Environmental Atomic Force and Confocal Raman Microscopies … · 2018-11-09 · Confocal Raman...

Environmental Atomic Force and Confocal Raman Environmental Atomic Force and Confocal Raman Microscopies in Pharmaceutical ScienceMicroscopies in Pharmaceutical Science

Greg Haugstad,1

Klaus Wormuth,3

Jinping Dong,1

Dabing

Chen2

and Raj Suryanarayanan,2

Eric Vandre,1

Jeannette Polkinghorne,4

John Foley,5

Robert Hoerr5

1Characterization Facility, University of Minnesota, Minneapolis,

Minnesota, USA2Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota, USA3Surmodics, Eden Prairie, Minnesota, USA4Boston Scientific, Maple Grove, Minnesota, USA5Nanocopoeia, St. Paul, Minnesota, USA

http://www.iprime.umn.edu/

This document was presented at PPXRD -Pharmaceutical Powder X-ray Diffraction Symposium

Sponsored by The International Centre for Diffraction Data

This presentation is provided by the International Centre for Diffraction Data in cooperation with the authors and presenters of the PPXRD symposia for the express purpose of educating the scientific community.

All copyrights for the presentation are retained by the original authors.

The ICDD has received permission from the authors to post this material on our website and make the material available for viewing. Usage is restricted for the purposes of education and scientific research.

ICDD Website - www.icdd.comPPXRD Website – www.icdd.com/ppxrd

http://www.icdd.com/

http://www.icdd.com/ppxrd

OutlineOutline

1.

Raman microscopy •

Brief introduction•

Two example polymer/drug systems; elution from coating

2.

AFM in conventional (AC/phase) and “pulsed force”

modes

•

Introduction•

Examples of AC mode for highest resolution and most delicate imaging at high temperature and humidity: two block copolymers, surface mobilization on crystalline drug

•

Examples of pulsed force mode, improved interpretation of materials contrast: two polymer/drug systems, phase segregation and elution

One motivationOne motivation: Drug: Drug--eluting coatings and biomaterialseluting coatings and biomaterials

Important:•

Conformal

coating; withstand balloon expansion of stent•

3D distribution of ingredientsSurface vs. bulkNanoscale segregation (size of drug domains)

•

Crystalline vs. amorphous drug

Raman EffectRaman Effect

•

Energy exchange occurs between incident photon and molecule

•

Energy difference is equal to the difference of the vibrational and rotational energy levels of the molecule.

-

Non-resonant excitation (Stokes) or annihilation (Anti-Stokes) of vibrational quantum states

-

Energy shift between the exciting photon and the scattered photon is characteristic for the molecules

involved in the scattering process

•

1 in 106

photons induces the Raman effect

•

Relative intensity of Rayleigh and Raman scattering dependant on

physical state, chemical composition, scattering angle

Raman spectrum

Chandrasekhara

Raman

XYZ stage

objective

holographicbeam splitter

Super Notch filter

multi mode fiber

APD

CCD

single mode fiber

laser

coupler

ConfocalConfocal

Raman microscopeRaman microscope

[Witec GmbH; www.witec.de]

Confocal Raman microscopy: high resolution chemical mapping (~250 nm lateral and ~500 nm vertical)

F F F F

F F F n

Si

CH3

CH3

O OSiSiH3C

H3C

H3C

CH3

CH3

CH3n

Fluoropolymer

PDMS

Fluoropolymer Spectrum

PDMS Spectrum

Fluoropolymer on PDMS Substrate Raman Depth Profile Fluoropolymer on PDMS Substrate Raman Depth Profile

[E. Vandre, J. Polkinghorne, J. Dong, U. Minnesota, 2007 IPRIME Meeting]

MatrixMatrix--drug system characterization, confocal Raman microscopydrug system characterization, confocal Raman microscopy

Matrix-Drug Collar

PDMS Spectrum

Si

CH3

CH3

O OSiSiH3C

H3C

H3C

CH3

CH3

CH3n

Dexamethasone Spectrum

C-H

2960cm-1

=C-H

3060cm-1

Si-O-Si

500cm-1

C-H

2915cm-1

2975cm-11665cm-1

C=O


Confocal Raman Depth Imaging & Cryofracture

•

Confocal

Raman depth scan lost signal probing into collar due to opacity.

•

Cryofractured collar provided a cross section for effective depth analysis using lateral Raman imaging scans.

