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Advancing FT-IR Imaging –Synchrotron IR Imaging in Your Lab
Dr. Mustafa KansizFTIR Microscopy & Imaging Product Manager
How can FTIR microscopy imaging help me?
An FTIR microscope has two essential purposes:1. To allow users to visually see small (micron) sized samples2. 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 point2. Single point mapping3. Linear array mapping4. 2-D Focal Plane Array (FPA) imaging
FTIR Microscope Measurement Modes
1 : Single Point Single or multiple spectra of different zones of a sample
2: Single Point MappingAutomated acquisition of spectra(one by one) defined by a grid. A hundred points can take severalhours.
FTIR Microscope Measurement Modes:
4: FPA ImagingWith an FPA detector, up to 16384 spectra can be recordedsimultaneously in a single measurement.
3: Linear array MappingAcquisition of spectra by a row(1x16 or 2x14) 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
FTIR Chemical Imaging and its applicationsMaterials/PolymerPolymer laminates (functionallayer & adhesive identification)Defect analysisPhase distributionComposites
Biomedical/Biological ResearchEarly disease diagnosis (Cancers)Study of diseases (Alzheimer’s, kidney)Plant/fungi tissue studiesLive cell chemical imaging (in water)Microbial identificationBone, teeth and cartilage
ForensicsCar paint layer & structure analysisTrace evidence analysis
PharmaceuticalsIngredient distributionCoating analysisDefect/particle analysis
ElectronicsDefect analysis
Art ConservationPainting components & layer identification
GeologyStudy of inclusions
Food/CosmeticsStudy of emulsions, eg cheese, mayonnaise,cream
A New Method of Magnification Enhancement
New Method of Magnification Enhancement
- The pixel size at the sample plane (pixel resolution) is a combination of:
- Native FPA detector element size- Intermediate optics magnification- Objective magnification
- It is important to note that, pixel resolution is therefore NOT ONLY governed by the objective.
Total system magnification
FTIR Microscope Magnification SchematicFPA
40x40 um pixel
Pixel size = 5.5 microns
Stage
Total system magnification = Native detector size/sample pixel size
Pixel size = (native detector size/obj mag) / “other mag”
FPA
40x40 um pixel
Total system magnification = Native detector size/sample pixel size
Pixel size = (native detector size/obj mag) / “other mag”
Pixel size = 1.1 microns
FTIR Microscope Magnification Schematic
It’s the “total magnification” that matters, which together with the native FPA pixel size, equates to final pixel size at the sample plane.Objective magnification alone, is only a factor in the overall “total magnification” equation
A big advantage of this approach is FULL PRESERVATION of the long objective working distance of 21mm, allowing a wide array of accessories and sample holders to be used
Stage
Microscope objectives: some comparisons
Pixel size:5.5 m (normal)
1.1 m (high mag)
Pixel size:1.1 m
Pixel size:19 m (normal)
3.8 m (high mag)
21 m
m
38 m
m
10 m
m
15x0.62 NA
4x0.2 NA
36x0.5 NA
Agilent
Agilent offer a wider range of pixel size options with better NA, to provide better spatial resolution or faster image collection over large areas – and with more useful working distances.
Pixel size:0.5 m
74x0.65 NA
Agilent’s new 15x objective
Agilent’s new 4x objective
Synchrotrons & Other Systems
1 m
mPixel size:
2.7 m
24 m
m
15x0.4 NA
Advancing FTIR Imaging with the Agilent Cary 620
February 3, 2015
12
Highest spatial resolution Largest Field of View
Superior signal-to-noise Fastest data collection
- What is a synchrotron?- A synchrotron is a huge particle accelerator that energizes electrons to
create bright beams of X-rays, infrared and ultraviolet light. Beams are taken off the main line and directed to detectors located on different nodes.
- Why use a synchrotron?- Great source of narrow intense beam of light (eg IR), which are great
for single point microscopy with apertures <10 microns.
Synchrotron FTIR microscopy
- FTIR imaging on a synchrotron?- Synchrotrons are great for very small FOVs (<10um), but to image
large FOVs (>hundreds of microns), requires multiple beam extractions and/or defocusing to spread the light out, removing all the brightness advantages of a synchrotron
- For large area imaging, simple optics rules tell us that a large area source is required to illuminate a large area detector, such as an FPA
- For this, the traditional FTIR globar is still the best option, as the large area globar is matched to the large area FPA detectors.
