Shining light on neurons

Adrian Negrean17/04/09

Outline

Neuro-basics

Patch-clamping

Optical readout of neuronal activity

Label-free imaging of live brain tissue

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

Neuro-basics

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 ?

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

Membrane potential sensitive dye at work

ANNINE6-plus results

the membrane potential is stepped to increasingly depolarized potentials

Nonlinear microscopy tools

Two-photon excitation microscopy

OPE TPE

excitation in the NIR

low scattering of tissue

deeper imaging

reduced phototoxicity/bleaching

tighter focus

condenserlens

Objectivelens

sample

incident

fluorescence SHGTHG

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

SHG and membrane potential sensitive dyes

SHG TPF

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

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…

Dr. Stefan WitteProf. Marloes Groot

Prof. Huibert Mansvelder Hans Lodder

as well as the other people fromthe “Neuro-Laser” think-tank

my supervisors

Dr.ir. Erwin Peterman Prof. Johannes de Boer

Dr. Mattijs de Groot Assist. Prof. Ruud Toonen

and many thanks to my colleagues from the electrophysiology dept.