Yellow=Dexamethasone

Blue=PDMS

Confocal Depth Scan

Loss of signal

Cryofractured Film Cross-Section


InIn--situ confocal situ confocal ramanraman

microscopy of microscopy of rapamycinrapamycin

in in arborescentarborescent

SIBSSIBS

Initial, ambient air

Z scan

Polymer drugx-y

scanLM at surface•Drug domain size

~2-6 µm; uniform distribution

[J. Dong et al., Langmuir, in press (available on line)]

Arborescent polyisobutylene-

polystyrene block copolymer

Rapamycin

C51

H79

NO13

Polymer drugx-y

scan

Z scan

LM at surface

•

After immersion for 10 hours, drug (bright) decreased significantly; drug domains no longer well defined

Final, in water after 10 hours[J. Dong et al., Langmuir, in press (available on line)]

InIn--situ confocal situ confocal ramanraman

microscopy of microscopy of rapamycinrapamycin

in in arborescentarborescent

SIBSSIBS

One motivationOne motivation: Drug: Drug--eluting coatings and biomaterialseluting coatings and biomaterials

Important:•

Conformal

coating; withstand balloon expansion of stent•

3D distribution of ingredientsSurface vs. bulkNanoscale segregation (size of drug domains)

•

Crystalline vs. amorphous drug

OutlineOutline

1.




2.


modes

•

Introduction•


•


Scanning or Scanning or ““AtomicAtomic””

Force Microscopy (AFM): The ConceptForce Microscopy (AFM): The Concept

Traditional microscopy: “far field”

AFM: “near field”PSD

Tip

~100 µm

chip

material

Blind microscopy: “feel the surface”

Sense cantilever movement via laser

Microfabricated, flexible cantilever

Piezoelectric tube scanner

Clip to secure cantilever chip

Photodetector

Presenter

Presentation Notes

Atomic force microscopy is blind microscopy. Unlike light microscopes (or electron microscopes or ion microprobes) optics are not used to image. That is, there is no lensing or steering of a particle beam to probe a material from macroscale distances (“far field”). Instead the probe - a pointed, microscale solid object - is vertically approached to within nanometers of a material surface and may even touch the surface; then, a nanoscale positioning device laterally scans the probe or material in a raster pattern (a 2D grid of locations). The probe or “tip” (a.k.a. stylus, needle, fiber) is very small and attached to a microfabricated cantilever that easily bends in the presence of forces. These forces are due to attractive and repulsive interactions in the “near field”, the tip-sample interface. (We will delve into the nature of these forces later, and indeed use them to obtain materials contrast and perform analytical work!) The cantilever bending is detected by bouncing a (focused) laser beam off of it and sensing the displacement of the reflected laser spot at a photodetector (far away). This position sensitive detector or PSD puts out two voltage signals quantifying the vertical and lateral displacement of the laser spot caused by the bending or twisting of the cantilever, in turn the result of normal or lateral forces. The sensitivity of this scheme is very high – a few tens of nanometers of vertical tip displacement produces a change of signal on the order of one volt. Thus atomic-scale surface corrugations are measurable (a few mV) via the normal bending, as are sub-nanonewton frictional forces (typical for a nanometer-scale contact) via twisting. But within this scheme, changes of height on the order of microns would extrapolate to ~100V, and in fact the photodetection would not work anyway because the laser spot movement would be too great to measure. So real AFMs work somewhat differently...

laser

sample surface

piezoelectric scanner

Position-sensitive photodetector

cantilever

Probe/tip

Atomic Force Microscopy: scanning under feedbackAtomic Force Microscopy: scanning under feedback

Scan tip OR sample

piezoelectric scanner

•

Image 3D surface topography digitally (measure heights, quantify roughness)

•

Image material composition via tip/sample interfacial forces (e.g., friction force)