Synchrotron FTIR microscopy
Synchrotron single point FTIR microscopy
10 um synchrotronsource
Single point MCT(100x100um)Transfer optics
(spectrometer & microscope)
1:1
In single point mode, beam does not need to illuminate full area of MCT detector.High brilliance of synchrotron beam is utilised
Synchrotron FPA FTIR Imaging
FPA (128x128)
5.1 mm
5.1
mm
10 um synchrotronsource
Transfer optics (spectrometer & microscope)
1:260000
In imaging mode, beam must illuminate full area of FPA detector, this therefore requires a 260,000x defocussing, losing the synchrotron intensity advantage
Agilent’s Thermal source FPA FTIR Imaging
FPA (128x128)
5.1 mm
5.1
mm
Transfer optics (spectrometer & microscope)
1:1
7.5 mm
5 m
m
Globar (double image)
With a globar (image doubled with retroreflector), the hot spot is area is a 1:1 match to the FPA area, maximising intensity usage, resulting in better sensitivity
2500 cm-1
700 microns
700
mic
rons
Standard Magnification 5.5 um pixel size
2500 cm-1
280 microns
280
mic
rons
High Magnification 1.1 um pixel size
Standard Magnification 5.5 um pixel size
280 microns
2500 cm-1
High IR Magnification – A Synchrotron on your bench!
AGILENT CONFIDENTAL
0.00
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0.50
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60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
3750…2500…
High Magnification Transmission3750 cm-1
2500 cm-1
0102030405060708090
100110
0 100 200 300 400
%T
Pixel
3750 cm-1
2500 cm-1
Rayleigh Criterion0.264
Lp/mm = 166Line Width = 3.0 m
Lp/mm = 205Line Width = 2.4 m
Group 6Group 7
Group 8
Con
trast
280 microns
280 microns
280
mic
rons
280
mic
rons
Modulation Transfer Function (MTF)
Pixel Size (obj/mode) Achieved Spatial Resolution3750 cm-1
Achieved Spatial Resolution2500 cm-1
Single FPA tile FOV (with 128x128FPA)
5.5 um (15x, normal) 6.9 um 7.6 um 700x700 um
1.1 um (15x, high) 2.4 um 3.0 um 140x140 um
19 um (4x IR, normal) 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 minutesSingle 128FPA tile
ADVANTAGES AND BENEFITS OF THE NEW FEATURES
Cary 620 top 4 advantages• New high mag optics• >400% IR energy• Synchrotron
comparable high resolution data
Highest Spatial Resolution
Highest Spatial Resolution
• Proprietary 4x IR objective
• Measure cm x cm areas in minutes
Largest Field of View
Largest Field of View
• >10x - other FPA’s• > 50x - linear array• >100x - single point
Fastest analysis time
Fastest analysis time
•Enhanced chemical contrast software mode
•Eliminate sample prep•Avoid damaging samples
Live FPA imagingLive FPA imaging
1. Highest spatial resolution• New high magnification optics provide pixel resolution down to 1.1 um in
transmission/reflection• Superior IR energy throughput from FTIR bench and new IR objectives
ensures highest quality data when measuring in high mag mode• Achieve data quality of high spatial resolution similar to that of a
Synchrotron
2. Largest Field of View (FoV)• Use proprietary 4x IR objective to measure samples cm x cm in area within
minutes• Change objectives in seconds to increase spatial resolution and zoom in
on smaller areas of interest• Switch to high magnification mode to resolve features as small as 2 um.
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 minutesSingle 128FPA tile
3. Fastest analysis time• Unparalleled IR energy from the FTIR bench (>400%) coupled with increased IR
throughput of new 15x objective (>40%) guarantees the highest quality data in the shortest period of time.
• Faster than any other FPA, Linear Array or single point detector available today• Measure the largest samples at the highest resolution in the shortest period of
time!
Linear array mapping – in 20 min, only 5% of image is collected.
Agilent FPA imaging – in 20 minutes 100% of image collected at 5.5um resolution
Agilent’s unique chemical contrast feature provides clear differences in live IR mages between no contact being made with the sample vs good ATR contact.
This provides a feedback mechanism on when to stop applying pressure to the sample, avoiding excess pressure that can damage the sample.
