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Picosecond fluorescence lifetime imaging microscopy as a new tool for 3D structure determination of macromolecules in cells


Simultaneous multi-parameter Picosecond Fluorescence Lifetime Imaging Microscopy (PFLIM) has been developed, based on the novel concept of Time- and Space-Correlated Single Photon Counting (TSCSPC), which was made possible by the recent introduction of time- and space-sensitive microchannel plate (MCP) photomultipliers with delay-line (DL) and quadrant anodes (QA). The QA-detector has a space resolution of 100 micron, resulting in 200x200-space channelsat < 10 ps time resolution and an unprecedented dynamic range of 107. Simultaneous acquisition of laser-correlation time, absolute arrival time, xy-coordinates, emission wavelength, and polarization of each individual photon is possible. From the stored list-mode data, any combination of parameters can be obtained resulting, e.g., in sub-second frame-rate movies of mobile fluorescent particles including picosecond dynamics along the trajectory of the moving object. This capacity led to the development of Vehicle-Based Micro-Spectroscopy (VBMS), allowing it to follow the movement and interaction of a suitably labelled virus in living cells. Photobleaching and photodynamic reactions do not distort the obtained data, since these effects can be separated in time, due to the movie-style acquisition. Biophysical information in living cells could be obtained under non-invasive conditions, such as ultra-low excitation light and ultra-low labeling, due to ultra-sensitive detection: (i) By observing the fluorescence dynamics at parallel and perpendicular orientation, fluorescence anisotropy decays of intercalated ethidium were obtained, revealing the chromatindynamics in the nucleus of living cells (Fig.1), (ii) DASPMI-labelled living cells allowed to obtain 3-exponential fluorescence dynamics (100 ps - 3 ns) of individual mitochondria (Fig.2), yielding new insights in the structural organization of the inner membrane, (iii) Fused GFP-proteins were expressed in living cells, whose fluorescence anisotropy dynamics revealed its 3D environment (Fig.3), and (iv) Understanding of photosynthesis in living plant leaves was advanced by comparing the 3-exponential fluorescence decays of individual chloroplasts in individual cells (Fig.4), at varying environmental conditions.