•

Characterize distance-

dependent interfacial forces (e.g., mechanical stiffness, molecular bonding)

animation

Presenter

Presentation Notes

Real AFMs use X, Y and Z to move the probe or material sample. (The simplest instruments scan the sample, but some scan the tip, particularly when extreme sample environments are sought, e.g., high humidity or sample temperature.) Whereas X and Y are programmatically scanned independent of what is happening at the tip-sample interface, Z is reactively displaced by a feedback circuit so as to keep a control signal constant. In “contact mode” AFM, where the tip continuously slides over the surface, the vertical cantilever bending or “deflection” is the control signal that drives the feedback. Thus the instrument attempts to keep the cantilever at some constant degree of bending by displacing Z (of probe or sample) up and down as the tip rides over the hills and valleys of the surface. The intrinsic speed of the feedback circuit, as well as operator-selected gain settings and scan speed, largely determine how precisely this Z displacement tracks the topography of the surface. The operator-selected cantilever deflection value determines the external load (the pushing force of tip to surface). The intrinsic interfacial forces between tip and sample add to the external load to determine the total load or contact force. Moreover this interaction results in frictional forces that can be used to contrast dissimilar materials and, of course, enable studies in tribology (friction/wear/lubrication).

Environmental AFM with digital pulsed force mode Environmental AFM with digital pulsed force mode

•

True phase•

Interlaced force curve mapping•

Switchable closed-

& open-loop scanning

•

Witec digital pulsed force mode•

Environmental control: 1-95% RH, -30 to 250ºC•

Remote control, web-based services, training

Molecular Imaging (Agilent) PicoPlus

SPM

Presenter

Presentation Notes

Different vendors’ AFMs look completely different! Some differences are purely due to the whims of the engineers designing the instrument. Others stem from fundamental needs, an obvious example being environmental control. The above system has an unscanned sample plate inside of a glass chamber, and ports for circulating air and electrical feedthroughs (e.g., for hygrometer or resistive heating stage). Humidity control systems usually include some owner customization and jerry-rigging as well. Our system has a humidity controller that toggles power to either an ultrasonic humidifier or to a blower connected to a dessication column. Here again feedback circuits are used, such that the operator can dial in set point humidity or sample plate temperature. Bungee cords (suspending heavy slabs) have been used to isolate AFMs from vibrations since the very first instrument designs in the late 1980’s. In the commercial system above, the entire AFM/bungee-slab/optical viewing system is enclosed in a sound isolation box with a door (not shown) that can be closed during operation. Nevertheless AFM often is done without special enclosures or gaseous control; just ambient air. On the flip side, most AFMs now come with liquid cells to immerse sample and probe completely, possibly the most stringent environmental control (e.g., pH, saline concentration). The above system (SPM4) also has special capabilities, including true phase, conducting AFM, digital pulsed force mode, closed-loop scanning and remote control, to be discussed in detail later. In short, this is our most advanced research system.

Set Point

“Top –

Bottom”

Error Signal

Z signal

Tip

XY scanner

Z scanner

Controller

Chip

PSD

Diode Laser / Lens

sample

1 2

3 4

(1+2) -

(3+4)

1+2+3+4x

(≈10 V)

Microcantilever

NanoScope dual image screen dump

AFM force feedback scheme, contact modeAFM force feedback scheme, contact mode

PVA thin film

PVA thin film

0 1 2 3 4 5

Time (microseconds)Dr

ive

sign

al

Response signal

~

Set Point

“Top –

Bottom”

Error Signal

Z signal

Tip

XY scanner

Z scanner

Controller

Chip

Microcantilever

PSD

Diode Laser / Lens

sample

AC Response

DC Amplitude

~

oscillator

AC Drive

φ

~

φ

AFM amplitude feedback scheme, AC (AFM amplitude feedback scheme, AC (““tappingtapping””) mode) mode

SIBS coatingSIBS coating

Differentiating materials via Differentiating materials via phase imagingphase imaging

within repulsive regime within repulsive regime

φ

Δφ ΔφPhase lag image

Solution-cast triblock

copolymer film (equilibrated)

ABA block copolymer, solvent-vapor annealed: poly (styrene-isobutylene-styrene) or SIBS(styrene cylinders standing or lying down)

Relatively bright regions: glassy phaseof triblock

copolymer → less dissipativeStiffness, adhesion, viscosity,…can give dissipation

100 nm

( )( )0202

1

cantdis AAsinkA

Q2E −φπ

≈

[J. P. Cleveland et al., Appl. Phys. Lett. 72, 2613 (1998)]

Amplitudes:

A = operating set pointA0 = free oscillation at resonance

Energy dissipation per “tap”, Edis :

