Bridging Single-Particle Characterisation Gaps of Optical Microscopy in the Nano-Submicron Regime

As the practical importance of particles in the nano-submicron size regime continues to increase in both biomedical applications and industrial processes, so does the need for accurate and versatile characterisation methods. Optical scattering microscopy methods are commonly used for single-particle characterisation as they provide quick measurements at physiologically relevant conditions with detection limits reaching down to individual biomolecules. However, quantitative particle characterisation using optical microscopy often rely on assumptions about the surrounding media and theparticle, including solution viscosity, boundary conditions, as well as particle shape and material. Since these assumptions are difficult to evaluate, particle characterisation beyond hydrodynamic radius and/or mass remains challenging.The aim of this thesis is to contribute to bridging the gaps that limit quantitative optical microscopy-based characterisation of individual particles in the nano-submicron regime by both developing new and improving existing microscopy methods. Specifically, in Paper I a method was developed to evaluate the relation between diffusivity and particle size to enable measurements of the hydrodynamic boundary condition. Papers II-V are based around the development of holographic nanoparticle tracking (H-NTA) and extensions thereof, with the intent of using the complex-valued optical field for material sensitive particle characterisation with minimal dependence on the surrounding media. In Paper II, H-NTA by itself was used to characterise suspensions containing nanobubbles and molecular aggregates. In Paper III, the combination of H-NTA with deep learning was used to achieve simultaneous quantification of size and refractive index directly from single microscopy images, which allowed detection of reversible fluctuations in nanoparticle aggregates. In Paper IV, H-NTA augmented with a low frequency attenuation filter, coined twilight holography, was used to investigate the interaction between herpes viruses and functionalised gold nanoparticles in terms of size, bound gold mass, and virus refractive index. In Paper V, the combination of twilight holography and interferometric scattering microscopy (iSCAT) was used to quantify both size and polarizability of individual nanoparticles without the need of detailed knowledge about the surrounding media. Taken together, the presented results in this thesis provide both new insights into heterogenous nanoparticle systems and contributes to narrowing the gap for detailed optical particle characterisation

Optical characterisation of subwavelength dielectric particles using particle tracking beyond the Stokes-Einstein relation

As the importance of nanoparticles continues to increase in both biology and industrial processes, so does the need for accurate and versatile characterisation methods. However, most light-based methods to quantify size and refractive index of individual particles are either limited to snapshot observations, particles larger than the wavelength of light, non-dynamic particle properties, or assuming the hydrodynamic boundary conditions without experimental evaluation. The aim of this thesis is to partially overcome these limitations by further developing two different characterisation methods based on optical microscopy combined with particle tracking, where the analysis goes beyond the ordinary Stokes-Einstein relation. The first method combines off-axis holographic nanoparticle tracking with deep learning (Paper I). By utilizing the optical signal, both size and refractive index of individual particles with a minimum size of R=150 nm were accurately determined using only five particle observations. The method was evaluated using particles of different sizes, refractive indices, surrounding media as well as for polystyrene nanoparticle clusters, for which reversible fluctuations of the number of monomers could be resolved while the fractal dimension remained constant. The second method is based particles tethered to a laterally fluid supported lipid bilayer and quantification of their diffusivity and flow-induced motion (Paper II). By separating the friction contributions from the tethers and the particle, simultaneous measurement of size and diffusivity enabled a comparison with theory using partial slip as a fitting parameter. This was used to quantify the slip length for different lipid vesicles, and to clarify the size-dependent mechanistic aspects concerning the mobility of membrane-attached nanoparticles

Label-free spatio-temporal monitoring of cytosolic mass, osmolarity, and volume in living cells

