Lung Imaging in Animal Models

Intravital Imaging of Pulmonary Immune Response in Inflammation and Infection

“…The selectivity for specific wavelengths facilitates the visualization of different fluorescent objects as a bright structure against a dark background (Lichtman and Conchello, 2005 ; Lindon et al, 2016 ). Although fluorescence microscopy has been widely used to study tissues in vivo (Lindon et al, 2016 ), its application in lung IVM is limited to superficial layers and low resolution (Witte, 1992 ; Presson et al, 2011 ; Lefrançais et al, 2017 ).…”

“…Both laser scanning and spinning disk confocal microscopes pass a single beam of laser light through the pinhole. It should be noted that while confocal laser scanning microscopy focuses the light through one small pinhole in order to sequentially scan the sample point by point, confocal spinning disk microscopy exploits multiple pinholes for simultaneous confocal illumination (Masedunskas et al, 2012 ; Sanderson et al, 2014 ; Lefrançais et al, 2017 ). Therefore, either X–Y-deflection of the laser or a spinning disk with a spatial array of pinholes and automated-focus (z-axis) control enables the visualization of sequential optical sections of the specimen and three-dimensional images.…”

“…Moreover, imaging with confocal microscopy requires a bright sample in order to negate the need for a strong excitation signal which may otherwise cause photodamage and photobleaching. In this regard, spinning disk confocal microscopy is superior as its high acquisition speed helps to restrict photobleaching (Croix et al, 2006 ; Masedunskas et al, 2012 ; Lefrançais et al, 2017 ).…”

“…Multiphoton microscopy involves application of an infrared laser light source and subsequent absorption of lower energy photons by a fluorophore inside the tissue (Tauer, 2002 ; Norman, 2005 ; Presson et al, 2011 ; Lefrançais et al, 2017 ). This method uses long-wavelength photons to penetrate farther into tissues and allow for imaging of thicker sections (Lefrançais et al, 2017 ). Using multiphoton microscopy, lungs have been imaged to a depth of ~500 μm (Croix et al, 2006 ).…”

“…This has the effect of restricting observation of dynamic processes which possess high temporal resolution (Niesner et al, 2007 ; Presson et al, 2011 ). Despite these drawbacks, multiphoton microscopy is nonetheless advantageous for IVM studies because the infrared excitation light is less prone to scatter, which allows for deeper penetration into the tissue (Croix et al, 2006 ; Lefrançais et al, 2017 ).…”