≈

100 eV

for k ≈

40 N/m ~ 10-4( )22

1 kAE dis

Presenter

Presentation Notes

Here we present a phase image of a triblock copolymer SIBS (styrene-isobutylene-styrene), operating in the repulsive regime. The color convention, brighter being lesser phase lag (below 90º), corresponds a greater shift of the resonant response to higher frequency. The preceding interpretative picture then would suggest that the brighter domains are stiffer, meaning higher in modulus (in the example, polystyrene as compared to polyisobutylene). But a more complete and rigorous understanding of phase also accounts for dissipative effects (i.e., not just changes in interaction stiffness, be it positive under net repulsion or negative under net attraction). Indeed an analytical expression has been derived based on power considerations (energy imparted by driving force minus lost to damping in the absence of tip-sample interaction, i.e., quantified by Qcant; see reference). As shown, this expression relates phase shift only to the energy dissipation, and nothing else related to tip-sample interaction. Analogous dissipation contrast is found in friction force or pulsed force mode adhesion imaging. So, then, does stiffness not play a role? Sure it does, because the stiffer a material the smaller the deformation volume and tip-sample contact area, and thus the less the energy dissipation (whether due to the anelasticity of deformation or interfacial irreversibility). But saying a phase image is simply an image of stiffness or modulus is incorrect in general. (You will find such glib interpretations in many papers.) One could have two materials that are identical in elastic modulus but different in lossiness, or charge, or Hamaker constant (polarizability), etc., and therefore exhibit significant phase contrast. In general, of course, two materials will differ in several physicochemical characteristics, all convolved into the phase value.

SIBS: two phases at surface, stiffer phase also just below surfaSIBS: two phases at surface, stiffer phase also just below surfacece

“tickling”: A/Afree

≈

0.99

Stiffers

cylinders of polystyrene standing or lying down

“pounding”: A/Afree

≈

0.5

Presenter

Presentation Notes

Phase images acquired at very different amplitude set points can highlight surface/bulk differences. This can be seen by comparing subregions of the above phase images. The top two boxes show a different number of bright polystyrene cylinders because every other one is covered by a thin skin of polyisobutylene (that has lower surface energy and thus favors the interface with air). The lower two boxes also show different levels of contrast: three distinct levels for the left image, two for the right. Again this is because some polystyrene cylinders are lying down just below a thin skin of polyisobutylene, whereas those standing on end seem to be protruding from the surface. Under “pounding” operation, the polystyrene is perceived equally by the tip whether right at the surface or just below a thin layer of polyisobutylene. We also note that the boundaries between bright and dark phase are more distinct in the left image, under very light tip-sample interaction. To understand this, we need to consider contact mechanics in terms of its effect on spatial resolution, the next slide.

humidity phase

92%

61%

30% 21º

35º

49º

62º

76º

90º

Poly (ethylene glycol)Poly (butylene

terephthalate)

An even finer block copolymer; humidity to enable contrastAn even finer block copolymer; humidity to enable contrast

1x1 micron phase image

Even delicate AC mode is sometimes not delicate enoughEven delicate AC mode is sometimes not delicate enough

Height

2.5 x 2.5 micron

Phase

Previously scanned

?

?

?

?

300-nm tall

AC attractive regime imaging of water droplets at ~90% RHAC attractive regime imaging of water droplets at ~90% RH

Water sorption on crystalline drug surfacesWater sorption on crystalline drug surfaces

[Kontny, et al. 1995]

RH0 is defined as the relative humidity over the drug’s saturated aqueous solution at temperature T.

RHi

< RH0 RHi

= RH0 RHi

> RH0

Equilibrium RH: AnhydrateEquilibrium RH: Anhydrate--Hydrate SystemHydrate System

- 5. 0

- 3. 0

- 1. 0

1. 0

3. 0

5. 0

7. 0

0 20 40 60 80 100RH ( %)

Weig

ht c

hang

e (%

)

Anhydrate is stable. Hydrate is stable.

OHAOHA 22 ⋅↔+Equilibrium RH for theophylline hydrate anhydrate system:

Presenter

Presentation Notes

After one day storage

What happens on a crystal surface above RHWhat happens on a crystal surface above RH00

??

•

Specific surface area of NaCl (RH0

: 75% RH, 25°C) decrease above 40% RH

Kontny

and Zografi, 1987

•

Surface Conductivity of NaCl increases above 40% RH

Hucher

and Hocart, 1967

The existence of “supersaturated solution ”

on the surface was hypothesized by Kontny

et al; however, solid state NMR did not detect any increase in molecular mobility.

