FTIR Microscopy and Imaging - Agilent · FTIR Microscopy and Imaging A world of applications and...
Transcript of FTIR Microscopy and Imaging - Agilent · FTIR Microscopy and Imaging A world of applications and...
FTIR Microscopy and Imaging
A world of applications and solutions
Dr. Mustafa Kansiz
FTIR Imaging and Microscopy Product Manager
Agilent Technologies
Email: [email protected]
FTIR Spectroscopy - What is it?
• Fourier Transform InfraRed Spectroscopy is the study of the
interaction of infrared light with matter.
• The vibrations of bonds between atoms in a molecule are
excited by IR light leading to absorbances that are specific
to chemical structure specific
How can FTIR microscopy imaging help me?
An FTIR microscope has two essential purposes:
1. To allow users to visually see small (micron) sized samples
2. Collect accurate FTIR spectra from small samples
FTIR Imaging takes this to another level by providing spatial
and spectral information from an area on your sample
FTIR microscopy can collect data in four modes:
1. Single point
2. Single point mapping
3. Linear array mapping
4. 2-D Focal Plane Array (FPA) imaging
FTIR Imaging gives spatial (WHERE) and spectral (WHAT) information, and FTIR
Chemical Imaging with a IR Focal Plane Array (FPA), gives this simultaneously
FTIR Microscope Measurement Modes
1 : Single Point
Single or multiple spectra of
different zones of a sample
2: Single Point Mapping Automated acquisition of spectra
(one by one) defined by a grid. A
hundred points can take several
hours.
FTIR Microscope Measurement Modes:
4: FPA Imaging
With an FPA detector, up to
16384 spectra can be recorded
simultaneously in a single
measurement.
3: Linear array Mapping Acquisition of spectra by a row
(1x16) of detectors. Faster than
single point mapping, but still
much slower than FPA imaging
Why use FPA chemical imaging?
Two reasons:
1. Provides rapid high spatial resolution chemical distribution – the where (spatial) and the what (spectral)
2. Allows for the measurement of defects as small as a ~2 microns
What do the Coloured Images Mean?
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ATR Chemical Image
Image @
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PET 7
0 u
m
70 um
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O
=
O
What do the Coloured Images Mean?
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um
70 um
ATR Chemical Image
Image @
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PET
Image @
3295 cm-1
Nylon
N
H
-
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=
O
O
What do the Coloured Images Mean?
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ance
ATR Chemical Image
Image @
1724 cm-1
PET
Image @
3295 cm-1
Nylon
Image @
2915cm-1
PP
70
um
70 um
C
O
=
O
N
H
-
C
H
-
FTIR Chemical Imaging and its applications Materials/Polymer Polymer laminates (functional
layer & adhesive identification)
Defect analysis
Phase distribution
Composites
Biomedical/Biological Research
Early disease diagnosis (Cancers)
Study of diseases (Alzheimer’s, kidney)
Plant/fungi tissue studies
Live cell chemical imaging (in water)
Microbial identification
Bone, teeth and cartilage
Forensics Car paint layer & structure analysis
Trace evidence analysis
Pharmaceuticals Ingredient distribution
Coating analysis
Defect/particle analysis
Electronics Defect analysis
Art Conservation Painting components & layer identification
Geology Study of inclusions
Food/Cosmetics Study of emulsions, eg cheese, mayonnaise,
cream
Infrared Microscopy Sampling Techniques
Transmission
Sample thickness:
10 – 20 mm
Reflectance
Sample thickness:
NA
Absorption/
Reflectance
Sample thickness:
5 - 10 mm
Objective
Sample
Condenser
Stage
θ θ θ
Micro - ATR
Sample thickness:
NA
“Kevley SlideTM”
θ
ATR - IRE
Large Sample Microscopy Sampling Techniques – Measure small areas on large samples
Large Sample
Reflectance
Sample thickness:
NA Sample thickness:
NA
Sample thickness:
NA
large
sample
External
sample
stage
large
sample
large
sample
Large Sample
Micro ATR
Large Sample
Grazing Angle
Obje
ctive
Obje
ctive
GA
O
External
sample
stage
External
sample
stage
Agilent’s patented1 Large Sample Microscope Objective allows you to measure unlimited sized samples in reflection or ATR single point or imaging mode
It’s as simple as:
