Biology Applications Nanonics

Nanonics Excellence
In Scanning Probe Microscopy
For Biological Imaging &
Functional Imaging
Transparent Optical
& Spectral Integration
for the
MultiView SPM Series
• MV4000
• MV2000
The Ultimate In Force Sensitivity
The Next Evolution in SPMTM

Nanonics Excellence in
Structural & Functional
AFM/SPM Imaging in Biology
Nanonics Imaging Ltd
www.nanonics.co.il

The Next Evolution In AFM
Fully Integrated Platforms To Address
The Simple To The Sophisticated
MultiProbe
MV1500
MV2000
Single Probe
Series The Hydra
Multiprobe

In General Other AFMs Have Given No
Deep Thought To The Importance of
Excellence In Optical Integration
Ultrasmall Z Range
Scanners As Small
as 6m in Z
Oft Repeated
Technology In
Conventional AFM
Probes Blocking
Optical
Axis
Generally
Worlds Apart

5
Nanonics Has The Only BioAFMs That
Can Transparently Integrate With Any
Upright Microscope Opening The World
Of>>
• Non transparent substrates
• Applications in microbiology
and virus research
• Applications in food, paper or
textile industry on fibers,
coatings or powders in air or
liquid
• Tissue culture
• Optical active compounds
or materials studies in
biosensors, capsules etc
• Biomaterial studies, biofouling,
And The There
Is Nanonics

The Next Evolution In NSOM
Scanners Probes & Feedback Allow
For Singular Excellence in AFM
And Glass Based Cantilevered
Probes That Do Not Obscure
The Optical Axis And Are
Exclusively Available For
Nanonics Customers Only
& &
Singular 3D Flat
Scanners
Feedback With
Unprecedented
Force Sensitivity Exclusive Glass
Probe

Exclusive Nanonics Probes Are The Most
Robust In The Industry As Proven In A
SEM Their Unique Structure Allow For
Unprecedented Imaging Horizons
Controlled High
Pressure
After Retraction
50nm
7

NanoOptical Light Source
Nanopipettes for
Ionic Conductance
NanoFountain Pens
for Liquid
& Gas Delivery
NanoVacuum
NanoHeaters or
Nanothermocouples
Plasmonic NanoProbes with
Single Gold NanoParticles
Glass Insulated Coaxial NanoElectrical &
Cantilevered NanoElectrochemical Probes
All Probes Are Electron
& Ion Optically Friendly
With Non-Obscuring
Cantilevers
& With Probe Tips Exposed
To The Optical Axis
Probes are
also Multiprobe
friendly
Many Functionally Important Optically
and Multiprobe Friendly Probes

But Such Use Of These Exclusive
Probes Is Complexed With The Ability
To Use Any AFM Probe On The
Market

Nonetheless Nanonics Systems Readily Allow
For Both Tuning Fork or Beam Bounce Feedback
& Any Probe Glass or Silicon
Tuning Fork Feedback
Advantages:
• Highest force
sensitivity
• No feedback laser
• Exact point of
contact &
• True non-contact
Beam Bounce
Feedback
Advantages:
• Contact mode
• Mount any 3rd
party probeLeading
To New Directions In Research
AFM Controlled Gas Delivery
& Associated Kelvin Probe Alterations
[Customer Publication]

And The AFM Force Sensitivity Is
So High That The Probes Can
Come Into Contact With One
Another
Critical for Transport Information

See What Customer’s Say In Papers
dx.doi.org/10.1021/nl501431y
Nano Lett. 2014
The Next Evolution in NSOMTM

Scanners Probes & Feedback Allow
For Singular Excellence in AFM
And Glass Based Cantilevered
Probes That Do Not Obscure
The Optical Axis And Are
Exclusively Available For
Nanonics Customers Only
&
The Next Evolution in SPMT
&
Singular 3D Flat
Scanners
Feedback With
Unprecedented
Force Sensitivity Exclusive Glass
Probe

The Singular 3D UltraFlat Scanner
Advantages

Open Optical Axis
Extension in Z Allow Effective Optical Sectioning
Nanonics 3D Flat Scanner Design
Open
Optical
Access
From
Above and
Below
7mm
Conventional
Vertical
Scanners Unlike
Nanonics Have
An Ultrasmall Z
Range Scanners
As Small as
6m in Z

Excellent 3D and Deep Trench
Capabilities With High Aspect
Ratio Glass Probes
Can Be Imaged By Nanonic
Due To Availability of::
• Large Z Scanning Range
85m
• The Long Tip Length of
100m
• The Very High 10:1
Aspect Ratio Of
Nanonics Tips
• Allows A Soft Touch AC
Mode
FIB Etched Trench

