# Comparing Imaging Depth of Intravital Lung Imaging Using Perfluorocarbon‐Based Liquid Ventilation With Tissue Clearing for Deep‐Tissue Imaging

## TL;DR

This study compares methods to improve imaging depth in the lungs, finding that perfluorocarbon ventilation doesn't help, but tissue clearing does.

## Contribution

The study reveals that tissue clearing with lipid removal improves imaging depth, while PFC ventilation does not.

## Key findings

- Partial liquid ventilation with PFC does not enhance imaging depth in the lung.
- Ex vivo tissue clearing with lipid removal improves optical imaging penetration depth.
- PFC ventilation allows homogeneous nanoparticle delivery to alveoli for real-time observation.

## Abstract

Intravital lung imaging has been employed to study physiological and pathophysiological processes related to nanoparticle deposition in the alveolar lung, particularly in the context of air pollution and drug delivery. However, optical imaging depth is limited, often attributed to the refractive index (RI) mismatch at the alveolar air‐tissue interface. To investigate this, we evaluated two complementary strategies. First, we demonstrated that eliminating the RI mismatch via partial liquid ventilation with oxygenated perfluorocarbon (PFC) did not enhance the imaging depth. A second approach, utilizing ex vivo optical tissue clearing (with RI matching), was only successful in improving imaging penetration depth if it included removal of scattering lipids such as pulmonary surfactant. Nevertheless, partial liquid ventilation with PFC in vivo enabled the homogeneous delivery of nanoparticles to the alveoli, allowing real‐time observation of their interactions with lung epithelium. This finding opens new avenues for studying inhaled particulates and optimizing inhalation‐based drug delivery.

Partial liquid ventilation with oxygenized perfluorocarbon (PFC) is used to assess the limitations of imaging depth, caused by the refractive index mismatch between air and surfactants in the mouse lung. PFC does not help to overcome this limitation. Only removing scattering lipids and decolorization ex vivo, with tissue optical clearing, enhance the penetration depth of optical imaging in the lung. Nevertheless, partial liquid ventilation with PFC allows for homogenously delivering and following nanoparticles to the alveoli.