sonder la structure des assemblages moléculaires de la ...100 120 140 160 180 1pf; kress et al.,...
TRANSCRIPT
Microscopie optique pour la biologie : sonder la structure des assemblages moléculaires de la
cellule aux tissus
Sophie Brasselet
J. Duboisset, P. Ferrand, H. Rigneault, J. Savatier
A. Kress, F.Z. Bioud, P. Gasecka
Institut Fresnel
Marseille, France
http://www.fresnel.fr/mosaic
Nonlinear microscopy
Fluorescence correlation
microscopy
Single molecule imaging
Polarized microscopy
Phase imaging
Nano-plasmonics
Thermo-plasmonics
Instrumentation and methodologies for biophotonics and nanophotonics
Detergent extracted to reveal actin cytoskeleton Scanning Electron Microscopy - lan Wells, M.D., D.Sc., DB Stolz, Ph.D.
5mm
Imaging structured bio-molecular assemblies
15mm
(DPPC) lipid membrane
http://www.sdsc.edu
2-10 nm
~ 3 nm
Resolution in optical microscopy: ~200nm
Molecular orientational order reports structural information
Fluorescence
Originates from dipoles excitation/radiation
Incoherent processes :
1PF, 2PF
mabs
mem
W
1PF: tuning the excitation - single molecule
r
X
Y
0 50 100 150 0
a
Flu
ore
scen
ce
r
exciting field Absorption dipole
Time and space averaging
0 50 100 150 0
a F
luo
rescen
ce
m
S. Brasselet et al. in Springer Series Fluoresc, Springer-Verlag (2013)
Many molecules
r
X
Y m
y
Polarimetric Fluorescence (1PF) imaging
Objective lens NA=1.2
Galvos
Dichroic mirror
Confocal pinhole APD
Motorized HWP
CW Laser
0 50 100 150 0
a
Inte
nsity
a
XY
X
Y E a
(A,B) (r,y) Gasecka et al. Biophys J 2009
Duboisset et al. JPC B 2012
Kress et al., Biophys J 2013
Probing lipid order in a cell membrane
di-8-ANEPPQ (lipid probe, 1mM)
inserted in the cell membrane of
COS7 cells, 37°C
x
y
Side view
20
40
60
80
100
120
140
160
180
Y ()
Top view
Psi 500 (deg)
20
40
60
80
100
120
140
160
180
Rho 600(deg)
20
40
60
80
100
120
140
160
180
Y
10-23-41COS7 ANEPPQ 625membrane contour, ROI = 3x3, 6000 < I < 23258cone
1000
2000
3000
4000
5000
Map of Psi 500, for Chi2 < threshold
50
100
150
0 20 40 60 80 100 120 140 160 1800
50
100
150
Rho
Psi
Rho = 101.976 54.9542
80 100 120 140 160 1800
10
20
30
40
50
60Psi500 = 121.58 17.9242
Map of Chi2 500
50 100 150
20
40
60
80
100
120
140
0
5
10
15
20
0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2
x 104
0
2
4
6
8
10
Chi2 vs. Nb Photons
<Y> = 123° ± 18°
Y
Cell membrane « macroscopic » folds
Psi 500 (deg)
20
40
60
80
100
120
140
160
180
y
x
Total fluorescence intensity 500
20 40 60 80 100 120 140
20
40
60
80
100
120
140 500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Psi 500 (deg)
20
40
60
80
100
120
140
160
180
1PF; Kress et al., Biophys J. 2013
Probing lipid order in a cell membrane di-8-ANEPPQ / COS7 cell
Y = 107 Y = 120 Y = 150
10 nm 30 nm
Subdiffraction wrinkles
VanRheneen et al. Mol Cell Biol (2002) 2PF : Gasecka et al. Biophys J. 2009
10 20 30 40 50 60 70
40
50
60
70
80
90
100
110
Rho (deg)
20
40
60
80
100
120
140
160
180
Psi (deg)
20
40
60
80
100
120
140
160
180
Y
Structural information in actin sress fibers
Alexa 488 – phalloidin in fixed COS 7 cells
Intensity
Mavrakis et al. Nat. Cell Bio (2014)
Probing the architecture of actin networks in tissues and its modulation by septin
Alexa Fluor 488-
Phalloidin
Septin
Wild type Septin mutant
Y
Y Fluorescence
Towards high frame-rate polarimetric imaging
Phase imaging of Tumor cells on collagen fibrils
J. Savatier, S. Monneret,M. Guilbert (UCRA)
Imaging local membrane tension?
