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 RI 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 μm 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.

Characterization of suspended nanoparticles in their native environment plays a central role in a wide range of fields, from medical diagnostics and nanoparticle-enhanced 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 and polystyrene 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.

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Nanobubbles, surprisingly stable submicrometer gas bubbles in water, appear to be common in water and biological fluids and are of great interest in technical applications ranging from ultrasound contrast agents to flotation in the mining industry. Nanobubbles on surfaces have been more researched than freely floating bulk nanobubbles, and the reason for their stability appears to be better explained. The stability of bulk nanobubbles is less well explained, several theories exist, and even their existence is sometimes questioned. In the present study, an attempt was made to generate nanobubbles through hydrodynamic cavitation as well as through vigorous shaking in test tubes, and it was found that none of these methods generated a detectable concentration of possible bulk nanobubbles if pure water was used, with or without a small addition of NaCl, the equipment was cleaned properly, and certain plastic materials were avoided. These results indicate that trace organic contaminants are necessary for nanobubble stabilization. Experiments were also made with the dissolution of a high concentration of inorganic salts, which generated bubbles by creating air supersaturation. Light scattering submicron particles were found in all solutions and appeared to be actual gas bubbles in at least one case. However, in many cases, these light scattering particles were unaffected by vacuum and pressure and appear, therefore, to be something else other than air bubbles. It is concluded that, in future research on nanobubble stability, it is very important to avoid contamination, as well as to ascertain that light scattering objects really are bubbles and not oil droplets or particles.

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