1. Taking your sample to the microscope
2. Placing it against the 90 degree objective
3. Press collect and get your results!
Agilent’s patented1 Large Sample Microscope Objective allows you to measure unlimited sized samples in reflection or ATR single point or imaging mode
Examples:
1. Investigation of new coating materials on helmets (military / aerospace / etc.)
Agilent’s patented1 Large Sample Microscope Objective allows you to measure unlimited sized samples in reflection or ATR single point or imaging mode
Examples:
1. Investigation of new coating materials on helmets (military / aerospace / etc.)
2. Large vehicle component analysis, ex: car door/panel
Large sample
15x Objective
Mirror assembly
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0.0 4000
cm-1
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s.
LS
Large Sample Objective
Single point analysis (Single-element detector analysis)
Single Point Analysis
1 detector element collects 1 spectrum per scan
Desired spatial resolution is attained by fixing aperture size to desired sampling size. This
eliminates spectral interference from the surrounding area.
Typical best achievable spatial resolution is 10 - 20 µm.
Transmission IR Analysis of Microtomed 3 layer Laminate
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Plastic Tubing – Defect analysis
ATR
ATR
Tube spectrum #2
Tube spectrum #3
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4x glass visible objective
15x reflective vis/IR objective
15x reflective vis/IR objective
2.5 mm
2.0
mm
660 mm
50
0 m
m
660 mm
50
0 m
m
4x glass visible objective
15x reflective vis/IR objective
ATR
Glue spectrum #1
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2.0
mm
660 mm
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0 m
m
Plastic Tubing – Defect analysis
Infrared Mapping
- Single Point
- Linear Arrays
Infrared Single Point Mapping
One data collect at each point gives one interferogram or spectrum
Move the sample with a motorized stage and collect multiple scans
Build up a Map of the sample 1 2
3 4
5 6
Automatic Single Point Mapping
The automated mapping experiment can be used to
obtain IR spectra over a sample.
• Define the field to be mapped.
• Define spacing of the points to be collected (x and y direction).
• Define aperture/spatial resolution (the area you are collecting data on an individual scan).
• You must have visual control of the experiment. You are visually locating areas of interest.
Infrared Linear Array Mapping
• A row of data from each pixel in the array (usually 1 x 16 ) collected
simultaneously
• Move the sample with a motorized stage and collect multiple scans
• Build up a Map of the sample by “stitching” linear array scan together
• Faster than single point mapping, but still much slower than FPA imaging
1 3 4 5 2 6
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Polyester resin
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Wavenumber (cm-1)
Infrared Mapping in Material Analysis:
Multi-layer Paint Sample
Infrared Mapping in Material Analysis:
Multi-layer Paint Sample
Feature image based on absorbance at 3692 cm-1.
Abs. at 3692 cm-1
Infrared 2-D Focal Plane
Array Imaging
FPA-FTIR detectors provide the ability to acquire a grid of spectra in the same
amount of time that it takes single point detectors to acquire one spectrum
One pixel (~5.5µm)
is an entire spectrum
Sample is imaged onto a FPA detector (n n pixel array)
n2 spatially resolved spectra are collected simultaneously
(e.g., 16 16 array 256 spectra)
Principle of Infrared Imaging
Pixel Size (obj mag, NA, mode) Achieved
Spatial
Resolution
3750 cm-1
Achieved
Spatial
Resolution
2500 cm-1
Single FPA tile
FOV (with
128x128FPA)
3.3 um (25x, 0.81NA, std mag) 4.3 um 5.0 um 420x420 um
0.66 um (25x, 0.81NA, high mag) 1.4 um 1.7 um 85x85 um
5.5 um (15x, 0.62NA, std mag) 6.9 um 7.6 um 700x700 um
1.1 um (15x, 0.62NA, high mag) 2.4 um 3.0 um 140x140 um
19 um (4x IR, 0.2NA, std mag) 20.4 um 20.0 um 2400x2400 um
Achieved Spatial Resolution Summary
USAF target imaged (280x280um) at 1.1 um
resolution (high mag mode) with 15x objective in
8 minutes.
