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Shining light on neurons Adrian Negrean 17/04/09 Outline Neuro-basics Patch-clamping Optical readout of neuronal activity Label-free imaging of live brain tissue Neuro-basics distinctive morphology common intracellular components specialized in transducing and conveying information to/from the environment have mastered the use of ion channels can modulate their membrane potential Neuro-basics equivalent circuit with voltage-dependent ion channels Patch-clamping credits: Rogier Poorthuis Optical readout of neuronal activity Ca2+ imaging is an indirect way of measuring the electrical activity of a neuron good S/N but slow fluorescence dynamics focus on membrane potential fluorescent sensors how does it work ? Membrane potential sensitive dye at work ANNINE6-plus stained cultured neuron grown on glia, widefield fluorescence imaging The neuron is patch-clamped and the voltage steps are increased gradually Besides the (hopefully) obvious “flickering”, a small and annoying motion artifact is present Membrane potential sensitive dye at work Neuron grown without glia to suppress background Same staining and imaging as before Membrane potential measurements from different locations ANNINE6-plus results the membrane potential is stepped to increasingly depolarized potentials Nonlinear microscopy tools Two-photon excitation microscopy OPE excitation in the NIR low scattering of tissue deeper imaging reduced phototoxicity/bleaching tighter focus TPE Nonlinear microscopy tools Second and third-harmonic generation microscopy SHG and THG are forwardly generated SHG requires non-centrosymmetric media SHG does not involve excitation of molecular Objective lens incident levels, but is enhanced when a two-photon sample transition can occur fluorescence SHG THG THG is generated at boundaries with a refractive index mismatch condenser lens SHG and membrane potential sensitive dyes SF9 cells with extracellular application of FM4-64 dye SHG at 470 nm, detected with bandpass filter SHG generated mainly in the outer membrane In progress Apply intracellularly new dyes and measure their SHG sensitivity to the membrane potential S-pol. P-pol. SHG TPF Label-free imaging of live brain tissue using THG at 3 x 420 nm (500 x 500 mm) Cell bodies of neurons Dead neurons Blood vessels “Stuck” red blood cells Axons and dendrites Label-free imaging of live brain tissue using THG at 3 x 420 nm (150 x 150 mm) Nucleus and nucleolus of neurons Unidentified cellular organelles Dead neurons Red blood cells Label-free imaging of live brain tissue using THG at 3 x 420 nm Sub-cellular structures Red blood cells Summary Neurons and electrophysiology Nonlinear microscopy tools Membrane potential sensitive dyes Label-free imaging of live brain tissue with sub-cellular resolution Acknowledgements and many thanks go to… as well as the other people from the “Neuro-Laser” think-tank my supervisors Prof. Marloes Groot Dr. Stefan Witte Prof. Huibert Mansvelder Hans Lodder Dr.ir. Erwin Peterman Dr. Mattijs de Groot Prof. Johannes de Boer Assist. Prof. Ruud Toonen and many thanks to my colleagues from the electrophysiology dept.