher-order cellular functions have called for fluorescence imaging under physiologically relevant conditions such as tissue systems in simulated native buffers. This endeavor has presented novel challenges for fluorescent probes initially designed for use in simple buffers and monolayer cell culture. Among current fluorescent probes, semiconductor nanocrystals, or quantum dots (QDs), offer superior photophysical properties that are the products of their nanoscale architectures and chemical formulations. While their high brightness and photostability are ideal for these biological environments, even state of the art QDs can struggle under certain physiological conditions. A recent method correlating electron microscopy ultrastructure with single-QD fluorescence has begun to highlight subtle structural defects in QDs once believed to have no significant impact on photoluminescence (PL). Specific defects, such as exposed core facets, have been shown to quench QD PL in physiologically accurate conditions. For QD-based imaging in complex cellular systems to be fully realized, mechanistic insight and structural optimization of size and PL should be established. Insight from single QD resolution atomic structure and photophysical correlative studies provides a direct course to synthetically tune QDs to match these challenging environments.

Quantitative morphological analysis of InP-based quantum dots reveals new insights into the complexity of shell growth.

The incorporation of quantum dots in display technology has fueled a renewed interest in InP-based quantum dots, but difficulty controlling the Zn chemistry during shelling has stymied thick, even ZnSe shell growth. The characteristic uneven, lobed morphology of Zn-based shells is difficult to assess qualitatively and measure through traditional methods. Here, we present a methodological study utilizing quantitative morphological analysis of InP/ZnSe quantum dots to analyze the impact of key shelling parameters on InP core passivation and shell epitaxy. We compare conventional hand-drawn measurements with an open-source semi-automated protocol to showcase the improved precision and speed of this method. Additionally, we find that quantitative morphological assessment can discern morphological trends in morphologies that qualitative methods cannot. In conjunction with ensemble fluorescence measurements, we find that changes to shelling parameters that promote even shell growth often do so at the cost of core homogeneity. These results indicate that the chemistry of passivating the core and promoting shell growth must be balanced carefully to maximize brightness while maintaining emission color-purity.

Minimizing the Reorganization Energy of Cobalt Redox Mediators Maximizes Charge Transfer Rates from Quantum Dots.

Light-induced charge separation is at the very heart of many solar harvesting technologies. The reduction of energetic barriers to charge separation and transfer increases the rate of separation and the overall efficiency of these technologies. Here we report that the internal reorganization energy of the redox acceptor, the movement of the atoms with changing charge, has a profound effect on the charge transfer rates from donor quantum dots. We experimentally studied and modelled with Marcus Theory charge transfer to cobalt complexes that have similar redox potentials covering 350 mV, but vastly different reorganization energies spanning 2 eV. While the driving force does influence the electron transfer rates, the reorganization energies had a far more profound effect, increasing charge transfer rates by several orders of magnitude. Our studies suggest that careful design of redox mediators to minimize reorganization energy is an untapped route to drastically increase the efficiency of quantum dot applications that feature charge transfer.

Ultrafast spectroscopy studies of carrier dynamics in semiconductor nanocrystals.

Semiconductor nanocrystals have become ubiquitous both in scientific research and in applied technologies related to light. When a nanocrystal absorbs a photon an electron-hole pair is created whose fate dictates whether the nanocrystal will be suitable for a particular application. Ultrafast spectroscopy provides a real-time window to monitor the evolution of the electron-hole pair. In this review, we focus on CdSe nanocrystals, the most-studied nanocrystal system to date, and also highlight ultrasmall nanocrystals, "standard nanocrystals" of different binary composition, alloyed nanocrystals, and core/shell nanocrystals and nanorods. We focus on four time-resolved spectroscopies used to interrogate nanocrystals: pump-probe, fluorescence upconversion, time-correlated single photon counting, and non-linear spectroscopies. The basics of the nanocrystals and the spectroscopies are presented, followed by a detailed synopsis of ultrafast spectroscopy studies performed on the various semiconductor nanocrystal systems.

Fluorescent Colloidal Ferroelectric Nanocrystals.

We report the appearance of ferroelectric behavior arising from a room-temperature cation exchange of cadmium-based semiconductor nanoparticles. Fluorescence retention was achieved through protective CdS shelling before cation exchange with tin(IV) by containing defects in the CdS shell rather than the fluorescent CdSe cores. Ferroelectric response, measured using a Sawyer-Tower circuit, was kept constant, while fluorescence retention increases with an increase in the number of CdS monolayers. At 8 monolayers, fluorescence retention reached 99%, allowing for the addition of ferroelectric applications to the already ever-growing list of quantum dot applications.

A Novel Biotinylated Homotryptamine Derivative for Quantum Dot Imaging of Serotonin Transporter in Live Cells.