The benefit is removing time consuming resin embedding sample preparation to increase productivity, as well as avoiding applying too much pressure to your sample that can cause damage.
4. Live FPA Imaging with enhanced chemical contrast
MARKETS AND APPLICATIONS
Materials Life Sciences Research Pharmaceuticals Food Forensics
Polymer laminates Early disease investigation (eg. Cancers, Alzheimer's, etc) Ingredient distribution Packaging quality Car paint layer and structure
Defect analysis Live cell imaging (in water) Coating analysis Study of emulsions Trace evidence analysis
Electronics, Semicon Plant/fungi tissue studies Defect/particle analysis Pathogen detection Art conservation/fraud
A world of applications…
Polymer laminates- Total elapsed time to
measure sample < 5min
- PE Layer ~3um thick
Live cell in water- IR image
measured in waterin ~6 minutes
- Data collected using high mag mode with a 128x128 FPA
Live Cell Imaging IN WATER
Visible image C-H image Lipid image Protein image Composite (RGB) imageLipid – Red
C-H – GreenProtein - Blue
Traditionally, FTIR is thought to be incompatible with measurements through waterAgilent’s new high throughput high magnification system allows for live cell imaging at biologically relevant time frames with spatial resolution now exposing organelles
100 microns100 microns 100 microns 100 microns100 microns
Conditions: Micrasteris Species, 8 micron pathlength, CaF2 liquid cell. 8 cm-1 resolution128x128 FPA, 1.1micron pixel size (21mm WD), 6 mins total collection time
Breast Carcinoma Tissue
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
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0.2
Wavenumber
Abso
rban
ce
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
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Wavenumber
Abso
rban
ce
Visible image
Total IR FOV = 1400x1400 um (2x2 mosaic 128FPA), pixel size = 5.5um, total data collect time ~ 6 mins,
Breast Carcinoma Tissue
Amide I image 1233 cm-1 image40x 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
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Wavenumber
Abso
rban
ce
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800
0.7
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0.5
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Wavenumber
Abso
rban
ce
Total IR FOV = 280x280 um (2x2 mosaic 128FPA), pixel size = 1.1um, total data collect time ~ 12 mins,
Normal Mag (5.5um) – High Mag (1.1um) comparison, CH image
280
um
280 um
280
um
280 um
3500 3000 2500 2000 1500 1000
0.6
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Wavenumber
Abs
orba
nce
3500 3000 2500 2000 1500 1000
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Wavenumber
Abs
orba
nce
Normal Mag (5.5um) High Mag (1.1um)
Normal Mag (5.5um) – High Mag (1.1um) comparison, Amide I image
280
um280 um
280
um
280 um
Normal Mag (5.5um) High Mag (1.1um)
Normal Mag (5.5um) – High Mag (1.1um) comparison, 1233 cm-1 image
280
um280 um
280
um
280 um
Normal Mag (5.5um) High Mag (1.1um)
Polymer Film Laminate FTIR Imaging
NEW - 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 razorStep 4. Place micro-vice (with sample) on
microscope stage & touch ATR
ATR Contact with sample
STEP 5. raise stage
to makecontact &
collect data
micro ATR
Microscope Stagemicro-vice
Sample100 micron wide (thick)
IR light inIR light out
micro ATR
micro-viceMicroscope Stage
Sample100 micron wide (thick)
IR light inIR 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 ContactNo 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 ContactNo 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 ContactNo 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 ContactNo 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 ContactNo 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 ContactNo 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
350u
mPolymer Laminate - Visible Images & ATR Imaging Sampling
Location
70 u
m
70 um
ATR
470um
15x obj. vis image –cross-section view
Polymer Laminate : Chemical Images & Extracted Spectra
46
15x obj. vis image70
um
70 um70
um
70 um
ATR Chemical Image
3500 3000 2500 2000 1500 1000
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Wavenumber
Abso
rban
ce
Image @ 2915cm-1 – PELeft side ~11 microns
Right side ~20 microns
3500 3000 2500 2000 1500 1000
0.3
0.2
0.1
Wavenumber
Abso
rban
ce
Image @ 1634cm-1 – Nylon~ 16 microns thick
Image @ 1735cm-1 Polyurethane
~ 2-3 microns thick