Microorganisms adapt their biophysical properties in response to changes in their local environment. However, quantifying these changes at the single-cell level has only recently become possible, largely relying on fluorescent labeling strategies. In this work, we utilize yeast (Saccharomyces cerevisiae) to demonstrate label-free quantification of changes in both intracellular osmolarity and macromolecular concentration in response to changes in the local environment. By combining a digital holographic microscope with a millifluidic chip, the temporal response of cellular water flux was successfully isolated from the rate of production of higher molecular weight compounds, in addition to identifying the produced compounds in terms of the product of their refractive index increment (dndc) and molar mass. The ability to identify, quantify and temporally resolve multiple biophysical processes in living cells at the single cell level offers a crucial complement to label-based strategies, suggesting broad applicability in studies of a wide-range of cellular processes

Fast and Accurate Nanoparticle Characterization Using Deep-Learning-Enhanced Off-Axis Holography

Characterization of suspended nanoparticles in their native environment plays a central role in a wide range of fields, from medical diagnostics and nanoparticleenhanced drug delivery to nanosafety and environmental nanopollution assessment. Standard optical approaches for nanoparticle sizing assess the size via the diffusion constant and, as a consequence, require long trajectories and that the medium has a known and uniform viscosity. However, in most biological applications, only short trajectories are available, while simultaneously, the medium viscosity is unknown and tends to display spatiotemporal variations. In this work, we demonstrate a label-free method to quantify not only size but also refractive index of individual subwavelength particles using 2 orders of magnitude shorter trajectories than required by standard methods and without prior knowledge about the physicochemical properties of the medium. We achieved this by developing a weighted average convolutional neural network to analyze holographic images of single particles, which was successfully applied to distinguish and quantify both size and refractive index of subwavelength silica andpolystyrene particles without prior knowledge of solute viscosity or refractive index. We further demonstrate how these features make it possible to temporally resolve aggregation dynamics of 31 nm polystyrene nanoparticles, revealing previously unobserved time-resolved dynamics of the monomer number and fractal dimension of individual subwavelength aggregates

Diffusion of Lipid Nanovesicles Bound to a Lipid Membrane Is Associated with the Partial-Slip Boundary Condition

During diffusion of nanoparticles bound to a cellular membrane by ligand-receptor pairs, the distance to the laterally mobile interface is sufficiently short for their motion to depend not only on the membrane-mediated diffusivity of the tethers but also in a not yet fully understood manner on nanoparticle size and interfacial hydrodynamics. By quantifying diffusivity, velocity, and size of individual membrane-bound liposomes subjected to a hydrodynamic shear flow, we have successfully separated the diffusivity contributions from particle size and number of tethers. The obtained diffusion-size relations for synthetic and extracellular lipid vesicles are not well-described by the conventional no-slip boundary condition, suggesting partial slip as well as a significant diffusivity dependence on the distance to the lipid bilayer. These insights, extending the understanding of diffusion of biological nanoparticles at lipid bilayers, are of relevance for processes such as cellular uptake of viruses and lipid nanoparticles or labeling of cell-membrane-residing molecules

Single-shot self-supervised object detection in microscopy

Object detection is a fundamental task in digital microscopy, where machine learning has made great strides in overcoming the limitations of classical approaches. The training of state-of-the-art machine-learning methods almost universally relies on vast amounts of labeled experimental data or the ability to numerically simulate realistic datasets. However, experimental data are often challenging to label and cannot be easily reproduced numerically. Here, we propose a deep-learning method, named LodeSTAR (Localization and detection from Symmetries, Translations And Rotations), that learns to detect microscopic objects with sub-pixel accuracy from a single unlabeled experimental image by exploiting the inherent roto-translational symmetries of this task. We demonstrate that LodeSTAR outperforms traditional methods in terms of accuracy, also when analyzing challenging experimental data containing densely packed cells or noisy backgrounds. Furthermore, by exploiting additional symmetries we show that LodeSTAR can measure other properties, e.g., vertical position and polarizability in holographic microscopy

Evaluation of Pressure Generated by Resistors From Different Positive Expiratory Pressure Devices

