Browsing by Author "Welsher, Kevin D"

Active-feedback single-particle tracking methods are increasingly being applied to in vivo studies. Cellular heterogeneity is both a contributor to anomalous biomolecule behavior (when compared to behavior in dilute solutions) and an ongoing challenge to stable observation using active-feedback methods. Here we develop combined online Bayesian estimation with windowed estimation of background and signal (COBWEBS) in an effort to achieve active-feedback tracking in heterogeneous background environments. In 2D and 3D simulations, COBWEBS stabilizes tracking in complex environments with changing signal and background intensities. Additionally, COBWEBS (and alternative post-processing methods) are applied to experimental data to extract improved particle localizations. Once appropriately calibrated, COBWEBS, along the imaging XY plane, shows accuracy improvements when compared to the current experimentally implemented particle localization algorithm. Evidence suggests that real-time implementation of COBWEBS is possible, and a framework for future development of real-time estimation is presented. An alternative Gaussian fitting post-processing leads to higher accuracy improvements than the COBWEBS approach but cannot be implemented as a real-time particle localization strategy. Next, it is demonstrated that the increased response speed of a galvo scanning mirror in place of the traditional piezoelectric feedback increases overlap of trackable particle speeds with diffusive speeds of intracellular proteins. Finally, 3D active feedback tracking is used to read out static FRET efficiencies from freely-diffusing single molecules, suggesting potential for applications in probing protein dynamics using FRET as a molecular ruler.

Optimization of sampling patterns in 3D Real-time Single Particle Tracking (3D-RT-SPT) systems is crucial due to the limited photon yield and irreversible photobleaching of fluorescent materials commonly used in biological imaging. This study focuses on the evaluation of different sampling patterns in a highly flexible and robust 3D Single Molecule Active Real-time Tracking (3D-SMART) microscope, a representative 3D-RT-SPT system, and further application of an information-efficient version of 3D-SMART. The investigation reveals that optimal sampling patterns in the XY-plane and Z-axis, are off-centered, leading to doubled precision. Theoretical analysis further demonstrates that information-efficient sampling patterns coincide with areas of high Fisher information. These findings not only enhanced the precision of the 3D-SMART system but also established a roadmap for achieving information-efficient sampling in similar 3D-RT-SPT methodologies. Utilizing the information-efficient 4-Corners sampling pattern implemented in the 3D-SMART system, further investigations focused on intracellular events, specifically filopodium dynamics, by employing silver nanoparticles (AgNPs) as probes. Filopodia are thin extracellular protrusions that have important functions in various biological events. Due to their small scale and highly dynamic nature, it is difficult to investigate their activities in a physiologically relevant condition. AgNPs offer advantages over conventional fluorescent materials, providing ample photon flux due to their unique photoluminescent mechanisms known as surface plasmon resonance. Upon incubation with live cells, AgNPs exhibited constrained cylindrical diffusion, revealing significant heterogeneity in residence time across the cylindrical surface. Notably, localized "hot spots" repeatedly visited by AgNPs were observed. Force analysis indicated that these hot spots corresponded to local potential wells, where diffusion speed remained relatively stable. These findings provided evidence of nanoscale structures (~50 nm scale) on filopodia, demonstrating charge-dependent interactions with AgNPs. Furthermore, this study presented a framework for extracting hidden information from 3D trajectories of particles interacting with cylindrical structures, surpassing the capabilities of conventional techniques.

The biochemical environment of the cellular interior is extremely complex andplays a critical role in the function of all biomolecules necessary to life. As the most abundant molecules in cytomatrix, intracellular water actively participates in a wide range of biochemical processes, thus the local water structure and solvation in different cellular compartments should strongly impact the local biochemistry. Despite the fundamental nature, it remains unclear to which extent the intracellular water differs from the familiar “bulk” water and there is a dearth of experimental evidence regarding the spatial variation of solvation inside the cell. To explore this question, we demonstrate a vibrational-shift imaging approach by combining the spectral-focusing hyperspectral stimulated Raman scattering technique with a water-sensing nitrile probe. This dissertation contributes to a complete picture of understanding the hydrogen bonding network and heterogenous solvation at the subcellular level, which has a profound impact on interpreting biochemical studies conducted outside of the native biological environment. This dissertation is structured as the following chapters. Chapter 1 provides an overview of the structure of intercellular water and the evolvement of this field. Chapter 2 introduces the basics of stimulated Raman scattering and elaborates the construction of a hyperspectral stimulated Raman scattering (hsSRS) microscope. Chapter 3 demonstrates the application of hsSRS to probe solvation heterogeneity at the microscopic level by coupling with vibrational solvatochromism of an environmentally-sensitive nitrile probe. The sensing ability is validated in the solution phase, microscopic droplets, and cellular environments. Finally, the subcellular solvation variance between the cytoplasm and the nucleus is quantitatively measured and the origin of the intracellular solvation heterogeneity is explored. To study protein-associated water, different nitrile probes with various sizes and hydrophobicity are investigated in the solution phase measurement. Chapter 4 illustrates a preliminary attempt to develop a real-time chemical imaging technique called Diffusive Enhanced Raman Nano-spectrometer (DERNS) by combining 3D real-time tracking technique with the advanced Raman techniques. The main focus is synthesizing the near-field fluorescent probes coupled with plasmonic structure and its pump-probe behaviors. Chapter 5 summarizes the key findings from the previous chapter and proposes a few future projects along vibrational-shift imaging.

The early stages of the virus-cell interaction have long evaded observation by existing microscopy methods due to the rapid diffusion of virions in the extracellular space and the large 3D cellular structures involved. Here we present an active-feedback single-virus tracking method with simultaneous volumetric imaging of the live cell environment to address this knowledge gap to present unprecedented detail to the extracellular phase of the infectious cycle. Additionally, we report the development of a solution for rapid imaging using a tessellating scan pattern that can be used as a novel way to perform rapid 3D imaging at up to 8x faster than conventional z-stack method. This new method is also capable of imaging with a moving stage, making it the ideal compliment to active-feedback tracking systems. We report previously unobserved phenomena in the early stages of the virus-cell interaction, including skimming contact events at the millisecond timescale, orders of magnitude change in diffusion coefficient upon binding, and cylindrical and linear diffusion modes along filopodia. Finally, we demonstrate how this new method can move single-virus tracking from simple monolayer culture towards more tissue-like conditions by tracking single virions in tightly packed epithelial cells. This multi-resolution method presents new opportunities for capturing fast, 3D processes in biological systems.