The Feedback Advantage

Oft Repeated
Method of Beam Bounce
Feedback Used In Commercial
Instruments
Nanonics VISTATM Method of
the Ultimate in Force
Sensitivity Without Any
Optical Interference
The Basis of VISTATM
Vivid Imaging AFM

Besides Optical Interference No
Sample Heating Beam Bounce
Feedback Has Significant
Problems

Jump To Contact & Ringing Instabilities Occur
In Beam Bounce Feedback
Jump to contact
Uncontrolled
ringing
• No Jump to
Contact Due To
High Force
Constants
• No Adhesion
Ringing
• Sharp
Frequencies or
High Q (Quaiity)
Factors With
Associated
Ultrasensitivity
Beam Bounce Feedback Problems
Resolving These
Critical Problems
Using Tuning
Forks Which Have:Probe
Approaches
From This
Point To
The Right
The
Technological
Limitations of
Soft
Cantilevers

High Q Low Q
High Q factors with ultrasharp tuning fork resonances
allow ultrasmall alterations in frequency to be detected
Resonance
Frequency
Amplitude
Resonance
Frequency
Amplitude
Sharp resonances
giving
ultrasensitivity to
monitor forces
Broad resonance
giving lower sensitivity
to monitor forces

Today Exact Equations Relate Tuning Fork
Frequency To Force Between The Tip & Surface

Amplitude Vs Distance Curve
Tuning Forks Uniquely Allow Knowledge of the
Point of Contact Impossible To Experimentally
Know With Certainty With Beam Bounce
Distance (d)
Amplitude
0
ApproachRetract
TR
M
FT
FR
FM
IF
Finteraction =
4
3
E*
R(d -d0 )3/2
+ Fadh
DMT Equation For Interaction of Tip With
Surface Using Tapping Mode
Tuning Forks Give All Values of
this Equation Experimentally
I = Start of Approach
FT = Force at Point of
Touching the Surface
FM = Maximum Force
FR = Force at Point of
Leaving The Surface
F = Final Retract Point

Hear From Professor Kit Umbach At
Cornell Of The Accuracy of the
Tuning Fork Method for Measuring
Forces Click On The Link Below
http://www.nanonics.co.il/user-testimonials

Cellular Wood Cells AFM Topography and Accurate
Elasticity Maps Without Need For Approximate
Digitization of Approach Curves and Assumptions
Height Elasticity (E*)
Great details and resolution in E* map is observed due to the different
elasticity of the materials
More information on the Lignin Protein distribution in these maps
will be seen in the Raman section of this presentation

True Non-Contact Demonstrated By The
Ability To Switch On-line With The AFM
Probe From AFM to Tunneling Feedback

The Feedback Allows For
True Non-Contact Demonstrated
By The Ability To Switch Between
AFM and STM Feedback
28

The History of Force Measurements
Peak Force
Force Volume<50 nN
Pulsed Force Mode
Dual AC
Single Harmonic
Tapping Mode
Phase Imaging
Harmonic X
<20 nN
<5 nN
<10 nN
<3 nN
<5 nN
<100 pN
The History of Force
Measurements Is Simply Better &
Better Algorithms To Account For
The Beam Bounce Instabilities.

Even The Mechanical Force Of A
Photon
(1.6 pN) Has Been Measured
Recently With A Nanonics Tuning
Fork System
With Nanonics The Tuning Fork
Provides The Ultimate In Force
Sensitivity

The History of Force Measurements
Peak Force
Force Volume<50 nN
Pulsed Force Mode
Dual AC
Single Harmonic
Tapping Mode
Phase Imaging
Harmonic X
<20 nN
<5 nN
<10 nN
<3 nN
<5 nN
<100 pN
VISTATM Vivid Imaging AFM <1.6 pN
The History of Force
Measurements Is Simply Better &
Better Algorithms To Account For
The Beam Bounce Instabilities.
Tuning Forks Provide The
Ultimate in Force Sensitivity at
1.6 pN Unachievable by Beam
Bounce Methods

Proven In The Literature By
Measuring Force Sensitivity
on Cells Down To 5pN

Apetureless Force Detection
Of Plasmons
With Extensions To the MidIR & Thz
Mechanical/Photon
Induced Force (PiFM)
Detection Of Plasmonic
Distribution

