Aerosol and droplet transmission is central to the spread of respiratory pathogens, which have profound impacts on human life, animals, and the global economy. The devastating COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has highlighted the urgent need for robust and rapid methods to detect, monitor, and diagnose airborne viruses. However, until now, there has been a lack of real-time and in-situ technology for observing airborne viral aerosol droplets [Arnaout et al., 2020; Morawska et al., 2022].

In response to the urgent need for effective detection and diagnosis methods, significant advancements have been made in the field of SARS-CoV-2 detection [Pal et al., 2023]. These advancements encompass a range of approaches, including molecular techniques, biosensors, and novel sampling methods [Pal et al., 2023; Pan et al., 2019]. Different types of polymerase chain reaction (PCR) have been used for diagnosis and saving lives during COVID-19 pandemic. However, these methods are non-in-situ technologies and require minimal 100 copies of viral RNA per milliliter of transport media [Arnaout et al., 2020]. Yet, accurately determining and investigating the real-time 4D physicochemical characteristics (viability, viral load/size distribution, shape, phase, and surface properties) of airborne viruses remains a challenge.

To address these challenges and contribute to the advancement of rapid airborne virus detection methods and diagnostics, we have developed Nano-Digital in Line Holography Microscopy (Nano-DIHM) [Pal et al., 2023; Pal et al., 2021]. Nano-DIHM enables the detection, diagnosis, and investigation of viruses' sizes, shapes, and surface properties within seconds, without the need for sample treatment like RT-PCR. Nano-DIHM utilizes direct imaging of airborne viral particles (Fig. 1), followed by particle-by-particle measurement, estimation of settling velocities using image analysis and a particle tracking algorithm. These analyses can be performed in real-time at the observation location or in offline mode using collected samples. Nano-DIHM operates as a two-stage process: 1) recording holograms, which are images of viral-loaded samples (Fig. 1a), and 2) numerically reconstructing these viral-loaded images to extract information about the viruses (Fig. 1b-c). The configuration of the Nano-DIHM technology is straightforward, consisting of a laser source (Pinhole) and a camera, as illustrated in Figure 1b. A pinhole laser (L) emits a wave at λ = 405 nm, which illuminates the objects and generates a highly magnified diffraction pattern (hologram) on a screen (CMOS).

Health risks associated with exposure to airborne virus particles are influenced by the shape and size distribution of aerosols containing infectious viruses [Comber et al., 2021; Morawska et al., 2022]. The novel Nano-DIHM technology enables the in-situ, real-time characterization of the physicochemical properties of viral-loaded aerosol particles (Fig. 2). To demonstrate the feasibility and reliability of Nano-DIHM, we conducted benchmark experiments focused on determining the 4D physicochemical properties of viral-loaded aerosols and droplets in both air and water samples. We selectively detected active MS2 bacteriophages (MS2), inactivated SARS-CoV-2 and RNA fragments, as well as an MS2 mixture containing metallic and organic materials [Pal et al., 2023]. Our findings revealed that the aerosolized MS2 viral-laden particles exhibited a bimodal distribution, with peaks at approximately 60-200 nm and 2-3 microns, respectively (Fig. 2). These experiments suggested an evaporation-condensation process of viral-laden particles in the air. Additionally, the introduction of a surfactant aerosol such as titanium oxides caused a shift towards larger sizes in the distribution of airborne viral-laden MS2 particles, leading to altered settling velocities and impacting transmission processes (Fig. 2).

In another example, we have shown that the physicochemical characterisation (size, shape and phase) of the heat inactivated SARS-CoV-2 viral laden aerosols in air in real time (Fig. 3). By utilizing artificial intelligence in combination with Nano-DIHM, we were able to detect and distinguish SARS-CoV-2 viral-laden particles from mixed samples of SARS-CoV-2 and MS2 bacteriophage (Table 1). The output results indicated "YES" for SARS-CoV-2 and "NO" for MS2 [Pal et al., 2023]. This suggests that our imaging-based method holds promise as an attractive approach for rapid testing and investigating real-time 4D physicochemical characterization of viruses. However, it is important to note that the current prototype does have certain limitations [Pal et al., 2023] including the accuracy of the output which may varies or decrease depending on the complexity of the sample matrix. Nevertheless, these challenges can be overcome by developing an extensive library or databank encompassing multiple sample matrices.

The currently developed Nano-DIHM offers rapid and comprehensive detection, classification, and determination of the physicochemical properties of SARS-CoV-2 in both air and water. Nano-DIHM can operate in static or dynamic mode on-site or in the laboratory, providing results in less than a minute with an accuracy exceeding +90%. In contrast, conventional COVID-19 testing methods are costly, time-consuming, and lack in-situ or real-time capabilities. A promising aspect of Nano-DIHM is its potential for simultaneous measurements of diverse particle types, enabling the identification of both active and past infections from multiple viruses. Real-time tracking of SARS-CoV-2 or any future viruses empowers policymakers with valuable knowledge for more informed responses in future epidemic management. Ultimately, these advancements will enable timely interventions, enhance outbreak management strategies, and protect public health in the face of emerging viral threats, and reducing substantial economic losses.

References

Arnaout, R., R. A. Lee, G. R. Lee, C. Callahan, C. F. Yen, K. P. Smith, R. Arora, and J. E. Kirby (2020), SARS-CoV2 Testing: The Limit of Detection Matters, bioRxiv : the preprint server for biology, doi:10.1101/2020.06.02.131144.

Comber, L., et al. (2021), Airborne transmission of SARS-CoV-2 via aerosols, Reviews in Medical Virology, 31(3), e2184, doi:https://doi.org/10.1002/rmv.2184.

Morawska, L., G. Buonanno, A. Mikszewski, and L. Stabile (2022), The physics of respiratory particle generation, fate in the air, and inhalation, Nat. Rev. Phys., 4(11), 723-734, doi:10.1038/s42254-022-00506-7.

Pal, D., M. Amyot, C. Liang, and P. A. Ariya (2023), Real-time 4D tracking of airborne virus-laden droplets and aerosols, Communications Engineering, 2(1), 41, doi:10.1038/s44172-023-00088-x.

Pal, D., Y. Nazarenko, T. C. Preston, and P. A. Ariya (2021), Advancing the science of dynamic airborne nanosized particles using Nano-DIHM, Commun. Chem., 4(1), 170, doi:10.1038/s42004-021-00609-9.

Pan, M., J. A. Lednicky, and C. Y. Wu (2019), Collection, particle sizing and detection of airborne viruses, J Appl Microbiol, 127(6), 1596-1611, doi:10.1111/jam.14278.