1. Shining light on neurons Adrian Negrean 17/04/09

2. Outline Neuro-basics Patch-clamping Optical readout of neuronal activity Label-free imaging of live brain tissue

3. 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

4. equivalent circuit with voltage-dependent ion channels Neuro-basics

5. Patch-clamping credits: Rogier Poorthuis

6. Optical readout of neuronal activity Ca 2+ 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 ?

7. 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

8. Neuron grown without glia to suppress background Same staining and imaging as before Membrane potential measurements from different locations Membrane potential sensitive dye at work

9. ANNINE6-plus results the membrane potential is stepped to increasingly depolarized potentials

10. Nonlinear microscopy tools Two-photon excitation microscopy OPE TPE excitation in the NIR low scattering of tissue deeper imaging reduced phototoxicity/bleaching tighter focus

11. condenser lens Objective lens sample incident fluorescence SHG THG 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 levels, but is enhanced when a two-photon transition can occur THG is generated at boundaries with a refractive index mismatch

12. SHG and membrane potential sensitive dyes SHGTPF S-pol. P-pol. 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

13. 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

14. 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

15. Label-free imaging of live brain tissue using THG at 3 x 420 nm Sub-cellular structuresRed blood cells

16. Summary Neurons and electrophysiology Nonlinear microscopy tools Membrane potential sensitive dyes Label-free imaging of live brain tissue with sub-cellular resolution

17. Acknowledgements and many thanks go to… Dr. Stefan Witte Prof. Marloes Groot Prof. Huibert Mansvelder Hans Lodder as well as the other people from the “Neuro-Laser” think-tank my supervisors Dr.ir. Erwin PetermanProf. Johannes de Boer Dr. Mattijs de Groot Assist. Prof. Ruud Toonen and many thanks to my colleagues from the electrophysiology dept.