BioAFM imaging
Nové směry v bioanalytické chemii
Jan Přibyl
CEITEC MU
Kamenice 5/A35, CZ-62500 Brno
pribyl@nanobio.cz
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Microscopy techniques - resolution
4
Scanning Probe Microscope
basic scheme
surface
probe
Movement
element
interaction
signal
Feedback
loop
Reconstruction
of image
5
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SNOM (=NSOM)
Scanning NearField Optical Microscopy
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SNOM – basic principles
Light diffraction limit - conventional optical microscopy:
λ/2 ~ 250 nm ( Abbe diffraction limit)
Real cases - optical resolution ~ λ, 500 nm
SNOM offers higher resolution around 50 nm
(or even < 30 nm), depending on tip aperture size.
Near-field = distance << wavelegth
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SNOM- simultaneous measurements of the:
- topography
- + optical properties (fluorescence)
direct correlation between surface nanofeatures and
optical/electronic properties.
Useful for the studying:
inhomogeneous material surfaces (nanoparticles, polymer
blends, porous silicon, biological systems)
History of NSOM
1928 roots trace back – letters between Edward Hutchinson
Synge and Albert Einstein
Technology developed in 1990’s:
Eric Betzig, et al. Science, 262, 1422-1425 (1993).
Prototype commercial available since 2000’s
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Scheme of SNOM
apparatus
Transmission
mode
probe
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NSOM images
Tissues images
Living cells
Epi-illumination
Near-field illumination
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Real instrument example
Ntgra Vita AURA, Ntgra Vita SPECTRA (NTMDT, Zelenograd, Russia)
Bringing light close to the surface
Optical fiber
+ tuning fork
AFM transparent
probe
+ light via
objective
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TERS
Tip Enhanced Raman
Spectroscopy
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Examples
Use TERS technology for DNA structure study
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Molecular Characterization of DNA Double Strand Breaks with
Tip‐Enhanced Raman Scattering
Angewandte Chemie International Edition
Volume 53, Issue 1, pages 169-172, 15 NOV 2013 DOI: 10.1002/anie.201307271
http://onlinelibrary.wiley.com/doi/10.1002/anie.201307271/full#fig1 15
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Tip-Enhanced Raman Spectroscopy of Combed
Double-Stranded DNA Bundles
J. Phys. Chem. C, 2014, 118 (2),
pp 1174–1181
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Scanning Tunnelling Microscopy
STM
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STM - the first member of SPM family
Developed in 1982 by Gerd Binnig and Heinrich Rohrer
members of IBM in Zurich (Phys. Rev. Lett., 1982, vol 49,
p57)
1986 - Nobel prize in physics for their brilliant invention
1982 - Triumph of Scanning
Probe Microscopy - image of
silicon surface 7x7
reconstruction.
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STM tip
STM tip - conductive (metals - Pt, W, Pt/Ir)
STM microscopy uses the very top (outermost) atom at the tip and the
nearest atom on sample
Tip is not necessarily very sharp in shape (different from AFM)
Tip preparation:
- Cutting with scissors
- Electrochemical etching
- Other techniques such as FIB (and combination)
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STM modes
Constant height / constant current
Si (111) 7x7, 40nm
empty states image,
room temperature, dark
spots represent missing
atoms or adsorbates
Ag-Si (111) 10nm
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Atomic Force Microscopy
AFM
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AFM microscope basic scheme
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Tip and cantilever
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Cantilever and tip
6 mm
2mm
tl. 1 mm
Cantilever holder
cantilever
hrot
100-200 um
50-100 um
5 um
• Cantilever holder is quite universal
• Cantilever and tip – a variety of various types
Reflex layer (metal)
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Cantilevers
Material properties
– Stiffness Force Constant [N/m]
Force const.[N/m] 10-130 1-10 0.1-1.0 0.005-0.1
Material cryst. silicon pol. silicon glass Si3N4
Res. f. [kHz] 200-500 100-200 15-100 1-20
Special applications – conductive, colloid, magnetic, tip less, …
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Cantilever field
choose the one you like/need
Cantilever
characterization you may
find on box
AFM probes (micro)fabrication is
quite complex
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Hydra2R-100N
Hydra6V-100N
Cantilever (silicon nitride)
transparent (length 100um, thick. 2um)
Tip-area (silicon)
non-transparent (reflective)
Tip-area (silicon)
non-transparent (reflective)
Cantilever holder (4x2 mm)
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Tip properties
Shape – Curvature Radius R [nm]
Supersharp Standard Special app. Tip less
R
R 1 nm 10 nm 100 nm NA
29 Plateau Tip
FIB (Focus Ion Beam) post-fabrication of AFM probes (tip)
Cantilever fabrication
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Curvature radius (R) effect
SuperSharp tip
= real image
Standard tip
= R ~ 5-10nm
Blunt tip
= affecting real shape
and size
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Curvature radius (R) effect
SuperSharp tip
= real image
Standard tip
= R ~ 5-10nm
Blunt tip
= affecting real shape
and size
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Laser, photodiode a cantilever
