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.