The serotonin transporter (SERT) is the primary target for selective serotonin reuptake inhibitor (SSRI) antidepressants that are thought to exert their therapeutic effects by increasing the synaptic concentration of serotonin. Consequently, probes that can be utilized to study cellular trafficking of SERT are valuable research tools. We have developed a novel ligand (IDT785) that is composed of a SERT antagonist (a tetrahydro pyridyl indole derivative) conjugated to a biotinylated poly ethylene glycol (PEG) via a phenethyl linker. This compound was determined to be biologically active and inhibited SERT-mediated reuptake of IDT307 with the half-maximal inhibitory concentration of 7.2 ± 0.3 µM. We demonstrated that IDT785 enabled quantum dot (QD) labeling of membrane SERT in transfected HEK-293 cultures that could be blocked using the high affinity serotonin reuptake inhibitor paroxetine. Molecular docking studies suggested that IDT785 might be binding to the extracellular vestibule binding site rather than the orthosteric substrate binding site, which could be attributable to the hydrophilicity of the PEG chain and the increased loss of degrees of freedom that would be required to penetrate into the orthosteric binding site. Using IDT785, we were able to study the membrane localization and membrane dynamics of YFP-SERT heterologously expressed in HEK-293 cells and demonstrated that SERT expression was enriched in the membrane edge and in thin cellular protrusions.

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking.

The role of lateral mobility and nanodomain organization of G protein-coupled receptors in modulating subcellular signaling has been under increasing scrutiny. Investigation of D2 dopamine receptor diffusion dynamics is of particular interest, as these receptors have been linked to altered neurotransmission in affective disorders and represent the primary target for commonly prescribed antipsychotics. Here, we applied our single quantum dot tracking approach to decipher intrinsic diffusion patterns of the wild-type long isoform of the D2 dopamine receptor and its genetic variants previously identified in several cohorts of schizophrenia patients. We identified a subtle decrease in the diffusion rate of the Val96Ala mutant that parallels its previously reported reduced affinity for potent neuroleptics clozapine and chlorpromazine. Slower Val96Ala variant diffusion was not accompanied by a change in receptor-receptor transient interactions as defined by the diffraction-limited quantum dot colocalization events. In addition, we implemented a Voronoї tessellation-based algorithm to compare nanoclustering of the D2 dopamine receptor to the dominant anionic phospholipid phosphatidylinositol 4,5-bisphosphate in the plasma membrane of live cells.

Enlightened: addressing circadian and seasonal changes in photoperiod in animal models of bipolar disorder.

Bipolar disorders (BDs) exhibit high heritability and symptoms typically first occur during late adolescence or early adulthood. Affected individuals may experience alternating bouts of mania/hypomania and depression, with euthymic periods of varying lengths interspersed between these extremes of mood. Clinical research studies have consistently demonstrated that BD patients have disturbances in circadian and seasonal rhythms, even when they are free of symptoms. In addition, some BD patients display seasonal patterns in the occurrence of manic/hypomanic and depressive episodes as well as the time of year when symptoms initially occur. Finally, the age of onset of BD symptoms is strongly influenced by the distance one lives from the equator. With few exceptions, animal models useful in the study of BD have not capitalized on these clinical findings regarding seasonal patterns in BD to explore molecular mechanisms associated with the expression of mania- and depression-like behaviors in laboratory animals. In particular, animal models would be especially useful in studying how rates of change in photoperiod that occur during early spring and fall interact with risk genes to increase the occurrence of mania- and depression-like phenotypes, respectively. Another unanswered question relates to the ways in which seasonally relevant changes in photoperiod affect responses to acute and chronic stressors in animal models. Going forward, we suggest ways in which translational research with animal models of BD could be strengthened through carefully controlled manipulations of photoperiod to enhance our understanding of mechanisms underlying seasonal patterns of BD symptoms in humans. In addition, we emphasize the value of incorporating diurnal rodent species as more appropriate animal models to study the effects of seasonal changes in light on symptoms of depression and mania that are characteristic of BD in humans.

Rate of change in solar insolation is a hidden variable that influences seasonal alterations in bipolar disorder.

The consensus in the literature is that bipolar disorder is seasonal. We argue that there is finer detail to seasonality and that changes in mood and energy in bipolar disorder are dictated by the rate of change of solar insolation.

Single Quantum Dot Tracking Unravels Agonist Effects on the Dopamine Receptor Dynamics.