Laser +
photodiode
Detection of
cantilever
bending
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Povrch vzorku
PZT skener
(PiezoElectric Tube)
PZT skener (PiezoElectric
Tube)
DFL
LF
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Automatic adjustment available
JPK Force Robot head
NTMDT Solver Next
Bruker Icon/FastScan
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AFM modes of operation
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Cantilever
deflection (DFL )
Contact mode
• Measured parameter - cantilever bending
(= deflection, DFL)
• Deflection ~ tip sample force interaction
• Hook`s law: F = - k * ∆h
F – force, k force constant (stiffness), ∆h – height (=deflection)
Semicontact mode (tapping mode, AC mode, oscillation mode, …)
• Measured parameter amplitude of oscillation (= magnitude, MAG, …)
A0
free amplitude
Aint
damped amplitude36
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PZT
Piezoelectric tubes
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Piezoelectric tubes PZT
Piezoelectrodes
• Hollow ceramic tubes
• Metal covered in selected parts
• Voltage application change of size
Notes + cautions
• Fragile
• High voltage applied
PZT – construction approaches of AFM
Scanning by probe
construction
• x,y,z axes movement by
head
• Oscillator in head
• Range x,y 100-150 um
• Range z 10-15 um
Scanning by sample construction
• x,y,z axes movement by sample
• Oscillator in head
• Range x,y 1-10 um
• Range z 1-3 um
•Low noise
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PZT: voltage-extension dependency
non-linear
Native (raw) AFM data are shifted.
Removed e.g. by polynomal regression of
data.
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Sample preparation
for AFM
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AFM sample preparation
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Concentration – surface density
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Substrates for preparation
of AFM samples
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Atomically flat surfaces
1. HOPG Highly Ordered Pyrolytic Graphite
•Kish’s graphite, waste in steel production
•Hexagonal planar structure
•C-C bond142 pm, layer-layer distance335 pm
•Conductive, highly hydrophobic
•Planar structure
•Synthetic form of graphite, high chemical purity
•Traditionally – substrate for SEM, STM i AFM (→
conductivity)
•Immobilization – spontaneous adsorption (→
hydrophobicity)
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Large areas
visible layers
Small areas
atomically flat
Atomically flat surfaces
1. HOPG Highly Ordered Pyrolytic Graphite
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•„Cat’s silver“, muscovite acc. to city
of Moscow
•Chem. structure: K2O·Al2O3·SiO2
•Hydrophilic surface
•Easy to be modified by chemical synthesis
•Immobilization by chemical bonding
as well as ionic interaction
•pKa ~ 3, physiological pH negative
surface charge
•Mica = silicate, hydrated SiO2 (~ Si-OH)
from the chemical point of view
Atomically flat surfaces
2. Mica (muscovite)
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Extremely flat on small and larger areas
Atomically flat surfaces
2. Mica (muscovite)
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•Inert metal
•Traditionally in
(bio)electrochemistry (i.e.
biosensors) - electrodes
•Conductive - STM + AFM
•Hydrophobic: spontaneous
non-selective adsorption of
molecules (proteins, DNA, …)
•Specific chemical binding of thiols
(-SH) – organic molecules +
cysteine
•Prepared usually by evaporation
•Adhesion layer for operation in
liquids (Al/Cr/Ti)
Sputtered gold layer
image by tapping
mode AFM
Other surfaces
3. Gold
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•Amorphous noncrystalline structure
•Lab glass composition: 75% SiO2 plus Na2O, CaO, borate and minor
additives
•Si-OH from chemical point of view
•Less hydrophilic comparing to mica
•Roughness much higher comparing to mica (production by pressing)
•Not suitable for individual molecules imaging with AFM
•Typically used together with optical microscopy – cell compartments,
whole cells
Other surfaces
4. Glass
AFM – optical image overlap
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Whole cells on glass
under AFM
glass
A. Thiooxidans
on glass
Human
sperm
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Fibroblast on
polystyrene
(PS)
• Most of lab supplies made of plastic (PP, PE, PS)
• No functional groups to be used in covalent binding
• PS – hydrophobic spontaneous non-specific adsorption of proteins
usually as underlying support (i.e. for cell attachment)
Non-modified polystyrene
(PS)
Other surfaces
5. Plastic materials
mica
Compare
roughness
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Immobilization procedures
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+ + +
1. Proteins
Surface: mica or HOPG (extremely flat)
P
pHIEP
no charge
P
+
+
+
+
+
+
+
+ P
-
-
-
-
-
-
-
OH
OH OH OH
SiSi
- - -
Si
Protein: charge is given by IEP + pH
Immobilization on mica: pKa (mica) < pH < IEP
P
+
+
+
+
+
+
+
+
- - -
Si
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Protein immobilization on HOPG
HOPG
A. Spontaneous (non-specific) adsorption of protein hydrophobic
surface (best results at zero charge pH = IEP)
P PP
Lysozyme
molecules on HOPG
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HOPG
COO-COO-
COO-
HOPG
Electrochemical oxidation
(E ~ 2 V vs. Ag/AgCl)
Adsorption of long chain
double-sided ions
(C16/C18)
•Ionic interation
•Covalent
binding (i.e. via
NHS-esters)
