shining light on neurons adrian negrean 17/04/09
TRANSCRIPT
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.