2x2 tile mosiac with 128FPA
Entire 2”x2” (50x50mm) USAF target imaged at
19 um pixel resolution with 4xIR objective in 90
minutes
(21x21 tile mosaic with128FPA)
USAF target (700x700um) imaged at 5.5 um pixel
resolution (normal mag. mode) with 15x objective in
2 minutes
Single 128FPA tile
1.4um
0.98um 25x obj
Advancing FTIR Imaging with the Agilent Cary 620
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Highest spatial resolution Largest Field of View
Superior signal-to-noise Fastest data collection
MARKETS AND APPLICATIONS
Polymer Film Laminate FTIR Imaging
Sample Preparation Free FTIR Chemical Imaging of Polymer laminates & Films
Step 1. Cut out small piece Step 2. Place cut-out piece in micro-vice.
Step 3. Cross-section sample with razor Step 4. Place micro-vice (with sample) on
microscope stage & touch ATR
ATR Contact with sample
STEP 5.
raise stage
to make
contact &
collect data
micro ATR
Microscope Stage
micro-vice
Sample
100 micron
wide (thick)
IR light in IR light out
micro ATR
micro-vice
Microscope Stage
Sample
100 micron
wide (thick)
IR light in IR light out
Standard Live ATR direct FPA IR Image – without correction
Live ATR direct FPA IR Image with Enhanced Chemical Contrast
No Pressure
(before contact)
“Live/Real-Time” ATR contact monitoring
No Pressure
(before contact)
No Pressure
(before contact)
“Live/Real-Time” ATR contact monitoring
First Contact No Pressure
(before contact)
Stage is
raised
Stage is
raised
First Contact
Standard Live ATR direct FPA IR Image – without correction
Live ATR direct FPA IR Image with Enhanced Chemical Contrast
No Pressure
(before contact) Increasing Pressure
Increasing Pressure
“Live/Real-Time” ATR contact monitoring
First Contact Complete Contact No Pressure
(before contact)
Stage is
raised
Stage is
raised
First Contact
Standard Live ATR direct FPA IR Image – without correction
Live ATR direct FPA IR Image with Enhanced Chemical Contrast
No Pressure
(before contact) Increasing Pressure
Increasing Pressure
“Live/Real-Time” ATR contact monitoring
First Contact Complete Contact No Pressure
(before contact)
Stage is
raised
Stage is
raised
First Contact
Standard Live ATR direct FPA IR Image – without correction
Live ATR direct FPA IR Image with Enhanced Chemical Contrast
No Pressure
(before contact) Increasing Pressure
Increasing Pressure
“Live/Real-Time” ATR contact monitoring
First Contact Complete Contact No Pressure
(before contact)
Stage is
raised
Stage is
raised
First Contact
Standard Live ATR direct FPA IR Image – without correction
Live ATR direct FPA IR Image with Enhanced Chemical Contrast
No Pressure
(before contact) Increasing Pressure
Increasing Pressure
“Live/Real-Time” ATR contact monitoring
First Contact Complete Contact No Pressure
(before contact)
Stage is
raised
Stage is
raised
First Contact
Standard Live ATR direct FPA IR Image – without correction
Live ATR direct FPA IR Image with Enhanced Chemical Contrast
No Pressure
(before contact) Increasing Pressure
Increasing Pressure
“Live/Real-Time” ATR contact monitoring
First Contact Complete Contact No Pressure
(before contact)
Stage is
raised
Stage is
raised
First Contact
Standard Live ATR direct FPA IR Image – without correction
Live ATR direct FPA IR Image with Enhanced Chemical Contrast
Sample Presentation
Confidentiality Label
June 12, 2015
39
A small piece of the sample was cut out
and place into a micro-vice sample holder
where it was cut flat with a sharp razor
The micro-vice is placed in a dedicated insert
on the motorised stage and contact is made with the
ATR crystal, using Agilent’s unique “Live ATR contact”
method, without any need for resin embedding
50
0 u
m
670 um
Sausage Packaging (Red): Visible images ATR Imaging Sampling Location
15x obj. vis image
ATR
spot 1
Collection Conditions:
Resolution: 4 cm-1
Scans (time): 32 scans (~30 sec) per spot
Spectral Range: 4000 – 850 cm-1
Collection Mode: Micro ATR & transmission (5 micron microtomed slices)
Pixel Size & FOV: 1.1 microns/pixel, 70x70 microns
ATR
spot 2
ATR
spot 3
Different collection mode comparison
1. Micro ATR (1.1 micron @ 70x70 micron FOV)
2. Transmission with 15x normal mag (5.5 micron pixel size & 700x700 micron FOV)
3. Transmission with 15x HIGH mag (1.1 micron pixel size & 140x140 micron FOV)
