Recent advances in dynamic intravital multi‐photon microscopy

Abstract

Standard multiphoton laser scanning microscopy (MPLSM) has revolutionized our view of physiologic and pathologic processes in living organisms by enlightening different aspects of cellular choreography in immune responses, that is, cellular motility and co-localization. To understand cellular communication on a molecular level, novel transgenic reporter mice have been generated. In parallel, MPLSM systems have been developed, which make it possible for this technique to be more widely used to address crucial immunological questions. Here, we review the latest progress concerning transgenic mouse technology and multiphoton imaging capacities and discuss further developments which will enable us to visualize all around monitoring and quantification of cellular function at a molecular level directly in vivo. ' 2011 International Society for Advancement of CytometryKey terms multi-photon laser scanning microscopy; intravital imaging IN the past, cytometry of cellular and molecular mechanisms underlying immune reactions was mainly performed ex vivo. These analyses focused on characterization of the players in the immune system as single entities, without taking tissue context into account. On the other hand, microscopic analyses of intact tissues were restricted to fixed samples. Thus, the dynamic nature of the immune response, with cellular interactions and migration being two of its most prominent features, could not be analyzed until recently.The development of multiphoton intravital microscopy and its application to address immunological questions has changed this over the past decade. For instance, typical motility patterns of lymphocytes could be tracked for the first time in timelapse movies taken in living tissue. Lymphocytes in secondary lymphoid organs were shown to move with a median instantaneous three-dimensional (3D) velocity of between 5 and 10 lm/min, and are therefore able to cover distances of several hundred micrometers within a few hours (1,2). Compared with other imaging techniques (summarized in Table 1), multiphoton microscopy is the only technology able to capture the dynamic nature of these processes in deep tissue, as it allows fast acquisition of 3D image stacks (for example: 300 3 300 3 30 lm 3 with a resolution of 512 3 512 3 11 voxels can be recorded every 15 s). Until now, most of the work in the field has focused on the analysis of motility parameters (e.g., the velocity at which cells migrate, their displacement, directed versus random migration) and the duration and type of cellular interactions using fluorescent reporter mice to distinguish between different cell subsets. These studies have already contributed tremendously to our understanding on how the immune system works in vivo, and at the same time have sparked the desire to visualize subcellular events by intravital microscopy. As novel photoactivatable fluorescent proteins and genetically encoded Förster resonant energy transfer (FRET)-biosensors of sufficient brightness for in vivo imaging

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