Jesse S. Aaron

Advanced Optical Imaging Reveals the Dependence of Particle Geometry on Interactions Between CdSe Quantum Dots and Immune Cells

The biocompatibility and possible toxicological consequences of engineered nanomaterials, including quantum dots (QDs) due to their unique suitability for biomedical applications, remain intense areas of interest. We utilized advanced imaging approaches to characterize the interactions of CdSe QDs of various sizes and shapes with live immune cells. Particle diffusion and partitioning within the plasma membrane, cellular uptake kinetics, and sorting of particles into lysosomes were all independantly characterized. Using high-speed total internal reflectance fluorescence (TIRF) microscopy, we show that QDs with an average aspect ratio of 2.0 (i.e., rod-shaped) diffuse nearly an order of magnitude slower in the plasma membrane than more spherical particles with aspect ratios of 1.2 and 1.6, respectively. Moreover, more rod-shaped QDs were shown to be internalized into the cell 2-3 fold more slowly. Hyperspectral confocal fluorescence microscopy demonstrates that QDs tend to partition within the cell membrane into regions containing a single particle type. Furthermore, data examining QD sorting mechanisms indicate that endocytosis and lysosomal sorting increases with particle size. Together, these observations suggest that both size and aspect ratio of a nanoparticle are important characteristics that significantly impact interactions with the plasma membrane, uptake into the cell, and localization within intracellular vesicles. Thus, rather than simply characterizing nanoparticle uptake into cells, we show that utilization of advanced imaging approaches permits a more nuanced and complete examination of the multiple aspects of cell-nanoparticle interactions that can ultimately aid understanding possible mechanisms of toxicity, resulting in safer nanomaterial designs.

Characterization of Differential Toll‐like Receptor Responses below the Optical Diffraction Limit

Many membrane receptors are recruited to specific cell surface domains to form nanoscale clusters upon ligand activation. This step appears to be necessary to initiate cell signaling, including pathways in innate immune system activation. However, virulent pathogens such as Yersinia pestis (the causative agent of plague) are known to evade innate immune detection, in contrast to similar microbes (such as Escherichia coli) that elicit a robust response. This disparity has been partly attributed to the structure of lipopolysaccharides (LPS) on the bacterial cell wall, which are recognized by the innate immune receptor TLR4. It is hypothesized that nanoscale differences exist between the spatial clustering of TLR4 upon binding of LPS derived from Y. pestis and E. coli. Although optical imaging can provide exquisite details of the spatial organization of biomolecules, there is a mismatch between the scale at which receptor clustering occurs (<300 nm) and the optical diffraction limit (>400 nm). The last decade has seen the emergence of super-resolution imaging methods that effectively break the optical diffraction barrier to yield truly nanoscale information in intact biological samples. This study reports the first visualizations of TLR4 distributions on intact cells at image resolutions of <30 nm using a novel, dual-color stochastic optical reconstruction microscopy (STORM) technique. This methodology permits distinction between receptors containing bound LPS from those without at the nanoscale. Importantly, it is also shown that LPS derived from immunostimulatory bacteria result in significantly higher LPS-TLR4 cluster sizes and a nearly twofold greater ligand/receptor colocalization as compared to immunoevading LPS.

Simultaneous, dual-color STORM imaging at the cellular interface.

Over the past decade optical approaches have been introduced that effectively break the traditional diffraction barrier. Of particular note were the introductions of Stimulated Emission and Depletion (STED) microscopy [1], Photo-Activated Localization Microscopy (PALM) [2], and the closely related Stochastic Optical Reconstruction Microscopy (STORM) [3]. Of these, STORM represents an attractive method for researchers, as it does not require highly specialized optical setups as is the case in STED, and can be more easily implemented for multi-color imaging.

Fluorescence Fluctuation Analysis of Mixed Chromophores from a Line-Scanning Hyperspectral Imaging System

Fluorescence fluctuation analysis of dilute biomolecules can provide a powerful method for fast and accurate determination of diffusion dynamics, local concentrations, and aggregation states in complex environments. However, spectral overlap among multiple exogenous and endogenous fluorescent species, photobleaching, and background inhomogeneities can compromise quantitative accuracy and constrain useful biological implementation of this analytical strategy in real systems. In order to better understand these limitations and expand the utility of fluctuation correlation methods, spatiotemporal fluorescence correlation analysis was performed on spectrally resolved line scanned images of modeled and real data from mixed fluorescent nanospheres in a synthetic gel matrix. It was found that collecting images at a pixel sampling regime optimal for spectral imaging provides a method for calibration and subsequent temporal correlation analysis which is insensitive to spectral mixing, spatial inhomogeneity, and photobleaching. In these analyses, preprocessing with multivariate curve resolution (MCR) provided the local concentrations of each spectral component in the images, thus facilitating correlation analysis of each component individually. This approach allowed quantitative removal of background signals and showed dramatically improved quantitative results compared to a hypothetical system employing idealized filters and multi-parameter fitting routines.

Advanced Optical Imaging of Endocytosis

Endocytosis is the highly controlled and complex process by which a portion of the plasma membrane, including its lipids, proteins, and local extracellular fluid becomes internalized in a cell. Endocytosis serves to mediate a multitude of interactions between a cell and its environment, including nutrient uptake, mitosis, motility, as well as adaptive and innate immune response, among many others. There are multiple routes of endocytotic uptake into cells, with the most studied being clathrin mediated endocytosis (CME). Although CME differs significantly on a molecular level from the clathrin-independent endocytosis mechanisms (e.g. macropinocytosis, phagocytosis), all of the endocytic mechanisms involve a sequence of changes in morphology, molecular composition, and protein interactions at the plasma membrane, as well as throughout the bulk of the cell. Further, each of these changes is tightly regulated in space and time. To fully characterize endocytic pathways and their intertwined relationship to other signalling pathways, there is a need to visualize the dynamics of multiple species at the plasma membrane and within the cell with high threedimensional spatial resolution.

Simultaneous, Dual-Color STORM Imaging of Membrane Reorganization during Early Immune Response

TLR-4 receptor reorganization in cell membranes was investigated using a novel STORM microscope. The increased resolution permits observation of receptor cluster formation following challenge with chemotypes of lipopolysaccharide.

Imaging Innate Immune Responses using Dual Color Stochastic Reconstruction Optical Microscopy (STORM)

Over the past decade, optical approaches have been introduced that effectively break the traditional diffraction barrier. Of particular note were the introductions of Stimulated Emission and Depletion (STED) microscopy [1], Photo-Activated Localization Microscopy (PALM) [2], and the closely related Stochastic Optical Reconstruction Microscopy (STORM) [3]. Of these, the STORM/PALM approach represents an attractive method for researchers, as it does not require highly specialized optical setups as is the case in STED, and can be more easily implemented for multi-color imaging. We have implemented a dual-color direct STORM system to simultaneously image the subresolution organization/colocalization of the TLR-4 receptor, a key mediator of innate immune response and lipopolysaccharide (LPS), a bacteria-specific antigen recognized by TLR4. We show that “super-resolution” imaging approaches such as this can provide information that is not attainable with other optical methods.

Differential Uptake and Trafficking of Nanoparticles by Living Cells

Engineered nanoparticles are becoming increasingly commonplace in commercial products as well as biomedical research settings. Surprisingly, however, systematic studies of the health hazards of engineered nanomaterials have severely lagged behind their development and application. It has been shown in some cases that nanomaterials can elicit unique and potentially deleterious physiological responses that are not observed with bulk materials of the same type [1].