NH2 NH2
NH2 NH2
NH2
HOOC HOOC HOOC HOOC HOOC
HOPG HOPG
B. Ionic (specific) binding of molecules creation of charge/chem.
groups on HOPG surface
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- - - - - - - - - - - - - - - - - - - - - -
- -
- - - - - - - - - - - - - - - - - - Immobilization
problem:
DNA (sugar-phosphate bone) as well as mica – negative charge
under physiological pH
→ surface introduction of positive charge
2. DNA
Surface: mica or HOPG (extremely flat)
mica
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SiO
O
O
CH3
CH3
CH3
R
OH
Si
R
OO
OH
- CH3
OH
N(Et)3
OH OH OH
OHOH OH OHOH OH OO O OO O
R-propyltrimethoxysilane
+
cat.
Si
Si Si
Silanization
= chemical (covalent) modification of mica surface
- Aim: introduction of functional group
- Applicable also for: glass, quartz, silicon, titanium, …
- Strong basis catalysis
- Procedure can monitored by water contact angle
measurement
silanization
hydrophobizatio
n
A. DNA on mica
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SiO
O
O
NH2
Si
O
NH2
SiO
O
O
SH
(3-Mercaptopropyl)trimethoxysilane
MPTS(3-Aminopropyl)trimethoxysilane
APTES
3-(Ethoxydimethylsilyl)propylamine
APDMES
Examples of alkoxysiloxanes
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Self-polymerization
practical complication
SiO
O
O
R2
R1
R1
R1
SiO
O
O
R2
R1
R1
R1
H O H
Si OO
R2
SiO
O
O
R2
R1
R1
Si
O
O
R2
+
-R1OH
• Especially with APTES during liquid silanization
• Even vapors of water can cause this effect
• Fixation for optical microscopy – expected factor
• In contrary – in fixation for AFM – very disturbing
• Solution:
- silanization in vapours under vacuum (i.e. in desiccators)
- monoalkoxysilanes – can not polymerize
Si
O
NH2
3-(Ethoxydimethylsilyl)propylamine
APDMES
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Self-polymerization
examples
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NH2 NH2
NH2 NH2
NH2
HOPG
HOPG
B. DNA on HOPG
Adsorption of long chain
double-sided ions
(C16/C18)
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Substrates for immobilization: mica / HOPG (smooth
surfaces), also gold, glass in selected cases.
Example: gold nanoparticles (AuNP) mercapto-silanized
mica (SH-mica):
OH
Si
SH
OO
OH
OH OH OH
SiO
O
O
SH
Si
(3-Mercaptopropyl)trimethoxysilane
MPTS
3. Nanoparticles
SH-mica
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Si
- - - - - - - - - - - - - - - -
Au
+
+
+
+
+
+
+
+
P P
P
P
P P
+
+
+
+ Au
+
+
+
+
+
+
+
+
P P
P
P
P P
+
+
+
+
Gold nanoparticles (AuNP)
conjugated with protein
molecules:
protein = immobilization bridge
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glass
+ + ++ +
+ +
+
++ ++
Protein adhesive layer, i.e. pLL
(poly-L-lysine introducing positive charge)
pLL
Standard coating on glass
glass
3. Bacteria,
spores
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A. Standard culturing on polystyrene dishes
Adhesive protein layers usually takes place
(i.e. pLL, RGD adhesion factors, fibronectin,
etc.)
5. Eukaryotic
cells
Cell culturing equipment
BioAFM incl.
Petri dish
heater for in-
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• Adhesion of cells out of incubator (37oC, 5% CO2) is mostly
problematic
• Allows study of cells in long term periods after removal from incubator
• Cell wall destruction
• Example: EtOH, acetic acid, paraformaldehyde, glutardialdehyde
B. Fixation agents
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AFM spectroscopy
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Motivation
Why to quantify elasticity of (living) objects?
