Table 2.

Advantages and limitations of visual and imaging methods and techniques used for measuring mucus properties

Method	Advantages	Limitations
Beads/microspheres visualization and tracking in vitro [87–89], ex vivo [20, 90] and in vivo [91]	- Easily visualized. - Potential for in vivo tracking.	- Most applicable in vitro and in small animal models ex vivo/in situ. - Data analysis can require careful application of modelled calculations that present opportunities for error. - Some of the in vivo applications may require expensive visualization set up.
Histology & Immunostaining (using specific antibodies, Lectins, PAS/AB) [90, 92, 93]	- Inexpensive, easily visualized. - Specific antibodies can provide precise mucin detection and localization or co-localization with other molecules. - Fluorescent lectins can be used for semi-quantitation by fluorescence intensity measurement and are inexpensive	- Applicable mostly in vitro and ex vivo. - When scoring systems are utilized, careful analysis by multiple individuals blinded to group treatments are necessary. - Fixation and washing steps might result in mucus being washed away. - Lectins bind to different carbohydrates in the oligosaccharide chains of glycoproteins and glycolipids and therefore are not mucin specific.
Electron microscopy [94]	- In depth view of micro anatomical structures of cells and gel-forming mucins.	- Difficult to detect more than one type of gel mucin at the same time. - The type and duration of fixation is very important for retention of mucin structures.
X-ray imaging analysis [1, 20, 95–98]	- Novel techniques provide in vivo ability to detect mucus - Very recent X-ray synchrotron [96] and quazi-monochromatic X-ray phase-contrast imaging techniques have been applied successfully to measure MCT in vivo, together with lung motion. - Can detect mucus plugs in humans in vivo.	- Expensive set-up and materials. - Potential for exposure to harmful rays. - At the moment, are limited in utility for longitudinal in vivo studies. - Highly specialized equipment and skills
Volumetric – submucosal gland bubble visualization [55, 99]	- Detect ex vivo/in vitro increased output from single cell or multiple glands under normal or treatment conditions. - The total volume technique gives a simple quantitation of total mucus secretion ex vivo/in vitro for a constant time period at baseline and/or after treatment.	- Volume output may not necessarily comprise only mucus but can also include changes in serous gland- and non-glandular cell-secretions. - Currently not applicable in vivo.