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:
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
4: FPA Imaging With an FPA detector, up to 16384 spectra can be recorded simultaneously in a single measurement.
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? ATR Chemical Image
O
=
Image @ 0.4 C 1724 cm-1 O
70 um70 0.2
PET Absorbance
0.0
3500 3000 2500 2000 1500 1000 70 um Wavenumber What do the Coloured Images Mean? ATR Chemical Image
O
=
Image @ 0.4 C 1724 cm-1 O
70 um70 0.2
PET Absorbance
0.0
3500 3000 2500 2000 1500 1000 70 um Wavenumber
0.3
H
Image @ - 3295 cm-1 0.2 N 0.1
Nylon Absorbance 0.0
3500 3000 2500 2000 1500 1000 Wavenumber What do the Coloured Images Mean? ATR Chemical Image
O
=
Image @ 0.4 C 1724 cm-1 O
70 um70 0.2
PET Absorbance
0.0
3500 3000 2500 2000 1500 1000 70 um Wavenumber
0.3
H
Image @ - 3295 cm-1 0.2 N 0.1
Nylon Absorbance 0.0
3500 3000 2500 2000 1500 1000 Wavenumber
0.15
H
Image @ - 0.10 C 2915cm-1 0.05
PP Absorbance 0.00
3500 3000 2500 2000 1500 1000 Wavenumber FTIR Chemical Imaging and its applications Materials/Polymer Biomedical/Biological Research Polymer laminates (functional Early disease diagnosis (Cancers) layer & adhesive identification) Study of diseases (Alzheimer’s, kidney) Defect analysis Plant/fungi tissue studies Phase distribution Live cell chemical imaging (in water) Composites Microbial identification Bone, teeth and cartilage Forensics Car paint layer & structure analysis Art Conservation Trace evidence analysis Painting components & layer identification Pharmaceuticals Geology Ingredient distribution Coating analysis Study of inclusions Defect/particle analysis Food/Cosmetics Electronics Study of emulsions, eg cheese, mayonnaise, Defect analysis cream Infrared Microscopy Sampling Techniques
Absorption/ Transmission Reflectance Micro - ATR Reflectance
Objective
θ θ θ θ Sample ATR - IRE Stage “Kevley SlideTM”
Condenser
Sample thickness: Sample thickness: Sample thickness: Sample thickness: 10 – 20 mm NA 5 - 10 mm NA Large Sample Microscopy Sampling Techniques – Measure small areas on large samples
Large Sample Large Sample Large Sample Grazing Angle
Reflectance Micro ATR
large large large
sample sample sample
GAO
Objective Objective External External External sample sample sample stage stage stage
Sample thickness: Sample thickness: Sample thickness: NA NA NA Large Sample Objective
Agilent’s patented1 Large Sample Microscope Objective allows you to measure unlimited sized samples in reflection or ATR single point or imaging mode
It’sExamples: as simple as: 1. TakingInvestigation your sample of new to coating the microscope materials on helmets (military / aerospace / etc.) 2. PlacingLarge vehicle it against component the 90 degree analysis, objective ex: car door/panel 3. Press collect and get your results!
0.4
0.2 Abs.
0.0 4000 3000 2000 1000 cm-1
Large sample
15x Mirror Objective assembly
LS 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
0.8
0.4 Absorbance
0.0
4000 3000 2000 1000 Wavenumber (cm-1)
0.8
0.8
0.4 0.4
Absorbance Absorbance 0.0 0.0
4000 3000 2000 1000 4000 3000 2000 1000 Wavenumber (cm-1) Wavenumber (cm-1) Plastic Tubing – Defect analysis
4x glass visible objective 15x reflective vis/IR objective
m m
ATR
2.0 mm2.0 500 500
2.5 mm 0.08 660 mm
0.06
15x reflective vis/IR objective 0.04
0.02 Tube spectrum #3
0.00
-0.02
m
Absorbance m
ATR -0.04 500 500 -0.06 Tube spectrum #2 -0.08
3500 3000 2500 2000 1500 1000 660 mm Wavenumber Plastic Tubing – Defect analysis
4x glass visible objective
0.10 Glue spectrum #1
2.0 mm2.0 0.08
0.06
2.5 mm 0.04 Absorbance 15x reflective vis/IR objective 0.02
0.00
m m 3500 3000 2500 2000 1500 1000
ATR Wavenumber 500 500
660 mm 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 2 3 4 5 6 Infrared Mapping in Material Analysis: Multi-layer Paint Sample
1.5 0.9
Layer 2 Layer 3
1.0 0.6
0.5 0.3
Absorbance Absorbance
0.0 0.0 4000 3000 2000 1000 4000 3000 2000 1000 Wavenumber (cm-1) Wavenumber (cm-1)
0.9 0.9
Layer 1 Layer 4
0.6 0.6
0.3 0.3
Absorbance Absorbance
0.0 0.0 4000 3000 2000 1000 4000 3000 2000 1000 Wavenumber (cm-1) Wavenumber (cm-1)
1.0
Polyester resin
0.5 Absorbance
0.0 4000 3000 2000 1000 Wavenumber (cm-1) 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 Principle of Infrared 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
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)
One pixel (~5.5µm) is an entire spectrum Achieved Spatial Resolution Summary 25x obj 0.98um Pixel Size (obj mag, NA, mode) Achieved Achieved Single FPA tile Spatial Spatial FOV (with Resolution Resolution 128x128FPA) 3750 cm-1 2500 cm-1 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 1.4um 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
Entire 2”x2” (50x50mm) USAF target imaged at USAF target (700x700um) imaged at 5.5 um pixel USAF target imaged (280x280um) at 1.1 um 19 um pixel resolution with 4xIR objective in 90 resolution (normal mag. mode) with 15x objective in resolution (high mag mode) with 15x objective in minutes 2 minutes 8 minutes. (21x21 tile mosaic with128FPA) Single 128FPA tile 2x2 tile mosiac with 128FPA Advancing FTIR Imaging with the Agilent Cary 620