X. Wang et al. Rev sci instrum (2013)
Parallel confocal imaging by spinning disk
Parallel confocal imaging (spinning disk unit Yokogawa, CSU-X1-M1)
Stack time :30*72ms=2.5s
5mm 5mm
See us at MiFobio 2014 !
Imaging vibrational modes with
Coherent Raman Anti-Stokes Scattering : CARS
Probing lipid order without staining?
Anti-Stokes
Pump wave wP
wAS= wP+WR
Stokes wS= wP-WR WR
Vertical
clamp
2PF imaging in the mouse spinal cord
Axon imaging (CFP, exc. 800nm)
EAE mice
F. Debarbieux, INT Marseille
100µm
2PF / CARS imaging in the mouse spinal cord
Axon imaging / myelin imaging
Fixed spinal cord,
EAE mouse
30mm depth
Score 0 (Healthy) Score 5 Score 2.5
Myelin (CARS) Axons (2PF, CFP)
2PF / CARS imaging in the mouse spinal cord
5µm 5µm
Remission
stage EAE Healthy
Score 0 (Healthy) Score 5 Score 2.5
Myelin (CARS) Axons (2PF, CFP)
2PF / CARS imaging in the mouse spinal cord
Immune cells (2PF, YFP)
5µm 5µm
Remission
stage EAE Healthy
A CARS polarization resolved experiment
Pump
Stokes
sample
Forward detector
40x
1.2
0.5
50x
AP
D
BS
x
z
Epi detector
AP
D
filters
2PF
FWM/CARS
SHG
Rotating half wave plate
40x40um
exciting fields
Linear and nonlinear susceptibilities
...)3(
0
)2(
0
)1(
0 = EEEEEEP
Measurement of molecular order parameters
2PF, THG, FWM, CARS
Ewp(a), Ews(a)
Y
r
Measurement of molecular order parameters
Sn : Order parameter
2PF, THG, FWM, CARS
jn : orientation of the order parameter
High S2: high order
Low S2: isotropy
High S4: Sharp order
Low S4: Smooth order
Measurement of molecular order parameters
2PF, THG, FWM, CARS
CD2 stretching (2100cm-1)
Polarized CARS in Multilamellar vesicles
S2
0
0.05
0.1
0.15
0.2
0.25
CARS : CH2 2845cm-1
S2 S2
10mm
Collaboration F. Debarbieux, INT Marseille
Fu et al. J Neurosci Res. 2007
EM
demyelination
Polarized CARS in spinal cord myelin
Conclusion
Revealing more and more details of molecular packing is
possible using nonlinear polarization resolved microscopy
- 3D information accessible
- Scattering in tissues can be
caracterized / circumvented
Prospectives :
Spinal cord, lipid bonds 20 40 60 80 100 120 140 160 180 200
20
40
60
80
100
120
140
160
180
200
S2 (molecular order)
0
0.05
0.1
0.15
0.2
0.25
Collaborations
Financing
ANR, CNRS, A*mideX, FBI, FLI
Région PACA
EU : Europhotonics EM, ITN FINON
D. Marguet (CIML Marseille Luminy)
M; Mavrakis (IBDM Marseille Luminy)
P. Jeannesson (Univ. Reims)
F. Debarbieux (INT Marseille)
M. Roche, P. Refregier (I Fresnel Marseille)
At mosaic - Institut Fresnel
J. Savatier
Thanks
H. Rigneault
P. Ferrand
J. Duboisset
F.Z. Bioud
P. Gasecka A. Kress