4. Transmission with NEW 25x normal mag (3.3 um pixel size & 420x420 um FOV)
5. Transmission with NEW 25x HIGH mag (0.66 um pixel size & 85x85 um FOV)
-
Confidentiality Label
June 12, 2015
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Confidentiality Label
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42
15x obj. vis image
70
um
70 um
ATR Image @ 2850cm-1
PE
~46 um thick
70
um
70 um
70
um
70 um
ATR Image @ 1607cm-1 PE (with additive)
~23 um thick
70
um
70 um
ATR Image @ 1725 cm-1 Polycarboxylic acid ester
polymer (possibly)
~5 um thick
70
um
70 um
ATR Image @ 1202cm-1 polyamide
ATR Chemical
RGBY Composite Image
Red: PE
Yellow: PE (with additive)
Green: Polycarboxylic acid ester (possible)
Blue: Polyamide
Layer 1&3
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ance
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Layer 5
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bsorb
ance
1 2 3 4 5
Sausage Packaging – ATR Chemical Images (Spot 1)
Sausage Packaging – ATR Chemical Images (Spot 2)
Confidentiality Label
June 12, 2015
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15x obj. vis image 7
0 u
m
70 um
ATR Chemical Image
RBGWC composite image
Red: PE
Green: Polycarboxylic acid
polymer (possibly)
Blue: Polyamide
White: Polypropylene
Cyan: EVOH
70
um
70 um
Ima
ge
@
28
50
cm
-1
Layer 4
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ance
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ance
Layer 6
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ance
Ima
ge
@
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25
cm
-1
Ima
ge
@
12
02
cm
-1
Ima
ge
@
29
51
cm
-1
Ima
ge
@
10
90
cm
-1 Layer 9
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ance
Layer 3, 7
& 11
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ance
PE (with additives)
~6 um thick (centre layer)
Polycarboxylic acid ester
polymer (possibly)
~5 um thick
Polyamide
~7um (left)
~2um (centre)
~3um (right)
Polypropylene
~20um
EVOH
~5um
(spectral overlap from
Polyamide present)
3 4
5 6 7
8 9 10
11
Confidentiality Label
June 12, 2015
44
15x high mag. obj. vis image
70
um
70 um 7
0 u
m
70 um
ATR Chemical Image PP (possible with an
additive as evident by
peak at 1060cm-1)
~37 um thick
RBC composite image
Red: PE
Blue: Polyamide
Cyan: EVOH
Sausage Packaging – ATR Chemical Images (Spot 3)
Layer 5, 8 & 10
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ance
Polyamide
~2um (left)
~3um (right)
Layer 9
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ance
EVOH
~5um thick
(spectral overlap
from Polyamide
present)
Layer 11
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ance
7 8 9 10 11
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June 12, 2015
45
Sausage Packaging – ATR Chemical Images
The sample was measured across its entire width with three
slightly overlapping ATR measurements, with a total
collection time of ~ 1.5 mins.
As extremely low pressures are applied, there is no sample
preparation (via Agilent’s “live ATR imaging” method) with
samples being measured “as is” and no risk of sample
surface deformation, which might otherwise make for
sequential side-by-side, or slightly overlapping
measurements impossible. There was also no evidence of
sample carryover between the measurements.
With the excellent signal-to-noise data collected, quite a
complex sample with at least 11 layers were revealed with
spectral library searches assisting in layer identification.
PE
~11
~130 um
FT
IR A
TR
im
ag
ing
de
term
ine
d id
en
tity
&
ap
pro
xim
ate
th
ickn
ess
(in
mic
ron
s)
PE
with
ad
ditiv
e
Poly
carb
oxylic
acid
este
r
poly
mer
(possib
ly)
P
A
PE
PP
PE
P
A
PA
E
VO
H
PE
~24 ~11 ~5 ~7 ~20 ~6
~3
~5
~3
~37
1 2 3 4 5 6 7 8 9 10 11
40x obj. vis image (polarized)
Pixel size: 1.1micron
Obj mag: 15x, 0.62NA
FOV: 70x70um
Total system mag: 36x
14
0 u
m
180 um
Confidentiality Label
June 12, 2015
46
Sausage Packaging – 25x Transmission High mag Chemical Images
- Even in transmission mode, the ultra high
NA and very small pixel size has allowed for
the resolution of the 3 micron PA layer,
hence rivalling the spatial resolution of Ge
micro ATR
- Most books and papers still talk of ~10um
spatial resolution for transmission imaging!
