Environmental Atomic Force and Confocal Raman Microscopies … · 2018-11-09 · Confocal Raman...
Transcript of 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
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
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
[E. Vandre, J. Polkinghorne, J. Dong, U. Minnesota, 2007 IPRIME Meeting]
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
[E. Vandre, J. Polkinghorne, J. Dong, U. Minnesota, 2007 IPRIME Meeting]
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.
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
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
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
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
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
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
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:
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.
Å
Steps ~ 12 Å
Crystal Steps on Theophylline Anhydrate Crystal Steps on Theophylline Anhydrate CrytalsCrytals
Å
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
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
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
In air
Height: In water
(> 14 hrs)
Height Adhesion
50 nm
PFM: PFM: Adhesion Adhesion contrast contrast and and elutionelution
Arborescent polyisobutylene-
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
Arborescent polyisobutylene-
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
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)
SpinSpin--coatedcoated
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
SpinSpin--coatedcoated
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.
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