BACKGROUND: Breathing exercises with positive expiratory pressure (PEP) are used to improve pulmonary function and airway clearance. Different PEP devices are available, but there have been no studies that describe the pressure generated by different resistors. The purpose of this study was to compare pressures generated from the proprietary resistor components of 4 commercial flow-dependent PEP valves with all other parameters kept constant. METHODS: Resistors from 4 flow-regulated PEP devices (Pep/Rmt system, Wellspect HealthCare; Pipe P breathing exerciser, Koo Medical Equipment; Mini-PEP, Philips Respironics [including resistors by R\ufcsch]; and 15-mm endo-adapter, VBM Medizintechnik) were tested randomly by a blinded tester at constant flows of 10 and 18 L/min from an external gas system. All resistors were tested 3 times. RESULTS: Resistors with a similar diameter produced statistically significant different pressures at the same flow. The differences were smaller when the flow was 10 L/min compared with 18 L/min. The differences were also smaller when the diameter of the resistor was increased. The pressures produced by the 4 resistors of the same size were all significantly different when measuring 1.5- and 2.0-mm resistors at a flow of 10 L/min and 2.0-mm resistors at a flow of 18 L/min (P < .001). There were no significant differences between any of the resistors when testing sizes of 4.5 and 5.0 mm at either flow. The Mini-PEP and adapter resistors gave the highest pressures. CONCLUSIONS: Pressures generated by the different proprietary resistor components of 4 commercial PEP devices were not comparable, even though the diameter of the resistors is reported to be the same. The pressures generated were significantly different, particularly when using small-diameter resistors at a high flow. Therefore, the resistors may not be interchangeable. This is important information for clinicians, particularly when considering PEP for patients who do not tolerate higher pressures

Size and Refractive Index Determination of Subwavelength Particles and Air Bubbles by Holographic Nanoparticle Tracking Analysis

Determination of size and refractive index (RI) of dispersed unlabeled subwavelength particles is of growing interest in several fields, including biotechnology, wastewater monitoring, and nanobubble preparations. Conventionally, the size distribution of such samples is determined via the Brownian motion of the particles, but simultaneous determination of their R1 remains challenging. This work demonstrates nanoparticle tracking analysis (NTA) in an off-axis digital holographic microscope (DHM) enabling determination of both particle size and RI of individual subwavelength particles from the combined information about size and optical phase shift. The potential of the method to separate particle populations is demonstrated by analyzing a mixture of three types of dielectric particles within a narrow size range, where conventional NTA methods based on Brownian motion alone would fail. Using this approach, the phase shift allowed individual populations of dielectric beads overlapping in either size or RI to be clearly distinguished and quantified with respect to these properties. The method was furthermore applied for analysis of surfactant-stabilized micro- and nanobubbles, with RI lower than that of water. Since bubbles induce a phase shift of opposite sign to that of solid particles, they were easily distinguished from similarly sized solid particles made up of undissolved surfactant. Surprisingly, the dependence of the phase shift on bubble size indicates that only those with 0.15-0.20 mu m radius were individual bubbles, whereas larger bubbles were actually clusters of bubbles. This label-free means to quantify multiple parameters of suspended individual submicrometer particles offers a crucial complement to current characterization strategies, suggesting broad applicability for a wide range of nanoparticle systems

Multidimensional Hybridization of Dark Surface Plasmons

Synthetic three-dimensional (3D) nanoarchitectures are providing more control over light matter interactions and rapidly progressing photonic-based technology. These applications often utilize the strong synergy between electromagnetic fields and surface plasmons (SPs) in metallic. nanostructures. However, many of the SP interactions hosted by complex 3D nanostructures are poorly understood because they involve dark hybridized states that are typically undetectable with far-field optical spectroscopy. Here, we use experimental and theoretical electron energy loss spectroscopy to elucidate dark SPs and their interactions in layered metal-insulator-metal disc nanostructures. We go beyond the established dipole SP hybridization analysis by, measuring breathing and multipolar SP hybridization. In addition, we reveal multidimensional SP hybridization that simultaneously utilizes in-plane and out-of-plane SP coupling. Near-field classic electrodynamics calculations provide excellent agreement with all experiments. These results advance the fundamental understanding of SP hybridization in 3D nanostructures and provide avenues to further tune the interaction between electromagnetic fields and matter