- The 25x, 0.81NA is a revolution in objective
design
40x obj. vis image (polarized)
EVOH, layer 3
PA, layer 2
PE, layer 1
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Wavenumber
Absorb
ance
Pixel size: 0.66 micron
Obj mag: 25x, 0.81NA
High mag: ON
FOV: 85x85um
Total system mag: 61x
Working distance: 12mm
1
2 3
14
0 u
m
180 um
Confidentiality Label
June 12, 2015
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The ~3 micron PA layers are only just resolved,
but the spectral differences between PA and
EVOH are now much less, owing to the lower
NA, and hence resolving power, of the 15x
objective compared to the 25x objective.
40x obj. vis image (polarized)
Pixel size: 1.1micron
Obj mag: 15x, 0.62NA
High mag: ON
FOV: 140x140um
Total system mag: 36x
Working distance: 21mm
Sausage Packaging – 15x Transmission High mag Chemical Images
1
2 3
EVOH, layer 3
PA, layer 2
PE layer 1
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Wavenumber
Absorb
ance
14
0 u
m
180 um
Confidentiality Label
June 12, 2015
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With the larger pixel size, now at 3.3um, even
with the high
NA 25x objective, the ~3um PA layers cannot
be spatially resolved
40x obj. vis image (polarized)
Pixel size: 3.3micron
Obj mag: 25x, 0.81NA
High mag: OFF
FOV: 420x420um
Total system mag: 12x
Sausage Packaging – 25x Transmission std mag Chemical Images
14
0 u
m
180 um
Confidentiality Label
June 12, 2015
49
14
0 u
m
180 um
40x obj. vis image (polarized)
Pixel size: 5.5 micron
Obj mag: 15x, 0.62NA
High mag: OFF
FOV: 700x700 um
Total system mag: 7.3x
Sausage Packaging – 15x Transmission std mag Chemical Images
As the pixel size gets larger, now at 5.5um,
layers that are ~5 um now start to blur out
Principal Component Noise Reduction (PCNR)
June 12, 2015
Agilent Confidential
50
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ance
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Absorb
ance
1 scan
1 scan with PCNR
128 scans
- Removes ONLY noise
- Typical S:N
improvement of
~5-10x
- Corresponding time
saving (for equivalent
S:N) is 25-100x !
- Only FTIR imaging
system to include
PCNR
Residual spectrum
1720 cm-1
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ance
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ance
640 um
480 u
m ATR
defect, image @ 1402 cm-1
polymer, image @ 1402 cm-1
70
mm
70 mm
Composite (Green/Red) image
GREEN – DEFECT
RED - POLYMER
At initial analysis, it appears that the defect is likely to be an
Inorganic material, most probably a carbonate, or a carbonate
containing mixture
15x obj. vis image – cross-section view ATR Chemical Image
Polymer Film Defects - Visible Images & ATR Imaging Sampling
Location
Micro ATR (FPA) imaging of defects in black rubber sample
Ima
ge
cre
ate
d
at 1
64
4 c
m-1
Im
age
cre
ate
d
at 2
84
8 c
m-1
Vis image IR image
70mm
70
mm
10 um
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ance
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ance
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Ab
so
rba
nce
Polyamide
PE
Polyisoprene (natural rubber)
ATR
640 um
480 u
m
25x, 0.81 NA Objective in “high mag” mode for Transmission Polymer bead analysis
June 12, 2015
Agilent Confidential
53
2915 cm-1 1442 cm-1 1118 cm-1
40x vis image
85mm
85
mm
5 micron polystyrene beads on BaF2 substrate are
clearly chemically distinguished.
