Post on 11-Jan-2016
description
Chemical Force Spectroscopy on single living cells
E. Dague, D. Alsteens, J.P. Latgé, Y. Dufrêne
Kaminsky et al., 2006. Mycological Research
Aspergillus hyphae and conidia as seen by SEM
Classical way of probing microbial hydrophobicity
• Hydrophobicity– MATH, MATS– HIC– Adhesion test– Contact angle
Not specific of hydrophobic character and under electrostatic interactions influence
(Mozes, Rouxhet 1987 ; van Loosdrecht et al., 1990 ;Gaboriaud et al., 2006)
vortex 1 min
Increasing CH3 surface fraction
Probing hydrophobic forces at the nanoscale
Alsteens, Dague et al., Langmuir 2007
0 2 4 6 8 100
10
20
30
100 % CH3
75 % CH3
50 % CH3
25 % CH3
0 % CH3
Fre
quen
cy (
%)
Adhesion force (nN)
Hydrophobicity calibration
Hydrophobicity quantification and calibration
0 20 40 60 80 1000
2
4
6
8
10
Surface fraction CH3 (%)
0 20 40 60 80 1000
2
4
6
8
10
Ad
he
sio
n f
orc
e (
nN
)
3 (%)
Single Aspergillus conidia trapped in a pore as seen by contact mode AFM
500 nm 500 nm
sample holder
membrane
water single cell
tape
-800 -600 -400-5
-4
-3
-2
-1
0
100 nm1000
pN
0
1
2
3
4
5
Adhesion force (pN)
0 1000 2000 3000 4000 5000 6000
Hydrophobic Force Microscopy of A. fumigatus
Freq
uenc
y (%
)
Dal : native
100 nm 100 nm
-300 -200 -100 0 100 200
X Axis Title
100 nm1000
pN
-800 -600 -400-5
-4
-3
-2
-1
0
100 nm1000
pN
Adhesion force (pN)
0
5
10
15
Freq
uenc
y (%
)
0
1
2
3
4
5
Hydrophobic Force Microscopy of A. fumigatus
0 1000 2000 3000 4000 5000 6000
Dal : native
Dal + NaOH 1M
100 nm 100 nm
100 nm 100 nm
-300 -200 -100 0 100 200
X Axis Title
100 nm1000
pN
-800 -600 -400-5
-4
-3
-2
-1
0
100 nm1000
pN
100 nm1000
pN
Hydrophobic Force Microscopy of A. fumigatus
Adhesion force (pN)
Freq
uenc
y (%
)
0 1000 2000 3000 4000 5000 60000
5
10
15
0
5
10
15
0
1
2
3
4
5
Dal : native
Dal + NaOH 1M
Mutant without hydrophobins
100 nm 100 nm
100 nm
100 nm
100 nm
100 nm
Dague, Alsteens et al., Nanoletters 2007
100 nm
Adhesion force map revealing highly correlated structural and hydrophobic
heterogeneities
100 nm 100 nm
Dague, Alsteens et al., Nanoletters 2007
Kaminsky et al., 2006. Mycological Research
Aspergillus hyphae and conidia as seen by SEM
Ger
min
atio
n
Real time imaging of conidia during germination
Dague et al., Biophysical Journal 2007
0 min 20 min
120 min 180 min
60 min
120 min200 nm200 nm
200 nm200 nm200 nm
200 nm
Structural dynamics of single germinating conidia
0 min 60 min
180 min
90 min
120 min 210 min
100 nm
200 nm
100 nm
200nm
100 nm
100 nm
Evolution of hydrophobicity with germination time
Dague et al., Biophysical Journal 2007
• Hydrophobicity can be probed and quantified at the nanoscale
• Direct visualization of correlated morphological and chemical evolution
• Biological process (germination), chemical modifications can be observed, in real time
Take Home Message
• Y. Dufrêne D. Alsteens J.P. Latgé
Chemical Force Spectroscopy on single living cells
E. Dague, D. Alsteens, J.P. Latgé, Y. Dufrêne
Toward a global bio-surface analysis :Combined use of AFM, XPS and SIMS
Dague et al., Langmuir 2008
A B
C D
N 1s/5
NameNH3+C-NH2, (C=O)-NH
Pos.401.31399.72
FWHM1.3391.339
L.Sh.GL(30)GL(30)
Area680.8
10982.1
%Area5.834
94.166
NameNH3+C-NH2, (C=O)-NH
Pos.401.68399.87
FWHM1.4561.456
L.Sh.GL(30)GL(30)
Area381.5
4428.0
%Area7.927
92.073
NameNH3+C-NH2, (C=O)-NH
Pos.401.51399.84
FWHM1.4031.403
L.Sh.GL(30)GL(30)
Area728.3
6492.8
%Area10.07989.921
NameNH3+C-NH2, (C=O)-NH
Pos.401.26399.75
FWHM1.3721.372
L.Sh.GL(30)GL(30)
Area756.9
9052.3
%Area7.712
92.288
0
10
20
30
40
50
60
70
80
Inte
nsity
(1
02
co
un
ts/s
)
410 408 406 404 402 400 398 396 394 392Binding Energy (eV)
Dal
Rod B
Rod A
Rod AB
Intensity (a. u.) XPS N peak
Dal Rod B Rod A Rod AB
0,00
0,01
0,02
Nor
mal
ized
inte
nsiti
es i/
i tot CH
4N+: Gly
C2H
6N+: Ala
C4H
8N+: Pro
C4H
10N+: Val
C5H
12N+: Leu
C9H
8N+: Trp
C8H
12N0+: Tyr
0.00
0.01
0.02
WT ∆RodB ∆RodA ∆RodA ∆RodB
No
rma
lize
d in
ten
sitie
s i/i
tot
C2H3O2-
NI, SIMS
CN- NI, SIMS