Stiffness (Young’s modulus) mapping
stiffness = basic parameter of any material
Elasticity-phenotype relation ship
Mechanobiological characterization
Driving of instrument properties (QNM, QI)
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Young’s modulus of materials
http://www-materials.eng.cam.ac.uk/
73 Acta Biomater. 2007 Jul; 3(4): 413–438.
Methods to measure Young’s
modulus
74
Mechanical Properties of Living Cells Using
Atomic Force Microscopy
J. Vis. Exp. (76), e50497, doi:10.3791/50497 (2013).
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1. Biomechanical characterization
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Primary CMCs
3 4 5 6
0.0
0.9
1.8
Force[nN]
Tim e [sec]
Noise ~ 10pN
Min. detectable movement ~
500 pm (acc. to Hook’s law)
Embryonic bodies - iPS cardiomyocytes
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Adrenergic reactivity - Metoprolol
Basic conditions
Increased arrythmic potential
Tyrode solution - control - 1,8 mmol Ca 2+
70 uM metoprolol
1 mM caffeine
Drug testing
studies
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2. Nanomechanical mapping
& relation to physical and phenotype properties
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Hertzian fit
Measured curves were fitted to following function:
Tip-sample separation = correction
of measured curve (height) for
cantilever bending
Adhesion
(SetPoint) Height
Height
Adhesion
Young modulus
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Height Young modulus
ADMSC cells
shTAZ cells
hTERT
ADM
SC
s
hTERT
AD
M
SC
s
PD
G
FP
PD
shTAZ
FC
hTERT
ADM
SC
free
0
10
20
30
40
50
60
70
80
Youngmodulus[kPa]
Mesenchymal
stem cells
(hTERT ADMSCs)
on
micropatterned
substrates
80
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Cells MCF7_PDLIMsiRNA
Optical image 4x
3.60 kPa
1.92 kPa
M
C
F7_C
TR
Lpl
C
F7_PD
ZIM
pl
M
C
F7_PD
LIM
siR
N
A
M
LF7_C
TR
LsiR
N
A
0
2
4
6
8
10
12
Youngmodulus[kPa]
Cancer cells stiffness
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Copyright 2012 Wisconsin Fast Plants Program
(Žádníková et al. 2010)
(Peaucelle et al., CB 2015)
Plant samples under AFM
spectroscopy investigation
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Water – YM 7.09 MPa Manitol YM 0.69 MPa
by Marçal Gallemí
Hypocotyl and root parts
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Fibroblaststhawingprocess
17:34
18:24
19:12
19:58
20:45
21:31
22:17
23:02
23:48
0:34
1:20
2:05
2:50
3:36
4:21
5:07
5:52
6:38
0
5
10
15
20
25
30
YM[kPa]
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158,8
2809,1
0
500
1000
1500
2000
2500
3000
3500
4000
YM(kPa)
Tuhost HYB Po MPO
Glycoaminoglycanes
structure & interaction with myeloperoxidase
Hyaluronane Hyaluronane-MPO
Hyaluronane-MPO
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Gel 2 Gel 3
Gel 1
With Vladimir Vinarsky, Giorgia Nardone, Giancarlo Forte (ICRC, FNUSA, Brno)
Flexible surfaces (gels)
as support for single CMs
PDMS based gels
High density
methacrylate
Low density
methacrylate
Average YM = 27.4 kPa
Average YM = 5.2 kPa
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3. (Semi)automatic driving of AFM
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TiO2 NT
Quantitative imaging (QI mode)
JPK NanoWizard 3
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Quantitative NanoMechanics (QNM)
PeakForce QNM = quantitative nanomechanical information (biological samples without
damaging)
Based on Peak Force Tapping technology - probe is oscillated (~TappingMode), res. freq
1 - 8 kHz (=sampling rate) depending on the tool).
Difference:
Tapping Mode – const. amplitude,
Peak Force Tapping maximum peak force on the probe (much lower comparing to contact
mode – biological samples)
attractive
forces
(capillary, VdW,
elstat)
negative forces >
cantilever’
stiffness indentation
Peak force
feed back control
withdrawing
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Lipozomes on graphite electrode
DNA on mica
With J. Vacek, UPOL
With H. Kolarova, H. Zapletalova,
UPOL
Bruker Dimension Icon/FastScan
BrukerNano supporting info
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AFM CoreFacility
CEITEC MU
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CEITEC AFM CoreFacility
JPK NanoWizard3
Bruker FastScan Bio
NTMDT NTgra Vita
NTMDT Solver Next
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AFM visualization of biomolecules and
bioobjects
Bare metal (gold) electrodesBacteria on glass slideSpermWhole cells (cell synapse)Individual biomolecules
IgG
Nanoparticles
(gold nanoparticles BSA modified)
DNA
J. Hejátko – YM mapping
P. Bouchal – YM mapping
J. Paleček - DNA
M. Pešl, V. Rotrekl CMCs
J. Sládková – CMCs
A. Meli - CMC
M. Kalbáčová – TiO2 NT
H. Kolářová - DNA
I. Crha - sperms
Cooperation:
94
Young modulus
Optical microscopy AFM Confocal microscopy
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