D2 dopamine receptors (DRD2s) belong to a family of G protein-coupled receptors that modulate synaptic dopaminergic tone via regulation of dopamine synthesis, storage, and synaptic release. DRD2s are the primary target for traditional antipsychotic medications; dysfunctional DRD2 signaling has been linked to major depressive disorder, attention-deficit hyperactivity disorder, addiction, Parkinson's, and schizophrenia. DRD2 lateral diffusion appears to be an important post-translational regulatory mechanism; however, the dynamic response of DRD2s to ligand-induced activation is poorly understood. Dynamic imaging of the long isoform of DRD2 (D2L) fused to an N-terminal antihemagglutinin (HA) epitope and transiently expressed in HEK-293 cells was achieved through a combination of a high-affinity biotinylated anti-HA antigen-binding fragment (Fab) and streptavidin-conjugated quantum dots (QD). Significant reduction (∼40%) in the rate of lateral diffusion of QD-tagged D2L proteins was observed under agonist (quinpirole; QN)-stimulated conditions compared to basal conditions. QN-induced diffusional slowing was accompanied by an increase in frequency, lifetime, and confinement of temporary arrest of lateral diffusion (TALL), an intrinsic property of single receptor lateral motion. The role of the actin cytoskeleton in QN-induced diffusional slowing of D2L was also explored. The observed dynamic changes appear to be a sensitive indicator of the receptor activity status and might also spatially and temporally shape the receptor-mediated downstream signaling. This dynamic information could potentially be useful in informing drug discovery efforts based on single-molecule pharmacology.

Seasonal effects on bipolar disorder: A closer look.

Bipolar disorders have an onset in late adolescence or early adulthood and patients may experience alternating episodes of mania and depression, with euthymic periods interspersed between these extremes of mood. Clinical research studies have shown that bipolar disorder patients exhibit disruptions in circadian and seasonal rhythms, even when they are symptom free. In addition, some bipolar patients display pronounced seasonal patterns in occurrence of manic and depressive episodes, time of year for disease onset, and age of onset. Several groups have emphasized the impact of seasonal changes in sunlight intensity on bipolar disorder, especially in locations farther from the equator. In this paper, we examine rate of change of solar insolation during the spring and fall in locations that vary in their distance from the equator and propose that seasonal changes in sunlight intensity may be tracked by the suprachiasmatic nucleus and affect disease onset and progression in seasonally susceptible bipolar patients.

Effect of indium alloying on the charge carrier dynamics of thick-shell InP/ZnSe quantum dots.

Thick-shell InP/ZnSe III-V/II-VI quantum dots (QDs) were synthesized with two distinct interfaces between the InP core and ZnSe shell: alloy and core/shell. Despite sharing similar optical properties in the spectral domain, these two QD systems have differing amounts of indium incorporation in the shell as determined by high-resolution energy-dispersive x-ray spectroscopy scanning transmission electron microscopy. Ultrafast fluorescence upconversion spectroscopy was used to probe the charge carrier dynamics of these two systems and shows substantial charge carrier trapping in both systems that prevents radiative recombination and reduces the photoluminescence quantum yield. The alloy and core/shell QDs show slight differences in the extent of charge carrier localization with more extensive trapping observed in the alloy nanocrystals. Despite the ability to grow a thick shell, structural defects caused by III-V/II-VI charge carrier imbalances still need to be mitigated to further improve InP QDs.

Quantitative Analysis of Single Quantum Dot Trajectories.

Single quantum dot tracking (SQDT) is a powerful technique for interrogating biomolecular dynamics in living cells and tissue. SQDT has particularly excelled in driving discovery at the single-molecule level in the fields of neuronal communication, plasma membrane organization, viral infection, and immune system response. Here, we briefly characterize various elements of the SQDT analytical framework and provide the reader with a detailed set of executable commands to implement commonly used algorithms for SQDT data processing.

Labeling Neuronal Proteins with Quantum Dots for Single-Molecule Imaging.

Single-molecule imaging has illuminated dynamics and kinetics of neuronal proteins in their native membranes helping us understand their effective roles in the brain. Here, we describe how nanometer-sized fluorescent semiconductors called quantum dots (QD) can be used to label neuronal proteins in a single QD imaging format. We detail two generalizable protocols accompanied by experimental considerations giving the user options in approach tailored to the materials and equipment available. These protocols can be modified for experiments to verify target specificity, as well as single molecule analysis such as single particle tracking and protein clustering.

Role of shell composition and morphology in achieving single-emitter photostability for green-emitting "giant" quantum dots.

