• 2P-Fluorescence life time imaging to quantify intracellular calcium

    We are currently using FLIM combined with 2-photon excitation to investigate the biophysical properties of endogenous calcium binding proteins within neurons. The advantage of this method is that it yields quantitative measures of absolute intracellular calcium and of the dye concentraion with very high precision. This allows us to use dye loading methods such as AM ester dyes or electroporation.
    The basic principle of the calibration procedure is that calcium free and calcium-bound dye each contribute a different decay time constant to the overall fluorescent decay. The relative amplitude of each component can then be used to infer the actual free calcium concentration.
    Our GaAsP-hybrid detector is very sensitive and together with a dedicated deconvolution approach enables us to track intracellular calcium in dendrites and spines with high temporal resolution.

  • 2-photon neurotransmitter uncaging

    If infra-red light of a Ti:Sa lasere (720 nm) is strongly focussed with an objective it can be used to photo-convert appropriate molecules in volumes as small as a few femtoliters. We use this thechniques together with a scanning microscope to activate caged-transmitter molecules such as glutamate and GABA. These transmitters can in this way be applied with very high spatial and temporal precision to neurons and glial cells in brain slices and closely mimick synaptic transmitter release. If the cells are held in patch clamp mode tiny currents of a few pA can be resolved and ascribed to the specific membrane area targeted with the infrared laser.

  • 2P-Glutamate uncaging & 2P-Imaging

    In certain types of hippocampal neurons we observe prominent and very localized calcium transients even if the electrical response is as small as a few millivolt.
    Imaging line scans along the dendrite allow as to extract temporal calcium handling properties, buffer capacity and the speed of calcium diffusion.

  • 3D-Electron microscopy

    The function of neuron-NG2 synapses and their fate upon differentiationg of NG2 cells is still unknown. Also, the functions of many presynaptic proteins, such as RIM1, in classical synapses are still poorly understood. We believe a good correlative functional-ultrastructural approach is mandatory for new insights in these fields. Classical electron microscopy, as used for many tens of years now, is limited in z-resolution by the sectioning capabilities of diamond knives and its limitation in obtaining larger series of z-sections.
    We use focused ion beam milling together with field emission SEM to acquire high resolution three dimensional image stacks (5 nm x 5 nm x 5 nm) of neuron-glial and neuronal synapses in wild-type and mutant mice (collaboration with Susanne Schoch, Valentin Stein, Alf Lamprecht). The example on the left shows a perforated synapse (encircled) in three orthogonal views and a cube-like rendering of a subvolume of the acquired data.

    See the video