Even at relatively long wavelengths (low
wavenumbers), 5 micron beads are clearly resolved
(3.4 microns) (6.9 microns) (8.9 microns)
Pixel size: 0.66 micron
Obj mag: 25x, 0.81 NA
High mag: ON
FOV: 85x85 um
Total system mag: 61x
Working Distance: 12 mm
35
0u
m
470um
Exposed green plastic – Visible images & ATR Imaging Sampling Location
15x obj. vis image
ATR
70
um
70 um
Sample Placement & Measurement
Confidentiality Label
June 12, 2015 55
The large green plastic block was placed directly “as-is” into
the micro-vice holder, which was then placed on the microscope
stage, followed by raising of the stage to make contact with the
ATR for data collection.
3500 3000 2500 2000 1500 1000
0.1
0.0
-0.1
Wavenumber
Absorb
ance
Results - Exposed Green Plastic
Confidentiality Label
June 12, 2015 56
40x high mag. obj. vis image
70
um
70 um
70
um
70 um
ATR Chemical Image Image @ 1736cm-1
A clear edge containing higher carbonyl content is
clearer visible along the expose side edge.
Pharmaceutical FTIR Imaging
57
Row = 51 Col = 28
3500 3000 2500 2000 1500 1000
0.3
0.2
0.1
0.0
Wavenumber
Absorb
ance
Row = 43 Col = 59
3500 3000 2500 2000 1500 1000
0.3
0.2
0.1
0.0
Wavenumber
Absorb
ance
Row = 12 Col = 10
3500 3000 2500 2000 1500 1000
0.4
0.3
0.2
0.1
0.0
Wavenumber
Absorb
ance
Row = 29 Col = 19
3500 3000 2500 2000 1500 1000
0.20
0.15
0.10
0.05
Wavenumber
Absorb
ance
Tablet – Contamination Location 15x Vis Image
48
0 m
m
640 mm
70
mm
70 mm
Clear and distinct domains from
4 constituents were detected:
- Active, compared to provided standard
- Lactose, identified from library search
- Starch, identified from library search
- Cellulose identified from library search
Active
Lactose
Starch
Cellulose
Defect (next slide)
Row = 10 Col = 26
3500 3000 2500 2000 1500 1000
0.20
0.15
0.10
0.05
Wavenumber
Absorb
ance
Row = 30 Col = 28
3500 3000 2500 2000 1500 1000
0.15
0.10
0.05
Wavenumber
Absorb
ance
15x Vis Image
48
0 m
m
640 mm
70
mm
70 mm
Clear and distinct domains from
4 constituents were detected:
- Contaminant #1 – Possibly a polyester
- Contaminant #2, - Possibly a polyamide
Tablet – Contamination Location 7
0 m
m
70 mm
Contaminant Composite
(RG), part 2
Polyamide
Polyester
From a single FTIR ATR imaging measurement and
without damaging the sample, 4 known constituents
and 2 unknown contaminants were imaged in 1 min
Electronics/Semicon FTIR Imaging
Spacer contamination on LCD filter
3500 3000 2500 2000 1500 1000
0.03
0.02
0.01
0.00
Wavenumber
Absorb
ance
3
50
um
350 um
70
um
70 um
5 mm
Total analysis time = 2 mins
Defects identified as dislodged
Spacers
No sample prep and no sample
damage
1647.960 12.936Row = 6 Col = 30
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
0.3
0.2
0.1
0.0
Wavenumber
Absorb
ance
LCD Defect – Protein image (1647 cm-1)
70 microns
70 m
icro
ns
Visible Image
IR image
at 1647cm-1 Total analysis time = 1 mins
Defects identified as protein, most
probably flake of dead skin
No sample prep and no sample
damage
480 u
m
640 um
3500 3000 2500 2000 1500 1000
0.20
0.15
0.10
0.05
0.00
Wavenumber
Absorb
ance
350 u
m
70
um
70 um
Contaminated Circuit Board – FTIR ATR Imaging
ATR contact damage
caused by other vendor
Total analysis time = 2 mins
Spectra library search
reveals contaminant to be
polyetherimide
Visible Image
Forensics FTIR Imaging
