HYPERSPECTRAL LINE-SCANNING MICROSCOPY FOR HIGH-SPEED MULTICOLOR QUANTUM DOT TRACKING

One of the challenges in studying protein interactions in live cells lies in the capacity to obtain both spatial and temporal information that is sufficient to extend existing knowledge of the dynamics and interactions, especially when tracking proteins at high density. Here we introduce a high-speed laser line-scanning hyperspectral microscope that is designed to track quantum dot labeled proteins at 27 frames/sec over an area of 28 um2 using 128 spectral channels spanning the range from 500 to 750 nm. This instrument simultaneously excites 8 species of quantum dots and employs a custom prism spectrometer and high speed EMCCD to obtain spectral information that is then used to distinguish and track individual probes at high density. These emitters are localized to within 10s of nm in each frame and reconstructed trajectories yield information of the protein dynamics and interactions. This manuscript describes the design, implementation, characterization, and application of a high-speed laser line-scanning hyperspectral microscope (HSM). The intended primary application is that of investigating the dynamics of transmembrane antibody receptors using quantum dot labeled immunoglobulin E (QD-IgE). Several additional examples demonstrate other advantages and applications of this method, including 3D hyperspectral imaging of live cells and hyperspectral superresolution imaging

Flow-Based Cytometric Analysis of Cell Cycle via Simulated Cell Populations

We present a new approach to the handling and interrogating of large flow cytometry data where cell status and function can be described, at the population level, by global descriptors such as distribution mean or co-efficient of variation experimental data. Here we link the “real” data to initialise a computer simulation of the cell cycle that mimics the evolution of individual cells within a larger population and simulates the associated changes in fluorescence intensity of functional reporters. The model is based on stochastic formulations of cell cycle progression and cell division and uses evolutionary algorithms, allied to further experimental data sets, to optimise the system variables. At the population level, the in-silico cells provide the same statistical distributions of fluorescence as their real counterparts; in addition the model maintains information at the single cell level. The cell model is demonstrated in the analysis of cell cycle perturbation in human osteosarcoma tumour cells, using the topoisomerase II inhibitor, ICRF-193. The simulation gives a continuous temporal description of the pharmacodynamics between discrete experimental analysis points with a 24 hour interval; providing quantitative assessment of inter-mitotic time variation, drug interaction time constants and sub-population fractions within normal and polyploid cell cycles. Repeated simulations indicate a model accuracy of ±5%. The development of a simulated cell model, initialized and calibrated by reference to experimental data, provides an analysis tool in which biological knowledge can be obtained directly via interrogation of the in-silico cell population. It is envisaged that this approach to the study of cell biology by simulating a virtual cell population pertinent to the data available can be applied to “generic” cell-based outputs including experimental data from imaging platforms

Biomolecule-Conjugated Quantum Dot Nanosensors as Probes for Cellular Dynamic Events in Living Cells

A single-molecule tracking/imaging technique with semiconductor quantum dot (QD) nanosensors conjugated with appropriate peptides or antibodies is appealing for probing cellular dynamic events in living cells. We developed a 2D analysis of single-molecule trajectories using normalized variance versus mean square displacement (MSD) to provide high-quality statistics sampled by nanosensors while preserving single-molecule sensitivity. This plot can be more informative than MSD alone to reflect the diffusive dynamics of a protein in its cellular environment. We illustrate the performance of this technique with selected examples, which are designed to expose the functionalities and importance in live cells. Our findings suggest that biomolecule-conjugated QD nanosensors can be used to reveal interactions, stoichiometries, and conformations of proteins, and provide an understanding of the mode of the interaction, stable states, and dynamical pathways of biomolecules in live cells

Electron-Transfer Mediated Photo-Switching in Nanoparticles

Previous publications from our lab demonstrated viable approaches to design a photo-switching nanoparticle with arguably superior brightness and photostability resulting in an improved resolution in localization-based microscopy, as compared to other photo-switching dyes or particles. As a follow-up, this dissertation is focused on two major tasks: first, developing nanoparticles with better photo-switching properties for super-resolution imaging; second, trying to achieve a better physical picture of the mechanisms involved in photo-switching, including polaron dynamics, charge transfer, and energy transfer. A new class of photo-switchable nanoparticles was developed by blending conjugated polymer semiconductor with fullerene-based electron acceptors, and further blending with polystyrene maleic anhydride could improve the bulk fluorescence quantum yield of the nanoparticle and increase the on/off contrast in single-molecule experiments, which is critical for better localization accuracy. To understand how the dynamics of polaron in nanoparticle affects apparent photo-switching properties, single-molecule fluorescence spectra were collected from nanoparticles with different blending ratios and under various excitation laser intensities. Further modulation of the excitation laser intensity led to a transient fluorescent response consistent with previously published behavior of nanoparticles in bulk suspensions. The results of those experiments further help the development of a better physical model connecting polaron generation/recombination dynamics and photo-switching properties

