Two-photon microscopy in pulmonary research

Abstract

As the lung is constantly exposed to both innocuous and potentially noxious antigens, a thorough understanding of both innate and adaptive immune responses in this organ is of the essence. Imaging modalities such as magnetic resonance imaging, positron emission tomography, and confocal microscopy have expanded our knowledge about various molecular processes and cellular responses in the lung. Two-photon microscopy has evolved into a powerful tool to observe cellular interactions in real time and has markedly expanded our understanding of the immune system. Recently, two-photon microscopy has also been utilized to image the murine lung. As immune responses in the lung differ from those in other non-lymphoid tissues, this technique holds great promise to advance our knowledge of the biology that underlies a wide spectrum of pulmonary diseases.

Fig. 1

Experimental setup for imaging of lung explants with custom-built 2P microscope. a Medium is pumped from the reservoir trough a feeding line to the imaging chamber and a vacuum line evacuates the medium from the chamber. The amount of fluid flowing through the chamber is controlled by the pump. The temperatures of the chamber and fluid are maintained at 37°C throughout the experiment with separate heaters. b The acquisition console is depicted with multiple components which control the intensity of the laser as well as the movement of the stage and the focus of the objective. Images of the tissue can be observed on the monitor in real time. c Lung tissue is immersed in warm medium within the imaging chamber. d Two different filters (570 and 458 nm, respectively) housed inside dichroic cubes. Filters can be exchanged based on experimental design

Fig. 2

2P microscopy of lung explants. Different fluorescent probes were used for this experiment to show the versatility of the system. T cells, isolated from a wild-type C57BL/6 mouse and labeled with fluorescent dyes CMTMR or CMAC, were injected into a C57BL/6 CD11c-EYFP host. The left lung was imaged 24 h after adoptive transfer. a Quantum dots (12 µl resuspended in 150 µl of phosphate-buffered saline), injected 10 min prior to sacrifice, label blood vessels in red (small clear arrow). T cells labeled with CMTMR are observed in orange (large white arrow), and T lymphocytes labeled with CMAC are seen in cyanide (white arrowhead). Host CD11c+ cells with dendritic morphology appear yellow-green (small white arrow). SHG depicts collagen in blue, which can be seen delineating air spaces (large clear arrow). b Alveolar macrophages display characteristic nuclear shadow (clear arrows). Peri-alveolar blood vessels have been labeled with quantum dots and appear red (white arrows). Space bar represents 30 µm