Browsing by Subject "Single particle tracking"

Single-molecule spectroscopic (SMS) measurements have revolutionized biological science due to their ability to directly observe exactly one molecule in the crowd. This single molecule observation removes the ensemble average, revealing molecular heterogeneities. However, traditional SMS techniques fail to study single molecules in the solution phase for a long observation time, because the molecules rapidly diffuse away from the small focal spot. Accordingly, it is typically required to isolate the molecules from the native solution to study time-dependent dynamical behaviors, by either tethering the molecules to a stationary surface or confining the molecules in a small space based on physical principles. This isolation of molecules barricades in-situ and in-vivo single-molecule research. To address this gap, real-time feedback single particle tracking (RT-3D-SPT) has been developed, with the ability to directly monitor individual freely diffusing particles in the solution phase less disruptively. Real-time tracking is realized by estimating the particle’s position using photon information and applying active feedback to keep the particle in a small detection center. This set of techniques is largely divided into two classes, each with its limitations. The first class of RT-3D-SPT techniques spatially separates the emission of the particle using a set of detectors. The signal variation detected by these detectors can be used to estimate the particle’s position in real-time. The second class of RT-3D-SPT uses a spatiotemporally patterned laser excitation to illuminate the particle. The detected photon arrivals can therefore be used to estimate the particle’s position within the dynamic laser excitation pattern. A feedback control actuator such as a scanning mirror or a piezoelectric stage is driven to compensate for the particle’s diffusion in real-time, keeping the particle in the focal volume. However, both methods have limited ability to track dim and fast diffusing objects, such as single molecules. Moreover, very few of these optical configurations provide simultaneous contextualization in three dimensions yet fail to observe rapid processes happening in the surrounding of the tracked objects.In this dissertation, we present a new real-time 3D tracking and imaging method to directly observe fast biological and chemical processes. These processes include the rapid protein adsorption onto nanoparticles when the nanoparticles are exposed to biological fluids. This adsorbed protein layer, called the protein corona, alters nanoparticles’ biological identity and their fate in vivo. Therefore, it is important to understand this critical roadblock in the biological application of engineered nanoparticles. Herein, we first introduce the construction of this microscope, called 3D real-time ultrafast local microscopy (3D-RULM), with its ability to track fast diffusing and lowly emitting objects while rapidly imaging the surroundings concurrently. Next, we show this RT-3D-SPT method can be applied to quantitatively characterize individual nanoparticle protein corona in situ particle by particle, with single protein sensitivity at signal-to-background ratios down to 1%. Finally, to expand this method to smaller NP-protein systems, we have further implemented a Galvo mirror as a control actuator with a response time five times faster than the currently used piezoelectric stage, opening the possibility to study transient NP-protein interactions and other fast biological phenomena in situ and in vivo.

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.