Single droplet-level aerosol deposition dynamics in functioning alveoli in mice during physiological ventilation

Introduction: The transport of aerosol droplets into the distal lung alveoli plays a crucial role in (i) drug delivery, (ii) pathogen transmission, and (iii) chronic respiratory diseases. While clinical imaging methods (e.g. CT and MRI) have observed the aerosol distribution within the bulk lung, their spatial and temporal resolution is too low to capture individual particles at the alveolar level. Histological techniques offer high spatial resolution, but only provide a static snapshot of the lung and cannot depict the dynamic transport of individual particles. Consequently, transport of single particles within alveoli has thus far been described by mathematical models without direct experimental visualization. However, there is a pressing need to characterize the dynamics of single particle transport to probe pathogen-immune interactions from the first moments of contact and to evaluate the efficacy of aerosol-based therapeutics.

Our innovative "crystal ribcage" imaging platform (Banerji & Grifno, et al., Nature Methods, 2023) addresses this challenge, enabling real-time microscopy of single alveoli as fluorescent aerosols (nebulized small molecules or live pathogens) are delivered to a functioning mouse lung during dynamic ventilation. Using ultrafast microscopy, we have visualized the arrival of single particles in individual distal alveoli, and quantified their impact and diffusion along alveolar walls with spatial heterogeneity that suggests that transport is not governed by diffusion alone, as was previously hypothesized. Additionally, we have probed the effect of gas density on alveolar-scale aerosol transport. These initial efforts will enable future studies of real time, cellular-level response of the lung to inhaled pathogens and therapeutics.

Materials and

Methods: The lung-heart bloc was extracted from mTmG-C57/BL6 mice tagged with a red fluorescent reporter gene and positioned within a crystal ribcage imaging device (Banerji & Grifno, et al., Nature Methods, 2023). Ventilation was administered using controlled positive pressure, as in clinical ventilation, or via negative pressure as in spontaneous breathing, by a custom-built ventilator and nebulizer using either room air or helium gas as the ventilation medium. Media or blood was circulated through the lung-heart system using cannulae inserted into the pulmonary artery and left atrium, enabling perfusion into the lungs and collection of the effluent exiting through the pulmonary veins. To investigate how particle transport depends on size, small molecular weight fluorescent dye (500 Da) was dissolved in saline and nebulized into the mouse lung trachea while actively ventilating the lungs within the crystal ribcage at physiological rates (2-3 Hz), to achieve delivery of fluorescent droplets representative of inhaled drug or pathogen aerosol transport. Utilizing ultrafast confocal fluorescence microscopy of the lung surface during particle delivery allowed real-time examination of the spatial and temporal distribution of micron-scale aerosol droplets within the distal alveoli at speeds up to 200 frames per second.

Results, Conclusions, and Discussions: We utilized the crystal ribcage to observe the transport dynamics of individual fluorescent aerosols nebulized into the lung during dynamic ventilation (A). Single aerosols (5-10 μm) appeared in the most distal lung alveoli only 50 milliseconds after starting inhalation. Fluorescent aerosols remained in the airspace for 60-80 milliseconds, before impacting onto the alveolar wall. After contacting the wall, droplets spread thinly across the alveolar surfactant layer in approximately 20 milliseconds. Moreover, particles exhibited spatial heterogeneity between adjacent alveoli. Certain alveoli received multiple droplets while neighboring alveoli received none. Additionally, droplets did not uniformly deposit on the alveolar surface. Instead, droplets preferentially deposited on the sides of the alveolar wall that were aligned with the motion of the bulk tissue during either exhalation or inhalation (B). This trend was conserved when particles traveled in room air or under low-density helium (C), indicating that particle inertia or convection, instead of diffusion, may dominate aerosol transport in alveoli, which was not previously thought possible based on computational predictions. Additionally, this heterogeneity suggests that epithelial and immune cells within the same alveolus may have different levels of exposure to pathogens, pollution, or drugs over the lifetime of the lung. Lastly, we found that most of the inhaled droplets entered the alveolar airspace during the exhalation phase of the breathing cycle (D), indicating that there is a phase lag of 50-100 milliseconds between the physical deformation of lung and the airflow that enters the distal alveoli (C). Under helium there was no correlation between droplet arrival and the ventilation cycle, meaning that aerosol temporal dynamics are dependent on gas density (E).

In conclusion, the crystal ribcage preserves the respiratory function of the isolated mouse lungs while facilitating precise imaging of micron-scale aerosol transport in the alveoli. We have used this technology to measure the spatiotemporal distribution of aerosol particles within individual alveoli in a functional lung for the first time. These findings offer a unique opportunity to investigate the physics of aerosol transport beyond computational modeling and to probe the first interactions between aerosolized drugs or pathogens and lung cells, in real time.

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