For Both Tuning Fork or Beam Bounce Feedback
& Any Probe Glass or Silicon
Tuning Fork Feedback
Advantages:
• Highest force
sensitivity
• Exact point of
contact &
Beam Bounce
Feedback
Advantages:
• Contact mode
• Mount any 3rd
party probeLeading

Therefore Nanonics Is Proven In The Literature
To Image With Excellent XY Morphological Fidelity
Even Compared To FESEM
The Tuning Fork Uniquely Provides A High Quality
Factor, Q, For Ultra Sensitivity In AFM And In AFM
Morphology. This Is Not Available With Any Beam
Bounce Feedback AFM (see green highlighted
customer description). Also True Non-contact Is
Achieved With The Nanonics Tuning Fork Systems.
Thus, Nanonics Provides The Only AFM Systems
That Allow For Switching Between AFM And STM
Feedback With The Same Probe. Proving Non-
contact AFM Operation

As Shown In This Multicenter Comparison
Tuning Fork Feedback Produces No Optical Artifacts
Critical in Single Molecule Imaging and NSOM FCS
The
CONCLUSION
MultiCenter Comparison Confirming
Nanonics Singular Capabilities
The Next Evolution in S

Ni
Si
Nanonics Results Without Beam Bounce
Laser Artifacts
Topographic Image
Si 0V
Ni Ni Ni
 Nanonics MultiProbe
MultiView 4000 SPM
 Electrical probe using a bias
of 0V
 Edge of Nickel capacitor on
n type silicon was scanned
 During scanning feedback
laser light was switched on
and off.
 Deminstrating that feedback
Light Off
Light Off
Light Off
Light On
Light Off
Light Off
Light On
Light On
Light On
Light On
Tuning Fork
Conductivity
Image With
and Without
Laser
Photocurren
t
Artifact

The Next Evolution in SP
Such Conductivity Images Without Interference Can
Scan State of the Art Transistors At High XY Resolution

Nanopipettes for
Ionic Conductance
NanoFountain Pens
for Liquid
& Gas Delivery
NanoVacuum
NanoHeaters or
Nanothermocouples
Plasmonic TERS NanoProbes with
With Non-Obscuring
Cantilevers & With
Probe Tips Exposed
To The Optical Axis
Probes are
also Multiprobe
friendly
NanoToolKitTM of Unique Optically, Electron
Optically & Multiprobe Friendly Probes

Glass probes also resolve
critical issues in biological
imaging
Silicon cantilever geometry is for from
ideal for biology. They:
 Generally are not angled or partially
angled & thus put pressure & easily
penetrate cell membranes
 Silicon probe tips close to straight &
squeeze out water layers and
protruding structures such as microvilli
 Produce images with shadow
 Lack high aspect ratios with long tips
compromising drastically deep
penetration into invaginations in cell
membranes & between cells
 Have flat cantilevers acting like a
paddle that reduces Q (quality factor)
in liquid to single digits
 Use of laser beam feedback causes an
~20% error in cellular elasticity due to
lack of information on point of contact
Glass cantilever geometry
is ideal for biology. They:
 Are angled for minimal cell
pressure, penetration and
shadowing
 Have high aspect ratios and long
tips allowing even imaging of
microvilli with different types of
cantilevers
 Allow today previously impossible
functional imaging on live cells
such as SECM, patch clamp,
NSOM etc
 Have cylindrical cantilevers with
essentially no liquid damping
 Accurately allow determining
point of contact with single pN
force sensitivity with tuning fork
feedback

Imaging Microvilli of MDCK Cells
Microvilli in live MDCK
cells with a cantilevered
glass probe attached to
tuning fork
Q factor in liquid 5000
normally 4 or 5 with beam
bounce methods silicon
cantilevers
Only in 2016 were very
specialized silicon
cantilevers able to image
this structure and even
these cantilevers could
not approximate the ideal
of glass probes and so
were not able to measure
the elasticity (see next
slide)

AFM With Tuning Forks Show For The
First Time MicroVilli Elasticity
11µm
0.22 Volts
-0.28 Volts
11µm
546.50 nm
-530.23 nm
Height Experimental
Amplitude
Low
Amplitude
Soft
Trampoline
High
Amplitude
Stiff
Trampoline
High
Amplitude
MicroVilli Are
Seen And
Shown To Be
Less Stiff Than
The Cell
Membrane
The Tuning
Fork Had A
Force
Constant of
5000

Liposome
Single Walled Liposome Imaging

AFM Scanning of Fibroblast Cells
Cantilevered AFM
Probe
Fibroblast Cells
3D Topographic Images
of the Fibroblast Cells
Watch The scan
https://www.dropbox.co
m/s/76h0g3wdwhweo70
/Cellular%20Imaging%
20Scan.mov?dl=0