Vehicle paint cross sectional analysis
36
96
33
99
30
38
29
50
1723.527 21.695
16
95
1602.985 1.303
1561.467 0.11515
10
14
65
13
75
11
33
10
55
Row = 75 Col = 160
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
0.8
0.6
0.4
0.2
0.0
Wavenumber
Absorbance
36
96 3
39
9
30
38
29
50
1723.527 37.245
16
95
1602.985 -1.255
1561.467 0.10015
10
14
65
13
75
11
33
10
55
Row = 24 Col = 146
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
0.6
0.4
0.2
0.0
Wavenumber
Absorbance
36
96
33
99
30
38
29
50
1723.527 20.244
16
95
1602.985 -0.465
1561.467 0.301
15
10
14
65 13
75 11
33
10
55
Row = 94 Col = 92
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
0.8
0.6
0.4
0.2
Wavenumber
Absorbance
36
96
33
99
30
38
29
50
1723.527 13.5691
69
5
1602.985 -2.144
1561.467 0.463
15
10
14
65
13
75
11
33
10
55
Row = 90 Col = 98
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Wavenumber
Absorbance
36
96
33
99
30
38
29
50 1723.527 15.150
16
95
1602.985 3.889
1561.467 0.031
15
10
14
65
13
75
11
33
10
55
Row = 164 Col = 123
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
0.6
0.5
0.4
0.3
0.2
Wavenumber
Absorbance
36
96
33
99
30
38
29
50
1723.527 21.636
16
951602.985 1.044
1561.467 0.080
15
10
14
65
13
75
1086.493 7.960
10
55
Row = 149 Col = 135
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
1.0
0.8
0.6
0.4
0.2
0.0
WavenumberA
bsorbance
3696 cm-1
3399 cm-1 1695 cm-1 1561 cm-1 1086 cm-1
3038 cm-1
MIXED POWDER MICRO ATR ANALYSIS – CALCIUM CARBONATE
CaCO3(1420.000)
STPP(1140.010)pyrophosphate(987.127)disodium phosphate(945.393)
Row = 37 Col = 32
3500 3000 2500 2000 1500 1000
0.8
0.6
0.4
0.2
0.0
Wavenumber
Absorb
ance
MIXED POWDER MICRO ATR ANALYSIS – DISODIUM DIPHOSPHATE ANHYDRIDE
CaCO3(1420.000)
STPP(1140.010)
pyrophosphate(987.127)
disodium phosphate(945.393)
Row = 38 Col = 48
3500 3000 2500 2000 1500 1000
0.20
0.15
0.10
0.05
0.00
Wavenumber
Absorb
ance
CaCO3(1420.000)
STPP(1140.010)
pyrophosphate(987.127)disodium phosphate(945.393)
Row = 61 Col = 56
3500 3000 2500 2000 1500 1000
0.2
0.1
0.0
Wavenumber
Absorb
ance
MIXED POWDER MICRO ATR ANALYSIS – SODIUM TRIPOLYPHOSPHATE
CaCO3(1420.000)
STPP(1140.010)
pyrophosphate(987.127)
disodium phosphate(945.393)
Row = 17 Col = 17
3500 3000 2500 2000 1500 1000
0.3
0.2
0.1
0.0
Wavenumber
Absorb
ance
MIXED POWDER MICRO ATR ANALYSIS – SODIUM PYROPHOSPHATE
Food/Cosmetics FTIR Imaging
Imaging of mayonnaise heterogeneity: measured in transmission on BaF2 slide
1648.271 24.136TmpltPk1(3614 0.5)
2999.749 -0.594
2925.612 0.119
2113.148 4.845
oil(1745 0.0)
water(1650 40.7)starch(1046 48.9)
Row = 69 Col = 63
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
1.5
1.0
0.5
0.0
Wavenumber
Absorbance
1648.271 1.474TmpltPk1(3614 0.0)
2999.749 1.613
2925.612 22.490
2113.148 0.086
oil(1745 9.6)
water(1650 2.3)
starch(1046 0.0)
Row = 28 Col = 106
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
1.4
1.2
1.0
0.8
0.6
0.4
0.2
Wavenumber
Absorbance
1648.271 24.505TmpltPk1(3614 0.5)
2999.749 -0.2642925.612 0.210
2113.148 4.503
oil(1745 0.1)
water(1650 39.8)
starch(1046 2.0)
Row = 70 Col = 66
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
1.5
1.0
0.5
0.0
Wavenumber
Absorb
ance
sugar @1046cm-1
oil @1745cm-1
water @1650cm-1
Visible CCD image
650 um
50
0 u
m
Measured on FTS7000+UMA600, using
a 128x128 FPA.
