Intravital two‐photon microscopy: focus on speed and time resolved imaging modalities

Summary: Initially used mainly in the neurosciences, two‐photon microscopy has become a powerful tool for the analysis of immunological processes. Here, we describe currently available two‐photon microscopy techniques with a focus on novel approaches that allow very high image acquisition rates compared with state‐of‐the‐art systems. This improvement is achieved through a parallelization of the excitation process: multiple beams scan the sample simultaneously, and the fluorescence is collected with sensitive charge‐coupled device (CCD)‐based line or field detectors. The new technique's performance is compared with conventional single beam laser‐scanning systems that detect signals by means of photomultipliers. We also discuss the use of time‐ and polarization‐resolved fluorescence detection, especially fluorescence lifetime imaging (FLIM), which goes beyond simple detection of cells and tissue structures and allows insight into cellular physiology. We focus on the analysis of endogenous fluorophores such as NAD(P)H as a way to analyze the redox status in cells with subcellular resolution. Here, high‐speed imaging setups in combination with novel ways of data analysis allow the generation of FLIM data sets almost in real time. The implications of this technology for the analysis of immune reactions and other cellular processes are discussed.