Optical methods and fluorescent proteins to probe and manipulate cellular and subcellular processes have been the driving force behind many recent breakthroughs in biology and medicine. Recent developments in photonics now allow the study of single identified synaptic contacts between neurons both ex vivo and in vivo. At the CNCR we combine optical methods with electrophysiology to study many aspects of brain function ranging from network connectivity and function in the living animal to transport and release of single secretory vesicles.

Live cell imaging Facilities at the CNCR

Multi-Photon Microscopy (2PLSM)
Two-photon platforms at the CNCR are used to image neuronal morphology and calcium dynamics in cortical networks and individual spines of the rodent brain. We image brain slices and are developing methods to image brain morphology and activity in a living animal.

Total Internal Reflection Fluorescent Microscopy (TIRFM)
TIRF microscopy selectively illuminates fluorescent molecules in a thin optical plane near the plasma membrane of cells that are grown on glass coverslips. This special feature is ideal to visualize the movement of secretory vesicles near their target membrane or insertion of receptors and other trans-membrane proteins into the membrane. TIRF microscopy provides vertical resolution and signal-to-noise ratio that is unmatched by any other technique using living cells. At the CNCR, we use TIRF microcopy to visualize dynamic events at the plasma membrane of neurons and chromaffin cells.

Tandem-Illumination Microscopy (TIM)
Many pressing questions in neuroscience require the possibility to simultaneously photo-manipulate cells and record the consequences at rapid time scales using optical methods in single neurons, acute brain slices as well as in the intact brain. We use tandem-illumination microscopy on TIRF, wide-field and multi-photon microscopes to photo-uncage compounds (like calcium, glutamate, diacylglycerol), photo-activate fluorescent proteins (like PA-EGFP, Keima, Dronpa), silence/activate neurons (rhodopsins) and conduct FRAP experiments on multiple locations in a single neuron or on multiple neurons within an intact network.

Non-Linear Microscopy
In close collaboration with the Laser Center VU we develop novel non-linear microscopy methods to detect brain activity, identify disease biomarkers and interfere with pathogenic pathways in brain tissue of animal models of human brain disease and ultimately in the human brain. We for instance use third-harmonic generation (THG) microscopy for high-resolution deep-tissue imaging without external contrast agents. THG microscopy provides an intrinsic probe of tissue structure and we are exploring the applicability of THG for its practical use during brain surgery.

Wide-field fluorescent microscopy
Wide-field microscopy in combination with electrophysiology is used to study single vesicle transport and release events in cultured neurons.

Electron Microscopy
To resolve synaptic organelles/structures as well as the subcellular distribution of neuronal proteins at nanometer level we apply electron microscopy together with immuno-(gold)-labelling techniques. Correlative light- and electron microscopy (CLEM) allows ultrastructural identification of organelles/proteins seen by advanced live-cell imaging techniques within the CNCR (see above). Our EM equipment is housed in the EM support facility of NCA (see EM support facility).