Simultaneous photo-manipulation and non-linear optical imaging of single synapses and neuronal networks in intact brain

Optical methods and fluorescent proteins to probe and manipulate cellular and subcellular processes have proven to be very powerful tools and have been the driving force behind many recent breakthroughs in biology and medicine. Recent developments in photonics have led to increased spatial and temporal resolution and now allow the study of single identified synaptic contacts between neurons as well as large-scale neuronal networks. Brain tissue strongly scatters light. Non-linear optical methods using near infra-red and infra-red wavelengths (700 – 1600 nm) have gained momentum in recent years and have shown to successfully penetrate living brain tissue and image neuronal activity with high spatial and temporal resolution. Recent technological advances in targeted photo-manipulation of single synapses and neurons now allow applying these techniques to intact living neuronal networks and the living brain. To simultaneously photo-stimulate single synapses or single neurons, and image the biologically relevant fluorescence changes that occur during or immediately following laser stimulation, we aim to establish a novel microscopy platform in the Netherlands, capable of combining photo-manipulation with simultaneous, uninterrupted non-linear optical imaging in acute brain slices as well as in the intact brain at the millisecond to hours time scale. The novel custom-built platform will contain two synchronized scanners that deliver two independent long-wavelength femto-second laser beams of different frequencies so that image observation is not interrupted during laser light stimulation. This system opens new avenues to ask essential fundamental and translational questions in cellular and systems neuroscience and address them in intact brain tissue, for instance, modulation of synaptic transmission at single synapses, synapse pathology in mental retardation, neuronal network connectivity in autism or synaptic membrane potential dynamics. In addition, novel non-linear methods will be developed and optimized, such as second and third harmonics generation, to visualize neuronal networks and neuronal activity. The expertise of the participating laboratories warrants optimal embedding of this novel technology in internationally recognized research, and the availability of many photonic tools, such as fluorescently tagged proteins, organelles and caged compounds. Furthermore, the host institute offers specialized personnel, technicians, PhD-students and staff, to perform and supervise the proposed projects.