The use of the varied chemical reactivity of precursors to drive the production of a desired nanocrystal architecture has become a common method to grow thick-shell graded alloy quantum dots (QDs) with robust optical properties. Conclusions on their behavior assume the ideal chemical gradation and uniform particle composition. Here, advanced analytical electron microscopy (high-resolution scanning transmission electron microscopy coupled with energy dispersive spectroscopy) is used to confirm the nature and extent of compositional gradation and these data are compared with performance behavior obtained from single-nanocrystal spectroscopy to elucidate structure, chemical-composition, and optical-property correlations. Specifically, the evolution of the chemical structure and single-nanocrystal luminescence was determined for a time-series of graded-alloy "CdZnSSe/ZnS" core/shell QDs prepared in a single-pot reaction. In a separate step, thick (∼6 monolayers) to giant (>14 monolayers) shells of ZnS were added to the alloyed QDs via a successive ionic layer adsorption and reaction (SILAR) process, and the impact of this shell on the optical performance was also assessed. By determining the degree of alloying for each component element on a per-particle basis, we observe that the actual product from the single-pot reaction is less "graded" in Cd and more so in Se than anticipated, with Se extending throughout the structure. The latter suggests much slower Se reaction kinetics than expected or an ability of Se to diffuse away from the initially nucleated core. It was also found that the subsequent growth of thick phase-pure ZnS shells by the SILAR method was required to significantly reduce blinking and photobleaching. However, correlated single-nanocrystal optical characterization and electron microscopy further revealed that these beneficial properties are only achieved if the thick ZnS shell is complete and without large lattice discontinuities. In this way, we identify the necessary structural design features that are required for ideal light emission properties in these green-visible emitting QDs.

Optical and electrochemical tuning of hydrothermally synthesized nitrogen-doped carbon dots.

Carbon dots (CDs) are a rapidly progressing class of nanomaterial which show promise towards applications in solar energy conversion due to their low toxicity, favorable electrochemical properties, and tunability. In recent years there have been a number of reported CD syntheses, both top-down and bottom-up methods, producing a diverse range of CDs with intrinsic properties dependent on the starting materials and utilized dopants. This work presents a citrate buffer-facilitated synthesis of nitrogen-doped carbon dots (NCD) and explores the impact of urea concentration on observed electrochemical and optical properties. Optical absorbance and quantum yield of NCDs were found to increase with the dopant concentrations present in the hydrothermal reaction mixture. Electrochemical analysis demonstrates that increased nitrogen content results in the shifting of carbon dot oxidation potentials without the need of post-synthesis surface modifications. Over the range of molar ratios of dopant-to-citrate tested, the oxidation potentials of NCDs shifted up to 150 mV towards more negative potentials. X-ray photoelectron spectroscopy confirms the addition of pyrrolic and pyridinic nitrogen at different levels in different batches of NCDs, which are likely the source of the observed changes.

Ligand-conjugated quantum dots for fast sub-diffraction protein tracking in acute brain slices.

Semiconductor quantum dots (QDs) have demonstrated utility in long-term single particle tracking of membrane proteins in live cells in culture. To extend the superior optical properties of QDs to more physiologically relevant cell platforms, such as acute brain slices, we examine the photophysics of compact ligand-conjugated CdSe/CdS QDs using both ensemble and single particle analysis in brain tissue media. We find that symmetric core passivation is critical for both photostability in oxygenated media and for prolonged single particle imaging in brain slices. We then demonstrate the utility of these QDs by imaging single dopamine transporters in acute brain slices, achieving 20 nm localization precision at 10 Hz frame rates. These findings detail design requirements needed for new QD probes in complex living environments, and open the door to physiologically relevant studies that capture the utility of QD probes in acute brain slices.

Quantum dots reveal heterogeneous membrane diffusivity and dynamic surface density polarization of dopamine transporter.

The presynaptic dopamine transporter mediates rapid reuptake of synaptic dopamine. Although cell surface DAT trafficking recently emerged as an important component of DAT regulation, it has not been systematically investigated. Here, we apply our single quantum dot (Qdot) tracking approach to monitor DAT plasma membrane dynamics in several heterologous expression cell hosts with nanometer localization accuracy. We demonstrate that Qdot-tagged DAT proteins exhibited highly heterogeneous membrane diffusivity dependent on the local membrane topography. We also show that Qdot-tagged DATs were localized away from the flat membrane regions and were dynamically retained in the membrane protrusions and cell edges for the duration of imaging. Single quantum dot tracking of wildtype DAT and its conformation-defective coding variants (R60A and W63A) revealed a significantly accelerated rate of dysfunctional DAT membrane diffusion. We believe our results warrant an in-depth investigation as to whether compromised membrane dynamics is a common feature of brain disorder-derived DAT mutants.

Real colloidal quantum dot structures revealed by high resolution analytical electron microscopy.

The development of bright and photostable colloidal quantum dots has been a truly interdisciplinary feat. Designing a specific composition of core and shell materials and then producing the desired nanoarchitecture through chemical routes require a blend of physical and inorganic chemistry, solid-state physics, and materials science. In a battle to separate charge carriers from a surface wrought with defect states, complex shell structures with precisely specified gradient compositions have been engineered, producing nanosized emitters with exceptional stability and color purity. However, much of the success has resided in II-VI materials, such as CdSe, and progress is only just being made on cadmium-free quantum dots. This perspective will discuss the primary challenges in engineering colloidal quantum dots and highlight how the advent of advanced analytical electron microscopy is revealing the structure-function relationships of these complex systems.