Christophe Leterrier

NeuroCyto lab
NICN, CNRS-AMU UMR7259

Photonic imaging
in neurobiology

M2 Neurosciences 2017 UE 15.1

Christophe Leterrier September 28 th , 2017 Master M2
 Visualize, localize, detect, measure

architecture function

structure dynamics physiology

confocal live cell imaging in vivo imaging
super-resolution FRAP, SPT probes (Ca2+, Na+, V)
resolution speed speed
avoid toxicity imaging depth
Christophe Leterrier September 28 th , 2017 Master M2
 photon budget

Eric Betzig

imaging
spatial depth
resolution

speed
Christophe Leterrier September 28 th , 2017 Master M2
 photon budget

structure
imaging
spatial depth
resolution

speed
Christophe Leterrier September 28 th , 2017 Master M2
 Zeiss Campus http://zeiss-campus.magnet.fsu.edu/articles/basics/fluorescence.html

Christophe Leterrier September 28 th , 2017 Master M2

 adhesions (vinculine)
actin (phallodin)
nucleus (hoechst)

Epithelial cell
Christophe Leterrier September 28 th , 2017 Master M2
 Numerical aperture (NA)

Refractive index (RI)

higher NA: smaller anti-point
higher RI: smaller anti-point

Lets introduce the PSF

Christophe Leterrier September 28 th , 2017 Master M2

 Point Spread Function (PSF) Resolution:
Rayleigh criterion

Y Z
X
X Airy disk

Rxy = /(2*RI*NA)
200 nm

Rz = 2/(RI*NA)2
500 nm

Christophe Leterrier September 28 th , 2017 Master M2

 single molecules

PSF
200 nm

dendritic spines

PSF
Izeddin 2011

1 m

Christophe Leterrier September 28 th , 2017 Master M2

 dendrite 1 - 4 m diameter actin microtubules
axon 0.5 - 2 m diameter
actin filaments 6 nm width
microtubules 25 nm width

S. Halpain
node of Ranvier 1 m length
dendrite spine 1 m length, 0.1 m neck diameter
post-synaptic density 0.5 m width, 30 nm cleft Caspr Nav Kv1

M. Rasband
axonal varicosity 1-3 m diameter
synaptic vesicle 40 nm diameter
synaptotagmin actin

Izeddin 2011
Willig 2006

Dani 2010

1 m 1 m

Christophe Leterrier September 28 th , 2017 Master M2

Christophe Leterrier September 28 th , 2017 Master M2
 Microtubules Dendritic spines Synapses
Conventional
Super-resolution

S. Kaech 5 m Izeddin 2011 Dani 2010 0.2 m

Christophe Leterrier September 28 th , 2017 Master M2

 Schermelleh 2010
STED

Testa 2012

Tonnesen 2011
YFP
GFP
organotypic slice
actin

Christophe Leterrier September 28 th , 2017 Master M2

 Schermelleh 2010
Conventional 3D-SIM

soluble fill
actin Fiolka 2012
Christophe Leterrier September 28 th , 2017 Master M2
Stochastic Optical Reconstruction Microscopy
single Gaussian localization
emitter t precision

1 m

200 nm 20 nm
wideeld blinking STORM
sequence

5 m

ATIP/AVENIRLeterrier
Christophe 2016 June 21st, 2016
September 28 th , 2017 Christophe Leterrier -Master
NeuroCyto
M2
 STORM, PALM

Schermelleh 2010
actin-bound probe Bassoon (presynaptic)
Homer1 (postsypatic)

0.2 m
Izeddin 2011 Dani 2010
Christophe Leterrier September 28 th , 2017 Master M2
 actin actin Xu 2013
Xu Science
widefield STORM

2 m 5 m
Christophe Leterrier September 28 th , 2017 Master M2
 actin Xu 2013

1 m

IV-spectrin NTer Xu 2013

IV-spectrin CTer Leterrier 2015

Christophe Leterrier September 28 th , 2017 Master M2

neurofascin
actin hotspots
AIS axon
trails

20 m 2 m

actin Z
Y

500 nm

+400 Z
Y
Z (nm)
--300
5 m
Ganguly 2015

Christophe Leterrier September 28 th , 2017 Master M2

 spectrins hotspots
ring hotspot trails
trail
2 m

actin Z
Y

500 nm

+400 Z
Y
Z (nm)
--300
5 m
Ganguly 2015

Christophe Leterrier September 28 th , 2017 Master M2

 II-spectrin actin actin 10 m
Z (nm)
--300 +400

2
1
actin (3D-STORM) 2 m
Z Z
Y Y

1 2 500 nm 500 nm

Christophe Leterrier September 28 th , 2017 Master M2

 replica EM

Korobova 2010

actin by STORM

Andersen 1999
Christophe Leterrier September 28 th , 2017 Master M2
 Dani 2010

Christophe Leterrier September 28 th , 2017 Master M2

 Dani 2010

Christophe Leterrier September 28 th , 2017 Master M2

 Dani 2010

Christophe Leterrier September 28 th , 2017 Master M2

 Dani 2010

Christophe Leterrier September 28 th , 2017 Master M2

 Three-color STED microscopy:
The periodic actin/spectrin mesh stops at presynaptic sites

