Intravital microscopy of the lung: minimizing invasiveness

Abstract

In vivo microscopy has recently become a gold standard in lung immunology studies involving small animals, largely benefiting from the democratization of multiphoton microscopy allowing for deep tissue imaging. This technology represents currently our only way of exploring the lungs and inferring what happens in human respiratory medicine. The interest of lung in vivo microscopy essentially relies upon its relevance as a study model, fulfilling physiological requirements in comparison with in vitro and ex vivo experiments. However, strategies developed in order to overcome movements of the thorax caused by breathing and heartbeats remain the chief drawback of the technique and a major source of invasiveness. In this context, minimizing invasiveness is an unavoidable prerequisite for any improvement of lung in vivo microscopy. This review puts into perspective the main techniques enabling lung in vivo microscopy, providing pros and cons regarding invasiveness.

A permanent window for the murine lung enables high-resolution imaging of cancer metastasis

“…This protocol is extremely invasive, prevents healing of the chest wall, potentially causes damage to the alveoli from overexpansion of the lung tissue, and runs a high risk of infection. These techniques fall in line with the conclusions presented in Fiole and Tournier’s recent review of the field, in which they observe that “strategies developed in order to overcome movements of the thorax caused by breathing and heartbeats remain the chief drawback of the technique and a major source of invasiveness” 15 . This leads them to the conclusion that, “In broad terms, greater invasiveness leads to better resolution” 15 .…”

“…These techniques fall in line with the conclusions presented in Fiole and Tournier’s recent review of the field, in which they observe that “strategies developed in order to overcome movements of the thorax caused by breathing and heartbeats remain the chief drawback of the technique and a major source of invasiveness” 15 . This leads them to the conclusion that, “In broad terms, greater invasiveness leads to better resolution” 15 . The WHRIL addresses these concerns, is (to our knowledge) the first permanent window to be developed for mice, and provides a solution that is both high resolution and minimally invasive.…”

“…This makes it possible for the mouse to recover from anesthesia, breathe independently (both between and during imaging sessions), and live with this ‘transparent ribcage’ for weeks. Immobilization of the lung tissue, accomplished with a thin (<10 μm) layer of adhesive, obviates the need for high-speed gated imaging 4 or other specialized ventilation 14 or image-processing techniques 5,15 . This results in a simplified experimental setup during imaging and makes the WHRIL accessible for use in a wide range of applications by any laboratory with a multiphoton microscope.…”

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

“…Wagner and Filley 27 studied the natural motion of the canine lung and designed their surgical protocol to locate their implanted window over a relatively stationary region while Wagner utilized vacuum in his window surgical preparation to immobilize the tissue 28 . Since that time, a variety of techniques have been utilized to image the lung including: bronchus clamping, sequential apnea and gated imaging, oversampled acquisition, gluing of the lung lobe and vacuum 34 . Each of these has its advantages and disadvantages and no one technique has emerged as superior to another 34 .…”

“…Since that time, a variety of techniques have been utilized to image the lung including: bronchus clamping, sequential apnea and gated imaging, oversampled acquisition, gluing of the lung lobe and vacuum 34 . Each of these has its advantages and disadvantages and no one technique has emerged as superior to another 34 . For example, bronchus clamping and sequential apnea alter the normal exchange of gases in the lung and may cause atelectasis.…”