6.0µm
Neuroblastoma Cells in
Medium
Re-
Trace
6.0µm
• Q factor = 2600
Comparing VISTATM
UltraSensitivity
Above with Similar
AFM Imaging With
Beam Bounce
Based AFM
Feedback Using An
Alternate AFM
Trace

Nikon
Nanonics Multiview
2000 Probe & Sample
Scanning
Readily Added To Any Beam
Scanning Confocal or Non-linear
Microscope

Two Photon Fluorescence
&Topographic of CLL Cells
Two Photon
Image
Scan Range
50 x50 µm
Topographic
Image
Scan Range
50 x50 µm
Two Photon Image
of GFP Label
CLL B cells
Accumulate in bone marrow and blood
They crowd out healthy blood cells
CLL is a stage of small lymphocytic lymphoma

With Tuning Forks These Geometric Restrictions of
Beam Bounce Tracking Are Removed Allowing Even
Water Immersion Objectives
Reflection of the
mount in liquid
Ultra low working distances as
small as 3.5 mm that provide
ultra high numerical aperture
objectives upto 0.8

Nanopipettes for
Ionic Conductance
NanoFountain Pens
for Liquid
& Gas Delivery
NanoVacuum
NanoHeaters or
Nanothermocouples
With Non-Obscuring
Cantilevers
& With Probe Tips Exposed
To The Optical Axis
Probes are
also Multiprobe
friendly
Many Functionally Important Optically
Friendly Probes

Apertured Near-field Scanning Optical
Microscopy Ultimately Resolves
Bleaching, Correlation & Resolution
NSOM illumination is
localized and hence NO
bleaching of fluorescent
molecules occurs outside the
local spot of illumination
Only those molecules under
the probe tip get excited
Pixel by pixel AFM gives
absolute correlation

NSOM Development Has Now Reached The
Pinnacle of Live Cell Imaging With Synergism
With Far-field Super-resolution Techniques

7.0µm
FLUORESCENCE GREEN EXCITATION AFM
ABSORPTION BLUE EXCITATION AFM
STEM Cell NSOM Imaging Correlated with
Topography With diI Membrane Staining
7.0µm
Excitation
514.5 nm
Excitation
457 nm
7.0µm
7.0µm
Absorption
Fluorescence

Absorption NSOM Of Live MDCK Cells Stained
With Di-4-AN(F)EPPTEA Shows Totally Different
Contrast To Fluorescent Confocal706.66 nm
0.00 nm
7.4µm
AFM
NSOM
Brightest point
3DAFM
Confocal
Fluorescence
Comparison
Mean
AFM Normal Force
 Solution: 60 µL
 Q-factor
in air: 2000
 Q-factor
in liquid: Approx
The Same
 Scan 40x40
micron
 12 ms/pixel
 488nm
 Dark dots (green arrow) due to
NSOM absorption of membrane
emanating microvilli filled with dye
 Large dark region is correlated
with a large cilliary protrusion in
the topography (blue arrow)

All Modes Possible
8.0µm
NSOM Absorption

Dye Staining Was With Voltage Sensitive
Dye That Undergoes A Stark Effect With
Such A Dye We Can Prove Live Cell Glass
Probe NSOM Imaging
The Membrane Binding
Dye Is Di-4-
AN(F)EPPTEA
Cell
Membrane With
Membrane
Voltage
With KCL
Membrane
Voltage
Reduction
Depolarization
l
-
+
+
-
Absorption Fluorescence
514nm

Imaging Membrane Potential With NSOM
Proving Live Cell Imaging
Proving The Imaging Was Of Live
MDCK Cells By Imaging
Depolarization With Addition of 5mM
KCl NSOM Fluorescence Imaging of
Membrane Potential With Di-4-
AN(F)EPPTEA Stained Membranes
Using a Large Probe [Live cell near-
field optical imaging and voltage
sensing with ultrasensitive force
control, OPTICS EXPRESS Vol. 25,
29 May 2017
https://doi.org/10.1364/OE.25.0121
31]

10µm 10µm
13.08 KHz
-0.00 KHz
37.18 KHz
-0.00 KHz
NSOM Before added KCl NSOM After added KCl
Before
KCl
After
KCl
Near-field Fluorescence Robust Change
With Membrane Voltage
 Robust change indicative of NSOM measurement
 NSOM depth of focus very low
 NSOM is membrane centric with little out-of-focus noise
 Thus, the brightest pixel in the image before and after KCl shows a considerable
reduction