Sidenstein 2016
Christophe Leterrier September 28 th , 2017 Master M2
 500 m 2 mm

5 m 20 m
Chen 2015

2.5 m 10 m

Christophe Leterrier September 28 th , 2017 Master M2

 Homer
Bassoon
YFP (fill)

500 nm
Chozinsky 2016

5 m

Christophe Leterrier September 28 th , 2017 Master M2

#1 1m #2
(at net)

#3 #4

Christophe Leterrier September 28 th , 2017 Master M2

Christophe Leterrier September 28 th , 2017 Master M2
 10 m
Leterrier 2004

CB1-GFP
HEK-293 cell
Christophe Leterrier September 28 th , 2017 Master M2
 photon budget

imaging
spatial depth
resolution

speed
Christophe Leterrier September 28 th , 2017 Master M2
 Cultured
photobleaching (fluorescence fades) neurons
photoxicity (cells suffer, then die)

phase

GFP

A given quantity of light will be tolerated: photon budget

Christophe Leterrier September 28 th , 2017 Master M2
Rat hippocampal neurons between one and three days in culture

Christophe Leterrier September 28 th , 2017 Master M2

Christophe Leterrier September 28 th , 2017 Master M2
 inhibitory synapses
cultured neurons from transgenic mice
imaged every hour for 12 hours

VGAT-Venus
gephyrin-mCherry
VGAT-Venus
gephyrin-mCherry

2 m
Kuriu 2012
Christophe Leterrier September 28 th , 2017 Master M2
 Isolate a thin area: optical section
confocal, spinning disk, deconvolution
Total Internal Reflection Microscopy (TIRF)
Living neuron
fluorescent label

Christophe Leterrier September 28 th , 2017 Master M2

Snell Descartes law n1sin(i1)=n2sin(i2)
Light from objective to sample: glass n11.5 > medium n21.33
Above a certain angle, no refracted light : total reflection
On the sample side, only a ~200 nm light sheet

intensity

Steyer 2001
Christophe Leterrier September 28 th , 2017 Master M2
Exocytosis of receptors: 2-AR Microtubule growth: EB3

Yudowski 2005 Leterrier 2011

Christophe Leterrier September 28 th , 2017 Master M2
 Isolate a thin area: optical section
confocal, spinning disk, deconvolution
Total Internal Reflection Microscopy (TIRF)
Living neuron
fluorescent label

intracellular
membrane

Isolate mobility of a subset of probes

Fluorescence Recovery After Photobleaching (FRAP)
Single Particle Tracking (SPT)
Christophe Leterrier September 28 th , 2017 Master M2
 Photobleaching: fluorophores destroyed by illumination light
FRAP: bleach a small region and measure recovery
Assess turnover, diffusion, trafficking. Variant: photo-activation

Christophe Leterrier September 28 th , 2017 Master M2

 Photobleaching: fluorophores destroyed by illumination light
FRAP: bleach a small region and measure recovery
Assess turnover, diffusion, trafficking. Variant: photo-activation

Christophe Leterrier September 28 th , 2017 Master M2

Single molecule labeling Individual trajectories
secondary Ab +
quantum dot
primary Ab
epitope
membrane
protein

Diffusion coefficients distribution Mean Square Displacement

frequency (%)

MSD (t)
diffusion coefficient
time (s)
Christophe Leterrier September 28 th , 2017 Master M2
 Manzo 2015

Christophe Leterrier September 28 th , 2017 Master M2

 photon budget

physiology
imaging
spatial depth
resolution

speed
Christophe Leterrier September 28 th , 2017 Master M2
 widefield confocal

Better contrast
~100 m deep kidney section
Christophe Leterrier September 28 th , 2017 Master M2
 Sakakibara 2014

Christophe Leterrier September 28 th , 2017 Master M2

 transfected neuron at the surface of an ex-vivo slice (E14.5 mice)
imaged every 20 minutes for 12 hours
centrosome
plasma membrane

Sakakibara 2014

How to go deeper? In vivo imaging?

Christophe Leterrier September 28 th , 2017 Master M2
 concentration
of photons

no bleach outside focal spot

can go up to ~1 mm deep
Christophe Leterrier September 28 th , 2017 Master M2
 even behaving ones!
Christophe Leterrier September 28 th , 2017 Master M2
 2

10 m 5

Christophe Leterrier September 28 th , 2017 Master M2

Christophe Leterrier September 28 th , 2017 Master M2
 dendrite
Chereau 2015

1 m

axon

1 m
spine growth after glutamate uncaging LTP

500 nm
Christophe Leterrier September 28 th , 2017 Master M2
 0' vs 25'

200 nm

0' vs 50'
Lauterbach 2016

non-stim
200 nm
1 m
Christophe Leterrier September 28 th , 2017 Master M2
 photon budget

imaging
spatial depth
resolution

speed
Christophe Leterrier September 28 th , 2017 Master M2