Single quantum dot tracking for quantitative molecular imaging

The application of semiconductor nanocrystals known as quantum dots (QDs) to biology in the past few decades has advanced the field of single-molecule biology by allowing for long time-scale tracking of individual biomolecules. However, QD single-molecule imaging studies have almost been exclusively limited to the extracellular space, due to limitations in intracellular delivery techniques and a limited understanding of how nanoparticles behave in the intracellular space. In the first half of this thesis, a new analysis methodology is developed to quantitatively assess the intracellular delivery of QDs. We present a method for using single-molecule imaging and subsequent single-particle tracking (SPT) of QDs delivered to the cytosol of living cells to assess delivery efficiency and uptake mechanisms. In this method, single quantum dot mobility information is used in conjunction with single-molecule brightness measurements to develop novel single-cell metrics of delivery efficiency. These metrics are used to investigate the impact of different nanoparticle surface properties on intracellular delivery and fate. We investigate the delivery of a series of QDs designed with diverse surface properties. This comparison revealed new insights into particle uptake and endosomal escape, as well as the discovery that zwitterionic surfaces are uniquely suited for intracellular mobility. Additionally, this new analysis methodology was validated by established experimental approaches and analysis of simulated single-particle trajectories. The second half of this thesis applies the aforementioned tools towards two applications. The first application is to quantitatively evaluate QD labeling of intracellular proteins in live cells. This was achieved by delivering QDs conjugated to biorthogonal functional groups to label a target protein and developing a new colocalization-based metric to quantify the degree of protein target labeling. We present evidence of protein target labeling by using this single-trajectory level colocalization metric in combination with nanoparticle mobility measurements. The second application is toward accurate measurement of hydrodynamic size of colloids with dimensions smaller than 100 nanometers (nm). We develop a new method using widefield fluorescence microscopy and SPT to measure nanoparticle size and demonstrate accurate single-molecule size measurements of a homogeneous nanoparticle population with hydrodynamic diameter of approximately 20 nm

Probing Cellular Uptake of Nanoparticles, One at a Time

Advanced fluorescence microscopy is the method of choice to study cellular uptake of nanoparticles with molecular specificity and nanoscale resolution; yet, direct visualization of nanoparticles entry into cells poses severe technical challenges. Here, we have combined super-resolution photoactivation localization microscopy (PALM) with single particle tracking (SPT) to visualize clathrin-mediated endocytosis (CME) of polystyrene nanoparticles at very high spatial and temporal resolution

Imaging Three-Dimensional Single Molecule Dynamics in its Cellular Context

Three-dimensional single molecule microscopy enables the study of dynamic processes in living cells at the level of individual molecules. Multifocal plane microscopy (MUM) is an example of such a modality and has been shown to be capable of capturing the rapid subcellular trafficking of single molecules in thick samples by simultaneously imaging distinct focal planes within the sample. Regardless of the specific modality, however, the obtained 3D trajectories of single molecules often do not fully reveal the biological significance of the observed dynamics. This is because the missing cellular context is often also needed in order to properly understand the events observed at the molecular level. We introduce the remote focusing-MUM (rMUM) modality, which enables 3D single molecule imaging with the simultaneous z-stack imaging of the surrounding cellular structures. Using rMUM, we demonstrate the 3D tracking of prostate-specific membrane antigen (PSMA) with a PSMA-specific antibody in a prostate cancer cell. PSMA is an important biomarker for prostate cancer cells. As such, it is a common target for antibody-based therapies. For example, of particular interest is the use of PSMA-specific antibodies that are conjugated with a toxin that kills prostate cancer cells. We analyze here the pathways of PSMA-specific antibodies, from prior to their first binding to PSMA at the plasma membrane to their arrival at, and continued movement in, sorting endosomes. By making possible the observation of single molecule dynamics within the relevant cellular context, rMUM allows, in our current application, the identification and analysis of different stages of the PSMA-specific antibody trafficking pathway