Microglia constitute a highly specialized network of tissue-resident immune cells that is important for the control of tissue homeostasis and the resolution of diseases of the CNS. Little is known about how their spatial distribution is established and maintained in vivo. Here we establish a new multicolor fluorescence fate mapping system to monitor microglial dynamics during steady state and disease. Our findings suggest that microglia establish a dense network with regional differences, and the high regional turnover rates found challenge the universal concept of microglial longevity. Microglial self-renewal under steady state conditions constitutes a stochastic process. During pathology this randomness shifts to selected clonal microglial expansion. In the resolution phase, excess disease-associated microglia are removed by a dual mechanism of cell egress and apoptosis to re-establish the stable microglial network. This study unravels the dynamic yet discrete self-organization of mature microglia in the healthy and diseased CNS.

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.

The photophysical properties of a new water-soluble fluorescent dye based on the perylene diimide chromophore are investigated at the ensemble and single molecule level. Water solubility was obtained by attaching hydrophilic substituents in the bay region. The fluorophore has an absorption maximum at 567 nm and a high quantum yield of fluorescence (0.6). In addition, the two-photon absorption cross section has higher or comparable values with the ones reported in the literature for other water-soluble dyes used in biological studies. The most important finding is its improved photostability in single molecule experiments when compared to other water-soluble dyes. This is demonstrated by the increased observation time of immobilized single molecules, which is almost 10 times higher than for commonly studied fluorophores. A possible reason is the lower intersystem crossing rate constant of perylene diimide in comparison, for example, to rhodamine derivatives. A similar conclusion can be drawn for this chromophore in an aqueous solution, because single molecule measurements using fluorescence correlation spectroscopy demonstrate a very low contribution of the triplet state in the autocorrelation function. The possibility of imaging the new molecule in living cells is demonstrated by one-photon confocal microscopy and by fluorescence lifetime microscopy with two-photon excitation.