3500 3000 2500 2000 1500 1000
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0.1
Wavenumber
Abso
rban
ce
3500 3000 2500 2000 1500 1000
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Wavenumber
Abso
rban
ce
Image @ 1735cm-1Polyurethane (possibly
pyrolyzate based)~ 5-6 microns thick
3500 3000 2500 2000 1500 1000
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Wavenumber
Abs
orba
nce
3500 3000 2500 2000 1500 1000
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Wavenumber
Abs
orba
nce
640 um
480
um
ATR
defect, image @ 1402 cm-1
polymer, image @ 1402 cm-1
70
m
70 m
Composite (Green/Red) image
GREEN – DEFECTRED - POLYMER
At initial analysis, it appears that the defect is likely to be anInorganic material, most probably a carbonate, or a carbonatecontaining mixture
15x obj. vis image – cross-section view ATR Chemical Image
Polymer Film 1 - Visible Images & ATR Imaging Sampling Location
Micro ATR (FPA) imaging of defects in black rubber sample
Imag
e cr
eate
d at
164
4 cm
-1Im
age
crea
ted
at 2
848
cm-1
visimage
IR image
70m
70
m
10 um
3500 3000 2500 2000 1500 1000
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Wavenumber
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orba
nce
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Abs
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nce
3500 3000 2500 2000 1500 1000
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Wavenumber
Abs
orba
nce
Polyamide
PE
Polyisoprene (natural rubber)
Art Conservation FTIR Imaging
Cross sections of paint layers for Art Conservation
• A typical object of studies for specialists of conservationchemistry,
• Chemically heterogenous,
• Taken usually during conservation process or examination of a work of art,
• FT-IR imaging used for non-destructive analysis of a paintcross section.
100 – 150 m
Oil painting from the Netherlands –XIX century ATR FTIR imaging
100 m
lithopone - white pigment(BaSO4 + ZnS)
oil binding medium zinc stearate/palmitateageing product
Selected IR images for identified substances:
cadmium oxalateageing product
barium sulphate - filler
calcite - filler proteinaceous material –impregnant of the canvas
ATR FT-IR imagingenables identification of painting materials of various functions as well as ageing products in paintfilms with a thickness of a few m
Z. Kaszowska, K. Malek, E. Panczyk, A. Mikolajska, Vib. Spec, (2013), 65, 1-11
70um
Pharmaceutical FTIR Imaging
52
Tablet – Contamination Location15x Vis Image
480 m
640 m
70
m
70 m
Aripiprazole (mixed with lactose), image @ 1673 cm-1(from 2nd derivative)
Cellulose (Avicel), image @ 1058 cm-1(from 2nd derivative)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
lactose, image @ 1168 cm-1(from 2nd derivative)
Starch(?), image @ 1147 cm-1(from 2nd derivative)
Row = 12 Col = 10
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Wavenumber
Abs
orba
nce
Row = 29 Col = 19
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Wavenumber
Abs
orba
nce
Row = 43 Col = 59
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Abs
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nce
Row = 51 Col = 28
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nce
Active
Lactose
Starch
Cellulose
Defect(next slide)
15x Vis Image
480 m
640 m
70
m
70 m
Contaminant #1 image @ 1730 cm-1(from 2nd derivative)
Clear and distinct domains from 4 constituents were detected:- Contaminant #1 – Possibly a polyester- Contaminant #2, - Possibly a polyamide
Contaminant #2 image @ 1651 cm-1(from 2nd derivative)
Tablet – Contamination LocationRow = 10 Col = 26
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Abs
orba
nce
Row = 30 Col = 28
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Abs
orba
nce
70
m
70 m
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
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0.00
Wavenumber
Abs
orba
nce
350
um
350 um70
um
70 um
5 m
Analysis time = 2 mins
Defects identified as dislodgedSpacers
No sample prep and no sampledamage
3500 3000 2500 2000 1500 1000
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Wavenumber
Abso
rban
ce
350
um
70 u
m70 um
Contaminated Circuit Board – FTIR ATR Imaging
ATR contact damage caused by other vendor
Analysis time = 2 mins
Spectra library searchreveals contaminant to bepolyetherimide
SUMMARYHighest Spatial
ResolutionHighest Spatial
Resolution
Largest Field of View
Largest Field of View
Fastest analysis time
Fastest analysis time
Live FPA Imaging