Bulk phase thermodynamics may not apply at the nanoscale on the crystal surface.

Presenter

Presentation Notes

Both ground and unground NaCl samples were exposed to certain RH and sealed in a NMR tube. Increase molecular mobility was observed in

Å

Steps ~ 12 Å

Crystal Steps on Theophylline Anhydrate Crystal Steps on Theophylline Anhydrate CrytalsCrytals

Å

Presenter

Presentation Notes

05/22/06 data

Surface valleys and islands progressively disappear, attributed to significant surface mobility.

5 ×

5 µm, 9 min 5 ×

5 µm, 108 min

Surface Rearrangement of Theophylline Surface Molecules Surface Rearrangement of Theophylline Surface Molecules at 60% RH (25at 60% RH (25˚̊CC), imaged in AC mode), imaged in AC mode

10 ×

10 µm, 117 min10 ×

10 µm, 0 min

Surface rearrangements also occur outside the repeatedly scanned

area (i.e., are not induced by the AFM scanning process).

Zooming Out: check for scanZooming Out: check for scan--induced effectsinduced effects

Presenter

Presentation Notes

The surface rearrangement is not merely facilitated by the tip By zooming out of the intensively scanned area, we can see that the same surface rearrangement also happened

OutlineOutline

1.




2.


modes

•

Introduction•


•


soft material

Desire to contrast material response within a selected interaction distance regime, instead of convolving over all regimes (as does AC/“tapping”

mode):

•

Long-range non-contact forces (e.g., electrostatic)•

Short-range non-contact forces (van der Waals, i.e., dipole-dipole)•

Initial contact (necking?)•

Compression (approach)•

Tension (retraction)•

Hysteresis between approach and retraction (e.g., viscoelasticity)•

Break of adhesive contact during retraction•

Chain molecule or fibril adhesion during retractionForc

e

Distance

Force Force --

distance mappingdistance mapping

FOR

CE

SIG

NA

L Non-contact

Imaging at Imaging at high pixel densityhigh pixel density

via force curvesvia force curves

Slow

(coarse-resolution mapping):

Conventional “force curves”

Triangular ramping of Z, typically ~0.1-10 cycles/second

Fast

(high-resolution imaging):

“pulsed force mode”

Sinusoidal ramping of Z, ~1 kcycles/s, contact time can be <0.1 ms; lateral movement then <1 nm during contact for small images

Contact

~1 kHz

~1 Hz

TIMELoss angle ≡

time location of Fmax

Viscoelastic memory: dependence of Fadh

on Fmax

Fadh

=

[courtesy Witec, www.witec.de]

Appr

oach

tipto

sam

ple

Retract tip

from sam

ple

Presenter

Presentation Notes

We digress briefly on pulsed force mode (PFM); a fuller treatment is left for an advanced training session. In analog PFM, only select features of the force-Z cycle are compiled into images, pixel by pixel. As already stated, one of these is tip-sample adhesion or snap-off force. Another is the time-location of the maximum force at turnaround, the so-called loss angle (i.e., relating to viscoelastic loss). A third is a stiffness measurement based on a deflection differential between two points preselected in time, whether during approach or retraction (in contact). One can also take images at different set point values corresponding to different maximum force during contact. In particular one may find very different tip-sample adhesion for higher forces and/or contact times due to viscoelastic memory effects. In digital PFM, one can additionally acquire the entire force curve at each pixel location using a second data acquisition computer. These can be gigabyte-regime data files (best stored on a portable USB hard drive that you can tuck in a pocket!). In post-processing, one can pull up individual force curves from selected pixel locations in the real-time acquired images; and one can render new images from different parts of each force curve cycle. Obviously data analysis time can reach new levels, but hopefully new analytical insights can be gained about one’s materials.

In air

Height: In water

(> 14 hrs)

Height Adhesion

50 nm

PFM: PFM: Adhesion Adhesion contrast contrast and and elutionelution


polystyrene block copolymer

Rapamycin

C51

H79

NO13

3. Room temp

200 nm

Pulsed force mode adhesion imaging at variable temperature Pulsed force mode adhesion imaging at variable temperature

→→

Reversible, temperatureReversible, temperature--dependent dependent changes; skin of isobutylene?changes; skin of isobutylene?

