Label‐Free Single Nanoparticle Identification and Characterization in Demanding Environment, Including Infectious Emergent Virus

Abstract

Unknown particle screening—including virus and nanoparticles—are keys in medicine, industry, and also in water pollutant determination. Here, RYtov MIcroscopy for Nanoparticles Identification (RYMINI) is introduced, a staining‐free, non‐invasive, and non‐destructive optical approach that is merging holographic label‐free 3D tracking with high‐sensitivity quantitative phase imaging into a compact optical setup. Dedicated to the identification and then characterization of single nano‐object in solution, it is compatible with highly demanding environments, such as level 3 biological laboratories, with high resilience to external source of mechanical and optical noise. Metrological characterization is performed at the level of each single particle on both absorbing and transparent particles as well as on immature and infectious HIV, SARS‐CoV‐2 and extracellular vesicles in solution. The capability of RYMINI to determine the nature, concentration, size, complex refractive index and mass of each single particle without knowledge or model of the particles’ response is demonstrated. The system surpasses 90% accuracy for automatic identification between dielectric/metallic/biological nanoparticles and ≈80% for intraclass chemical determination of metallic and dielectric. It falls down to 50–70% for type determination inside the biological nanoparticle's class.

Enhanced Quantitative Wavefront Imaging for Nano-Object Characterization

“…FWC = 40 ke − ), can result in an SNR gain that is approximately twice larger than what would be achieved with a state-of-the-art high FWC camera (FWC = 2000 ke − ). 22 Importantly, this sensitivity enhancement does not depend on NP properties or dimensions. It holds true for NPs typically smaller than a few hundred nanometers of diameter (see Supplementary Section 2), thus making Z-WIM highly suitable for the blind characterization of unknown NP mixtures, including viruses and extracellular vesicles.…”

“…While standard image averaging usually performs well and remains relevant with static samples, it does not allow the characterization of small NPs moving in solution. Recent approaches based on extensive image postprocessing have proven effective in this context . However, they require a sufficient SNR on each individual image to enable the detection, precise 3D superlocalization, and 3D registration of the NP before averaging.…”

“…This QLSI system yields performances comparable to those of modified Hartmann− Mask-based sensors. 22 Description of the Spatial Filters. Most of the experiments presented in this article have been performed using a semiopaque filter for Fourier-plane filtering consisting in a 100 nm-thick gold disc deposited on a glass substrate.…”

“…Based on such analyses, several recent works have demonstrated the potential of QPI to estimate the mass or chemical nature of unknown NPs, viruses, or vesicles in solution and even identify them in a label-free manner. 17,22,23 A recent study has even demonstrated the ability to characterize nanoobjects down to the scale of a single protein, albeit at the cost of increased experimental complexity. 29 The main roadblock to the detection of weak scattering/ extinction cross sections from single nano-objects arises from the very little amount of light scattered when compared to the nonscattered light.…”

Determination of the absolute concentration of Rayleigh particles via scattering microscopy

“…Holographic NTA, leveraging a k-space holographic sensing platform, has extended the observation volume and allowed for 3D single-particle tracking (8). Additionally, Rytov microscopy for nanoparticles identification (RYMINI) microscopy, utilizing a modified Hartmann mask, has enabled label-free imaging of NPs with more than 90% accuracy in classification (9). These advancements have promoted label-free detection of NPs in solution, as detailed in Table S1.…”