NSOM Illumination without Background Is Also Very
Important for FCS
Attoliter Illumination Volumes
Alloswing 3 Orders of Magnitude Increased Sensitivity
1E-3 0.01 0.1 1 10 100
1.00000
1.00002
1.00004
1.00006
G(t)Amplitude
10nM
100nM
1m
correlation time (s)

Super-resolution Fluorescence
Single Molecule Biology Of
Amyloid Fibrils With Nanonics

Synergistically Confirmed 3 years Later Using
Far-field Single Molcule Super-resolution
Fluorescence by Moerner the
2014 Nobel Prize
winner in Chemistry

AFM image
Lifetime image with
decay analysis
Lifetime image
without analysis
Integrated NSOM Super-resolution
Fluorescence Lifetime Imaging of Liposomes
Stained with Di-4-AN(F)EPPTEA

And Many Other Functional
Imaging Tasks Capable of
Being Accomplished Only With
Nanonics Probes

Nanopipettes for
Ionic Conductance
NanoFountain Pens
for Liquid
& Gas Delivery
NanoVacuum
NanoHeaters or Thermal
Conductivity or Nanothermocouples
With Non-Obscuring
Cantilevers & With
Probe Tips Exposed
To The Optical Axis
And We Have NanoToolKitTM of Optically, Electron
Optically & Multiprobe Friendly Probes

From:
D. Ossola, M.-Y. Amarouch, P. Behr, J. Vö rö s, H. Abriel, and T.
Zambelli, “Force-controlled patch clamp of beating cardiac cells,”
Nano Lett. 15(3), 1743–1750 (2015).
Pioneers In Force Sensing
Nanopipettes

Fountain Pen
Nanochemistry To Write
and NanoDispense as
NanoDrops a Variety of
Molecules Including
Proteins, DNA etc From
Solution or Dispersion
The Nanopipette Aligns
As It Draws
Watch A Movie Of Drawing With An AFM
Fountain Pen
https://www.dropbox.com/s/puk5hzjp8ge
nhm9/Drawing%20SWCNTs%20on%20Si
O2.mp4?dl=0

The crucial factor is that
the 'fountain pen' can
have different inks
channeled into it
automatically, simply by
connecting it up to
standard high-
performance liquid
chromatography
instrumentation. This
should make writing a
multi-protein nanoarray
much easier than by
using DPN, and without
the need for any
complex pre-treatment
of the substrate.
NanoFountain Pen Protein Printing

32.521.510.50
24
22
20
18
16
14
12
10
8
X[µm]
Z[nm]
Deposition of a Copper Line
of ~15nm Between Two
Electrodes
The
Ultimate In
Resolution

For Both Tuning Fork or Beam Bounce
Feedback
& Any Probe Glass or SiliconTuning Fork
Feedback
Advantages:
• Highest force
sensitivity
• Exact point of
contact &
Beam Bounce
Feedback
Advantages:
• Contact mode
• Mount any 3rd
party probeLeading

Scanning Ion Conductance
Microscopy Advantages
• SICM cantilevered nanopipette probes monitors
the ion conductance & topography in separate
channels
• High resolution SICM with 10-30 nm apertures
• AFM Feedback holds the tip in ultrasensitive
feedback with the sample and monitors the
topography as a separate channel
• AFM of the cells can be obtained with the highest
sensitivity
• Largest Z range available: <85µ

AFM Controlled
Conductance Microscopy
Porous
Sample or
Biological
Membrane
with Pores
or Channels
Cantilevered
NanoPipette
Electrode in
NanoPipette with
counter electrode
in Solution in which
Porous Sample is
Sitting
Measuring the Porosity of MicroPores
Non-Destructively with AFM Controlled
Conductance Imaging

Scanning Ion Conductance Microscopy
With A Cantilevered NanoPipette With
Normal Force Topography Correlated With
SICM Signal Of Nucleopore Filters
2.0µm2.0µm
Height SICM

Similar Imaging
Correlation between (a) Topographic and (b) Current image.

Direct Correlation of Peak
Contact With Current

Nanonics Atomic Force For Real
Samples Opens New Horizons:
C. elegans

AFM C. elegans Only
Achievable With Nanonics

All modes of optical microscopy on-line
including fluorescence
& DIC (differential interference contrast)
To Make Things Easier to View

Differential Interference Contrast (DIC)
& Phase Imaging
Glass AFM
probe in
feedback on
a cell surface
in
physiological
media as a
function of
the focus of a
50 X
objective in a
DIC
microscope.
Notice that
when the cell
is in focus
(lower right)
the probe is
completely
transparent &
invisible.