1. Room temp

2. 50°C


polystyrene block

copolymer

Rapamycin

C51

H79

NO13

InIn--waterwater

poly poly (butyl (butyl methacrylate) / methacrylate) / dexamethasone dexamethasone (50:50, (50:50, spin spin coatedcoated))

1. Initial dry 2. 10 min in water

3. 150 min in water

n 4. 23 hrs in water

500 nm

Z Skewness (cubic dev.)1. 0.192. 0.583. -0.424. -0.52

Presenter

Presentation Notes

A more complete statistical analysis of surface roughness is over a two-dimensional XY domain (rather than just along a single scan line, previous slide). In this example, we compare roughness metrics for a coating that consists of a glassy polymer and a drug, as prepared and after different degrees of drug elution (release) into an aqueous liquid cell. In addition to the standard deviation of height (RMS or root-mean-square), other metrics like skewness (cubic deviation), kurtosis (quartic deviation) and excess surface area are shown. The dominance of protrusions or holes results in positive or negative skewness, respectively. Kurtosis weights the extremities or tailings of the height distribution; in our case a positive number means greater extremes of high and/or low and fewer values near the mean (i.e., a pointy distribution with a wider base, more of a “witch hat” distribution). For a Gaussian or normal distribution, normalized along the abscissa to give a standard deviation of 1, the raw kurtosis is 3. Our value of kurtosis is relative to 3 (i.e., 3 is subtracted to give the displayed numbers). One must be mindful that some programs will instead provide the raw kurtosis (i.e., not subtract 3). The excess surface area is the percentage by which the actual, surface-integrated area exceeds the plan-view area. For example, in a 10x10 micron image, a value of 4.6% (A) means 104.6 square microns of actual surface area.

20 40 60 80 100 120 1400

50

100

150

Num

ber o

f Gra

ins

Equivalent Disk Radius (nm)

SpinSpin--coatedcoated

poly (butyl methacrylate) / dexamethasone (50:50)poly (butyl methacrylate) / dexamethasone (50:50)

Island size distribution via watershed algorithm(freeware Gwyddion)

500 nmN

umbe

r of i

slan

ds

Equivalent disk radius (nm)

Height

Height Adhesion

Loss Angle(Stiffness)


PBMA / PLMA / dexamethasone (43.5:13:43.5)PBMA / PLMA / dexamethasone (43.5:13:43.5)

500 nm

Differential Height

≈22

min in water ≈230 min in waterInitial dryHeight

Adhesion

0

5000

10000

15000

20000

25000

30000

0 10 20 30 40 50 60 70

Adhesion (arb. units)

Cou

nts

≈22 minutes≈230 minutes

Height

Adhesion

Height

Adhesion


PBMA / PLMA / PBMA / PLMA / dexdex. (43.5:13:43.5): water exposure. (43.5:13:43.5): water exposure

500 nm

Amorphous

-

crystalline

drugPLMA-PBMA

5 µm

In water ~1 hour;In water ~1 hour;spinspin--coatedcoated

PBMA / PLMA / PBMA / PLMA /

dexamethasone, AFM and dexamethasone, AFM and confocal Raman microscopyconfocal Raman microscopy

Loss angleHeight

Before After 100°C flash

40x40-μm AFM image

Light microscopy: effects of high temperature Light microscopy: effects of high temperature on on sprayspray--coatedcoated

PBMA / PLMA / dexamethasone (43.5:13:43.5)PBMA / PLMA / dexamethasone (43.5:13:43.5)

(distortion from thermal expansion of substrate)

Increasing contact force

Increasing contact force

SpraySpray--coatedcoated

PBMA / PLMA / dexamethasone (43.5:13:43.5) PBMA / PLMA / dexamethasone (43.5:13:43.5)

Height

Adhesion

Error signal

Stiffness

15 µm

Derivative Height

SpraySpray--coatedcoated

PBMA / dexamethasone (50:50): 100PBMA / dexamethasone (50:50): 100°°CC

100°C

Nanosegregation:

•

Appears while at high temperature but only in thin domains

•

Remains after cooling

Viscoelastic loss (dark: soft)

15 µm

Initial room temperature

Outline / SummaryOutline / Summary

1.




2.


modes

•

Introduction•


•


Environmental Atomic Force and Confocal Raman Microscopies … · 2018-11-09 · Confocal Raman...

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Transcript of Environmental Atomic Force and Confocal Raman Microscopies … · 2018-11-09 · Confocal Raman...