Stabilized imaging of immune surveillance in the mouse lung

Abstract

Real-time imaging of cellular and sub-cellular dynamics in vascularized organs requires image-resolution, image-registration, and demonstrably intact physiology to be simultaneously optimized. This problem is particularly pronounced in the lung in which cells may transit at speeds > 1 mm/sec, and in which normal respiration results in large-scale tissue movements that prevent image registration. Here, we report video-rate, two-photon imaging of a physiologically intact preparation of the mouse lung that is at once stabilizing and non-disruptive. The application of our method provides evidence for differential trapping of T cells and neutrophils in mouse pulmonary capillaries and enables observation of neutrophil mobilization and dynamic vascular leak in response to stretch and inflammatory models of lung injury in mice. The system permits physiological measurement of motility rates of > 1 mm/sec, observation of detailed cellular morphology, and could be applied to other organs and tissues while maintaining intact physiology.

Intravital microscopy of the lung: minimizing invasiveness

“…The animal preparation prior to imaging is performed according to this consensual protocol [11,18,[23][24][25][26][27][28][29]:…”

“…The lung is maintained by suction with a force great enough to lessen the physiological motion into a level compatible with high-resolution dynamic microscopic imaging [18,24,28,29,[43][44][45][46][47][48][49][50]. In particular, the resolution reached using this technique recently permitted observation of dendritic cell dendrites (Figure 2d) [44] and patrolling monocytes controlling tumor metastasis to the lung [51].…”

“…While mouse physiology requires a breathing frequency of 106 bpm to 230 bpm, depending upon other factors such as strain and weight [57], it is possible to modulate this frequency to 130 bpm [28], 120 bpm [11,27,29], 100 bpm [25], or even 60 bpm for the recording period after which ventilation is returned to 100 bpm [26]. This decrease of the breathing frequency noticeably increases stability of the images.…”

“…This decrease of the breathing frequency noticeably increases stability of the images. In some cases, mice are ventilated with pure oxygen in order to compensate the likely hypoxia resulting from a slower breathing frequency [28,29]. Animals may also be treated with pancuronium bromide to avoid all muscle-related chest motion during assisted mechanical ventilation [24,58].…”

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

“…With this system, the stomach, liver, intestine, rectum, pancreas, spleen, lymph nodes, testis, muscle, and skin of anesthetized mice have been successfully imaged (35,46) and unpublished data). Another group has also reported a simple aspiration fixation system for lung imaging (47). Therefore, although they are not suitable for longitudinal applications (as discussed below), we predict that aspiration fixation systems will become the method of choice for tissue stabilization in intravital imaging.…”