Thus Controlled Nanopipette Penetration &
Injection of C. elegans With
Nanonics MultiView System
Is Made Very Easy
Nanopipette
Controlled By
Atomic Force
Microscopy Injecting
a Fluorescent Dye
Around A Specific
Neuron With On-line
Fluorescence
Microscopy
Nanonics Patented
Cantilevered
Nanopipette
Controlled Point of
Penetration with Nanonics
Atomic Force Microscope
Stained Neuron

Readily Moved Onto The Stage
Of Any Renishaw Microscope
Atomic Force
Microscopy
Raman
MicroSpectroscopy
AFM Raman
The Next Evolution in SPMT

AFM Sample Z Feedback Allows For Accurate
Raman Spectral Intensity Comparisons At
Different Sample Positions With AFM
Autofocus & Nanonics AFM Large Z Range Is
Required For The Large C. elegans Dimensions

On-line Raman Chemical Indentification
Obtained on C. Elegans AFM Controlled
Nanopipette Lipid Injection
Laser 785 nm
Power 2mW
Exposure time 600s
CCD image of the point of AFM
injection of lipid & Raman
investigation in C. elegans with
on-line chemical identification
Raman Spectrum of C-H vibration of lipid
injected into C. elegans collected at region
indicated in the image to the left

Multiprobe AFM
Force Excitation & Detection
One Probe Periodically Excites While A
Second Probe Monitors C. elegans Oscillation
0 2 4 6 8 10 12 14 16 18 20
0.1
0 2 4 6 8 10 12 14 16 18 20
0.2
0.4
0.6
0.8
Time, msec
Voltage,V
0 2 4 6 8 10 12 14 16 18 20
0
200
400
600
800
1000
1200
Time, msec
Voltage,mV
0.2
0.3
0.4
Time, msec
Voltage,V
0 2 4 6 8 10 12 14 16 18 20
0
200
400
600
800
1000
1200
Time, msec
Voltage,mV
0 2 4 6 8 10 12 14 16 18 20
0.1
0.2
0.3
0.4
Time, msec
Voltage,V
0 2 4 6 8 10 12 14 16 18 20
0
200
400
600
800
1000
1200
Time, msec
Voltage,mV
Set Point = 15 nN, Tip size =
0.2 um
First 1 sec
Bottom Excitation Probe
& Top Detection Probe
Set Point = 15 nN, Tip
size = 0.2 um
After 10 secs
Bottom Excitation
Probe
& Top Detection Probe
Both After 10 and 15 secs
(below) of AFM Probe Periodic
Excitation Adaptation Is Seen
Of The Mechanical Response
Detected With A Second AFM
Probe On-line dissipates
Touch Receptors with GFP mec-2
Chimeras in Mechanosensory ALM Touch
Receptors
Next Evolution in SPMTM

Cantilevered SECM probes for simultaneous
normal force sensing with full SECM functionality
A continuous nanowire of platinum
is embedded in glass and as can be
seen in the electron micrograph
below the wire is flush with the
nanometric glass end
SEM of the
nanowire at the
nanometric tip
70 nm
Diagrammatic
representation of
the SECM probe
Platinum
nanowire
Glass

Nanonics Founded By Aaron Lewis
With Contributions to Both Optics
and Electrochemistry

Exemplary approach curve of a Nanonics Nanoelectrode
SECM Probe To A Non-conducting Surface
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 2 4 6 8 10 12 14 16 18 20
NormalizedCurrent
L= d/r
Nanoelectrode_1.3
Neg_4
Neg_3
Theor_Neg_RG5
Theor_Neg_RG10
Theor_Neg_RG20
Neg_4
Neg_3
Theor_Neg_RG5
Theor_Neg_RG10
Theor_Neg_RG20
Negative feedback approach curve of a Nanonics Nanoelectrode SECM Probe
The experimental approach curve (smooth lines) was fitted with theoretical approach
curve (points)
A determination of the effective radius is 180-200nm
Approach Speed: 0.05µm/ 0.0167sec

0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
0 2 4 6 8 10 12 14 16 18 20
NormalizedCurrent
L= d/r
Nanoelectrode_1.3
Theor_Post
Post_2ndtime
Positive feedback approach curve of previous 180-200nm electrode
Exemplary Approach Curve of a Nanonics
Nanoelectrode SECM Probe Relative To A
Conducting Surface Without Sample Voltage

