Two-photon intravital imaging of leukocyte migration during inflammation in the respiratory system

Abstract

Two-photon intravital imaging is a powerful method by which researchers are able to directly observe biological phenomena in live organisms. Researchers in various biomedical research fields have applied two-photon imaging to a variety of target organs by utilizing this technology’s ability to penetrate to significant depths with minimal phototoxicity. The mouse respiratory system in inflammation models is a good example, as two-photon intravital imaging can provide insights as to how the immune system is activated in response to inflammation within the respiratory system. Inflammation models can be generated via influenza viral, bacterial, or lipopolysaccharide injection. To exteriorize the lungs or trachea, thoracotomy or tracheotomy is performed, respectively; the appropriate combination of inflammation induction and organ exposure is selected depending on the study purpose. On the other hand, visualizing the movement of leukocytes is also an important component; to this end, immune cell populations of interest are either labeled via the genetic attachment of fluorescent proteins or stained with antibodies or dyes. With the proper selection of methods at each step, twophoton intravital imaging can yield visual evidence regarding immune responses to inflammation.

Integration of immune cells in organs-on-chips: a tutorial

“…Thus, the molecular mechanisms by which immune cells transmigrate depend on their microenvironment, specifically the vascular characteristics of the respective tissue. Unfortunately, live imaging of extravasation in internal organ vessels can only be carried out after a complex surgical procedure or with expensive equipment such as a two-photon microscope to perform intravital imaging ( Kim et al, 2019a ). Surgery not only causes stress for the animals, which may activate immune cells, but anesthesia also influences vascular dilation and immune cell activation, making it crucial to identify alternative methods to study the mechanisms of inflammation and infection in vitro ( Hickey and Westhorpe, 2013 ).…”

Intravital Imaging of Pulmonary Immune Response in Inflammation and Infection

“…With recent advances of fluorescent probes, fluorescent proteins and exogenous fluorophores, IVM allows the detection of molecular events with subcellular resolution in real time in the intact animal (Presson et al, 2011 ). Tracking the movement, morphology and behavior of leukocytes and blood vessels in the lung requires labeling methods such as the use of fluorescent dyes, transgenic mice, or fluorescently stained antibodies (Kim et al, 2019 ). Moreover, knockout mice can be used to study the cellular mechanisms involved in pulmonary pathophysiological conditions (Aird, 2003 ; Gill et al, 2015 ) ( Table 1 ).…”

“…Changes in microvessel diameter and blood flow under various physiological and pathophysiological conditions is widely visualized via intravenous administration of fluorophore-conjugated dextran or albumin (Kim et al, 2019 ). Higher molecular weight dextran (70 kDa) is particularly useful for visualization of microvessels because extravasation through the intact endothelium is minimal.…”

“…Administration of lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, induces an inflammatory response by activation of immune cells through Toll-like receptor (TLR)-4 (Aderem and Ulevitch, 2000 ; Opal, 2010 ; Kim et al, 2019 ). Following LPS administration as well as bacterial infection, neutrophils are the first group of leukocytes that respond to inflammation.…”