Characterization of optical clearing mechanisms in muscle during treatment with glycerol and gadobutrol solutions.

The recent increasing interest in the application of radiology contrasting agents to create transparency in biological tissues implies that the diffusion properties of those agents need evaluation. The comparison of those properties with the ones obtained for other optical clearing agents allows to perform an optimized agent selection to create optimized transparency in clinical applications. In this study, the evaluation and comparison of the diffusion properties of gadobutrol and glycerol in skeletal muscle was made, showing that although gadobutrol has a higher molar mass than glycerol, its low viscosity allows for a faster diffusion in the muscle. The characterization of the tissue dehydration and refractive index matching mechanisms of optical clearing was made in skeletal muscle, namely by the estimation of the diffusion coefficients for water, glycerol and gadobutrol. The estimated tortuosity values of glycerol (2.2) and of gadobutrol (1.7) showed a longer path-length for glycerol in the muscle.

Broadband spectral verification of optical clearing reversibility in lung tissue.

The increase of tissue transparency through sequential optical immersion clearing treatments and treatment reversibility have high interest for clinical applications. To evaluate the clearing reversibility in a broad spectral range and the magnitude of the transparency created by a second treatment, the present study consisted on measuring the spectral collimated transmittance of lung tissues during a sequence of two treatments with electronic cigarette (e-cig) fluid, which was intercalated with an immersion in saline. The saline immersion clearly reverted the clearing effect in the lung tissue in the spectral range between 220 and 1000 nm. By a later application of a second treatment with the e-cig fluid, the magnitude of the optical clearing effect was observed to be about the double as the one observed in the first treatment, showing that the molecules of the optical clearing agent might have converted some bound water into mobile water during the first treatment.

Tissue Processing.

Tissue processing is the technique by which fixed tissues are made suitable for embedding within a supportive medium such as paraffin, and consists of three sequential steps: dehydration, clearing, and infiltration. In most clinical and research settings, tissue processing is accomplished using an automated tissue processor, with or without microwave-assistance. To ensure high-quality results, processing protocols should be tailored to tissue size and composition by modifying variables such as reagents used and the timing of the various steps. Herein, we provide an overview of tissue processing theory and outline a basic tissue processing method for use with a conventional automated fluid transfer/enclosed processor. The principles described will assist readers in optimizing tissue processing for their own projects.

Three-Dimensional Whole-Organ Characterization of the Regional Alveolar Morphology in Normal Murine Lungs.

Alveolar architecture plays a fundamental role in the processes of ventilation and perfusion in the lung. Alterations in the alveolar surface area and alveolar cavity volume constitute the pathophysiological basis of chronic respiratory diseases such as pulmonary emphysema. Previous studies based on micro-computed tomography (micro-CT) of lung samples have allowed the geometrical study of acinar units. However, our current knowledge is based on the study of a few tissue samples in random locations of the lung that do not give an account of the spatial distributions of the alveolar architecture in the whole lung. In this work, we combine micro-CT imaging and computational geometry algorithms to study the regional distribution of key morphological parameters throughout the whole lung. To this end, 3D whole-lung images of Sprague-Dawley rats are acquired using high-resolution micro-CT imaging and analyzed to estimate porosity, alveolar surface density, and surface-to-volume ratio. We assess the effect of current gold-standard dehydration methods in the preparation of lung samples and propose a fixation protocol that includes the application of a methanol-PBS solution before dehydration. Our results show that regional porosity, alveolar surface density, and surface-to-volume ratio have a uniform distribution in normal lungs, which do not seem to be affected by gravitational effects. We further show that sample fixation based on ethanol baths for dehydration introduces shrinking and affects the acinar architecture in the subpleural regions. In contrast, preparations based on the proposed dehydration protocol effectively preserve the alveolar morphology.