-9.00E-12
-8.00E-12
-7.00E-12
-6.00E-12
-5.00E-12
-4.00E-12
-3.00E-12
-2.00E-12
-1.00E-12
0.00E+00
1.00E-12
2.00E-12
-400-350-300-250-200-150-100-500
Current,Amp
Potential, mV (vsHg/Hg2SO4)
10mM Ferrocyanide & 0.1M Na2SO4
CVFC# 4
Radius= 4nm
Cyclic voltammogram of 4 nm radius Nanonics Nanoelectrode
SECM probe in presence of 10mM ferrocyanide and 0.1M Na2SO4
Exemplary approach curve of a
Nanonics Nanoelectrode SECM Probe
This is the
ultimate
and should
not be
taken as a
standard

ΔX=250 nm
Line Scan of the SECM Image An
Order of Magnitude Better Resolution
Than All Other SECMs Along With
Topography Due To On Line AFM

Unique Designs of Electrochemical Cells With
Environmental Control & Optical Integration
Reference Electrode

Details of Electrochemical Cell
Counter Electrode Completes Circuit Allowing Current
To Flow From Probe and/or Sample or Vice Verse
Back Electrical
Contact For Allowing
Voltage To The
Sample Relative To
The Reference
Electrode
SECM Probe or Working Electrode With Voltage
Relative To Reference Electrode
Sample
Voltage Between
SECM Probe &
Reference
Electrode Reference
Electrode
Voltage
Between
Sample &
Reference
Electrode
Counter
Electrode
To Probe &
Sample

An Electrochemical Liquid Cell
Designed To Protect From Spillage

Top Left: Exposed SECM Cell. Sample is placed in
ring in the middle. Changeable wire electrodes on
the sides.
Top Right: SECM Cell that is partially covered to
minimize evaporation.
Left: SECM Cell from backside. Contains
back contact for applying voltage to the
sample.
SECM Cell

Thus All Nanonics Systems Permit
On-line Raman Chemical Characterization
Atomic Force
Microscopy
Raman
MicroSpectroscopy
AFM Raman

The Nanonics MultiView 2000 System Are Flexible
Under The Lens of A Bruker microRaman
MV2000

MultiView 2000 with the Renishaw InVia System
Combinations with Raman
& Fluorescence Spectroscopy
Especially Effective with Water Immersion
Objectives from Above on Opaque Materials
• Chemically image the electrochemical process
MV2000

STFMTM Probes Allow For Water Immersion
Objectives From Above to Be Used
Reflection of the
mount in liquid
Ultra low working distances as
small as 3.5 mm that provide
ultra high numerical aperture
objectives upto 0.8
Ultra high
Raman
signals

Substrate:
Thin layer
of Cu on Si
Working Electrode
and AFM probe:
When voltage is
applied on it, it
creates oxidants
which oxidize the
Cu.
When Cu is etched, the Si
is exposed. A spectro-
chemical signal of Si is
detected.
SECM-Raman
SECM Applications: Ionic Dissolution +
Electron-Kinetic Transfer

Results SECM-
Raman
Optical image Raman signal
BEFORE
SECM Applications: Ionic Dissolution + Electron-
Kinetic Transfer

IN REAL TIME
Raman signal in real time
SECM Applications: Ionic
Dissolution + Electron-Kinetic
Transfer
Results SECM-
Raman

Thermal conductivity imaging of
Sematech produced voids
150100500
75
70
65
60
55
50
45
40
35
X[nm]
Z[mV]
ΔX=30 nm
The line-scan across the thermal conductivity image
demonstrates lateral resolution 30 nm on a test
sample of voids in Silicon Provided By Sematech
AFM / Thermocouple
or Thermoresistive
Probe

Thermal Coonductivity
& Topography Imaging of Nanotubes
& Quantum Dot Decorated DNA
Imaging Semiconductor Quantum Dots
With Thermal Conductivity Image
(Above)
SiGe Structures on
Silicon Imaged
With
Topography, Phase
and Thermal
Conductivity (Aux
Channel)

NanoThermal Imaging of Polymer
Blend & Thermal Analysis
Select a Point for Thermal
Analysis
&
Choose The Experimental
Parameters

ThermoMechanical and
Differential Scanning Calorimtery

ThermoMechanical Analysis
of Nylon

• Resolution In X Y & Z Mapping
• UltraLow Noise
• Low Drift & High Resolution
• Soft Sample Imaging
• Phase Imaging Heterogeneous Thin Film
• Deep Trench Imaging of Real Samples
• Magnetic Force Imaging
• Fine Nanomanipulation
Other AFM Imaging Tasks