IR measurement area = 700x700 microns
Time of measurement = ~ 2min
Resolution = 8 cm-1
Biological & Biomedical FTIR Imaging
Breast Tissue section
CH image (2943-2893 cm-1) Amide I image
Note: red box, indicates area for single tile high mag collect on next slide
1233 cm-1 image
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
1.2
1.0
0.8
0.6
0.4
0.2
Wavenumber
Absorb
ance
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Wavenumber
Absorb
ance
Total IR FOV = 1400x1400 um (2x2 mosaic), pixel size = 5.5um, total data collect time ~ 6 mins,
Amide I image 1233 cm-1 image 40x obj vis image
CH image (2943-2893 cm-1)
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800
1.0
0.8
0.6
0.4
0.2
0.0
Wavenumber
Absorb
ance
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Wavenumber
Absorb
ance
Breast Tissue section (high mag mode)
Note: red box, indicates area for single tile high mag collect on next slide
Total IR FOV = 280x280 um (2x2 mosaic), pixel size = 1.1um, total data collect time ~ 12 mins,
Confidentiality Label
June 12, 2015
75
Normal Mag (5.5um) – High Mag (1.1um) comparison, CH image
28
0 u
m
280 um
28
0 u
m
280 um
3500 3000 2500 2000 1500 1000
0.6
0.4
0.2
0.0
Wavenumber
Absorb
ance
3500 3000 2500 2000 1500 1000
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Wavenumber
Absorb
ance
Normal Mag (5.5um) High Mag (1.1um)
The 3 features
within this oval
shape are each
about ~3 um
in diameter!
3341.250 71.728
1733.180 -0.889
1654.450 22.9651651.180 28.040
1539.600 7.408
1044.970 27.715
Row = 26 Col = 89
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
0.8
0.6
0.4
0.2
Wavenumber
Absorb
ance
3341.250 162.961
1733.180 7.765
1654.450 6.1331651.180 8.118
1539.600 -0.261
1044.970 74.983
Row = 68 Col = 104
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
1.2
1.0
0.8
0.6
0.4
Wavenumber
Absorb
ance
3341.250 104.920
1733.180 2.899
1654.450 9.8421651.180 14.684
1539.600 2.721
1044.970 54.253Row = 57 Col = 100
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
1.0
0.8
0.6
0.4
Wavenumber
Absorb
ance
Protein – 1654 cm-1
Lipid – 1733 cm-1
Carbohydrate – 1200-950 cm-1
Wheat stem cross section FTIR Image See next set of
data for med.
res. close up
See next set of
data for med.
res. close up
See next set of
data for med.
res. close up
See following set of
data for high.
res. close up
See following set of
data for high.
res. close up
See following set of
data for high.
res. close up
1043.570 11.216
Row = 12 Col = 21
3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Wavenumber
Absorb
ance
Visible Image
(red box show field of
view ATR)
2-D IR image
at 1043 cm-1
3-D IR image
at 1043 cm-1
IR spectrum from
cross-hairs above
ATR Imaging has provided
for a 5 fold increase in
magnification and REAL
spatial resolution
enhancement.
~5 micron cells wall have
been CLEARLY resolved
with 10 micron light (~1000
cm-1) and have produced
high quality spectra free of
artefacts due to diffraction
and scattering.
70 microns
70 m
icro
ns
June 12, 2015
78
Visible image C-H image Lipid image Protein image
Composite (RGB) image
Lipid – Red
C-H – Green
Protein - Blue
100 microns 100 microns 100 microns 100 microns 100 microns
Live cell FTIR imaging in water!
Liquid transmission cell pathlength = 7microns
Total measurement time ~ 10mins.
Pixel size : 1.1 microns
Summary of Cary FTIR Imaging Highest Spatial Resolution
with new 25x, 0.81NA obj.
Re-defines, what is possible with actual spatial resolution of <1.5 microns possible in transmission mode!
Largest Field of View
Measure up to 2.4x2.4mm in a single shot
Fastest analysis time
Reduce analysis times by >100x , with higher light throughput & PCNR
June 12, 2015
79
Live FPA Imaging
Removes need for complex, time consuming sample prep & allows for
delicate analysis