Quantum Dot
Decorated DNA
With Tuning
Fork Quality
Factors of
Thousands
Resolution X Y: Uncompromised
NanoImaging

• UltraLow Noise
• Magnetic Force Imaging

Resolution Z: UltraLow Noise Image Atomic Steps
Strontium Titanate (001)
7006005004003002001000
10
9
8
7
6
5
4
3
X[nm]Z[Å]
ΔZ=0.28 nm
Tuning-fork based feedback with Nanonics glass cantilevered AFM probe
Noise in this image is 0.01 nm peak to peak
with the RMS generally three times lower
Noise Is
At The
0.01nm
Level

Low Noise & Ultrahigh Resolution
Comparative Examples
Comparing tuning Fork UltraSensitivtty
with Similar HOPG Beam Bounce Image
Omnicrom
Single atomic steps on Highly Oriented Pyrolytic Graphite
HOPG is a standard of choice for AFM resolution in Z

Single atomic steps on Highly Oriented Pyrolytic Graphite
HOPG is a standard of choice for AFM resolution in Z
Low Noise & Ultrahigh Resolution
Comparative Examples
Comparing tuning Fork UltraSensitivtty
with Similar HOPG Beam Bounce Image

UltraLow Drift Two of the Same
Area Taken 20mins Apart
Only Single Nanometers
Per Hour

Resolution Test Fischer
Structure
0.05
mm
Heightnm
1
2
3
4
5
4nm
0.1

Monolayer Polymer Film
Decorated With Gold Nanoparticles Showing
Packed Polymer Strands
600nm

Soft Sample Imaging: Molecular Pentacene
Dendrimers

Soft Sample Imaging: Monolayer of
Associated Insulin
Associated Insulin Is Used As Exemplary of Other Associated Proteins
Such As Amyloid Proteins
Y: 1 mm X: 1 mm

Soft Sample Imaging: BioMolecular
Imaging of Collagen Protein Fibers
Comparing tuning Fork UltraSensitivtty with Similar
Collagen Beam Bounce Images
A Tuning Fork Image
of Collagen As
Compared With Similar
Images From Other
Vendors With Beam
Bounce Feedback
The Next Evolution in SPM

Phase Image Overlaid on Topography
Phase Imaging NanoParticle Impact-Modified
Heterogeneous PMMA Thin Film Coating of A
Thermoplastic and Transparent Coating

Excellent 3D and Deep Trench
Capabilities With High Aspect
Ratio Glass Probes
Can Be Imaged
Only By
Nanonics Due To
Availability of::
• Large Z
Scanning Range
85m
• The Long Tip
Length of 100m
or greater
• Very High 10:1
Aspect Ratio
Glass Probes
• VISTATM Soft
Touch AC Mode

Glass Probes Combined with Unprecedent
Z Range 20 Micron PMMA
Polymer Microspheres
Large 100 micron z scanning
range of the Nanonics 3D Flat
Scanning System and the ability to
use glass cantilevered AFM probes
with 100 micron or more tip
length allow such large
topographic alterations to be
readily imaged.
AFM Imaging Only
Nanonics Can Provide
+

The large Z range of the Nanonics 3D Flat Scanner allows us to readily
measure the height of these microspheres.
20 Micron PMMA
Polymer Microspheres

Even MFM Has A Unique
Nanonics Contribution
Single Co Particle Probes In
Glass Nanopipettes Like Our
Plasmonic Probes
Only At The Tip Of The Probe
Without Magnetic
Interference From Coated
MFM Probes

Magnetic Force Microscopy (MFM)
AFM Topography
A close up AFM Height image of Co
nanoparticles (bar is 200nm)
A close up MFM image of Co nanoparticles
presented at left (bar is 200nm)
MFM

Magnetic Force Microscopy (MFM)
Videotape
AFM Topography MFM
AFM Topography MFM

• UltraLow Noise

Fine Nanomanipulation and Imaging
of Chromosomes with Fine Glass
ProbesGlass probes with their slender
profiled tip are ideal for imaging
and nanomanipulation of soft
structures such as this human
chromosome
Silicon AFM Probes
Cannot Compete
With Glass AFM
Sensors Both In
Imaging &
Nanomanipulation

Placing A Single Gold Nanoparticle
Onto a Single Single Walled Carbon Nanotube
AFM (Left) and SEM (Right) Image93.71 nm
580nm
AFM Image
SEM Image

138
Placement of a Nanoparticle On a Plasmonic
Structure
200n
m
200n
m
100n
m

Biology Applications Nanonics

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