Highest spatial resolution Largest Field of View
Superior signal-to-noise Fastest data collection
June 12, 2015 27 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 4. Place micro-vice (with sample) on Step 3. Cross-section sample with razor microscope stage & touch ATR ATR Contact with sample
IR light out IR light in IR light out IR light in
micro ATR micro ATR
STEP 5. Sample raise stage 100 micron wide (thick) Sample to make 100 micron micro-vice wide (thick) contact & collect data Microscope Stage micro-vice Microscope Stage “Live/Real-Time” ATR contact monitoring Standard Live ATR direct FPA IR Image – without correction
No Pressure (before contact) Live ATR direct FPA IR Image with Enhanced Chemical Contrast
No Pressure (before contact) “Live/Real-Time” ATR contact monitoring Standard Live ATR direct FPA IR Image – without correction
Stage is raised
No Pressure First Contact (before contact) Live ATR direct FPA IR Image with Enhanced Chemical Contrast
Stage is raised
No Pressure First Contact (before contact) “Live/Real-Time” ATR contact monitoring Standard Live ATR direct FPA IR Image – without correction
Stage is raised
No Pressure First Contact Increasing Pressure (before contact) Live ATR direct FPA IR Image with Enhanced Chemical Contrast
Stage is raised
No Pressure First Contact (before contact) Increasing Pressure Complete Contact “Live/Real-Time” ATR contact monitoring Standard Live ATR direct FPA IR Image – without correction
Stage is raised
No Pressure First Contact Increasing Pressure (before contact) Live ATR direct FPA IR Image with Enhanced Chemical Contrast
Stage is raised
No Pressure First Contact (before contact) Increasing Pressure Complete Contact “Live/Real-Time” ATR contact monitoring Standard Live ATR direct FPA IR Image – without correction
Stage is raised
No Pressure First Contact Increasing Pressure (before contact) Live ATR direct FPA IR Image with Enhanced Chemical Contrast
Stage is raised
No Pressure First Contact (before contact) Increasing Pressure Complete Contact “Live/Real-Time” ATR contact monitoring Standard Live ATR direct FPA IR Image – without correction
Stage is raised
No Pressure First Contact Increasing Pressure (before contact) Live ATR direct FPA IR Image with Enhanced Chemical Contrast
Stage is raised
No Pressure First Contact (before contact) Increasing Pressure Complete Contact “Live/Real-Time” ATR contact monitoring Standard Live ATR direct FPA IR Image – without correction
Stage is raised
No Pressure First Contact Increasing Pressure (before contact) Live ATR direct FPA IR Image with Enhanced Chemical Contrast
Stage is raised
No Pressure First Contact (before contact) Increasing Pressure Complete Contact Sample Presentation
A small piece of the sample was cut out The micro-vice is placed in a dedicated insert and place into a micro-vice sample holder on the motorised stage and contact is made with the where it was cut flat with a sharp razor ATR crystal, using Agilent’s unique “Live ATR contact” method, without any need for resin embedding
June 12, 2015 Confidentiality Label
39 Pixel Size & FOV: Collection Mode: Spectral Range: Scans (time): Resolution: Collection Conditions: Sausage Packaging (Red): Visible (Red): images Packaging Sausage ATR Imaging
1.1 1.1 microns/pixel, 70x70 microns Micro ATRtransmission& (5 micron 4000 32 scans (~30 sec) per spot 4 cm
- – 1
850 cm 500 um - 1
Sampling Location Sampling spot spot 1 ATR spot spot 2 ATR
15x obj spot spot 3 ATR
670 670 um
.
vis
image microtomed
slices)
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)
-
June 12, 2015 Confidentiality Label
41 Sausage Packaging – ATR Chemical Images (Spot 1) PE (with additive) ATR Image @ 2850cm-1 ATR Image @ 1607cm-1 ~23 um thick 0.15 PE
Layer 1&3 0.15 Layer 2 ~46 um thick
0.10
0.10
0.05
Absorbance
70 um70 70 um70 Absorbance 0.05 0.00
0.00 3000 2000 1000 Wavenumber 3500 3000 2500 2000 1500 1000 70 um 70 um Wavenumber ATR Chemical 15x obj. vis image RGBY Composite Image 4 1 2 3 5 Red: PE
Yellow: PE (with additive) Green: Polycarboxylic acid ester (possible)
70 um70 Blue: Polyamide
ATR Image @ 1725 cm-1 70 um ATR Image @ 1202cm-1 0.06 Polycarboxylic acid ester polyamide
polymer (possibly) 0.15 Layer 4 Layer 5 ~5 um thick
0.04
0.10
0.02
70 um 70
70 um70 Absorbance
Absorbance 0.05
0.00 0.00 3500 3000 2500 2000 1500 1000 3500 3000 2500 2000 1500 1000 70 um Wavenumber 70 um Wavenumber
June 12, 2015 Confidentiality Label
42 Sausage Packaging – ATR Chemical Images (Spot 2) 15x obj. vis image ATR Chemical Image
PE (with additives)
1 1 Layer 3, 7 - ~6 um thick (centre layer) 0.10
& 11 70 um 70
70 um 70 0.05
Absorbance
Image @ @ Image 2850 cm 2850 0.00
70 um 70 um 3500 3000 2500 2000 1500 1000
0.06 Wavenumber
Layer 4 Polycarboxylic acid ester 1 1 - 0.04 polymer (possibly) 0.02
Absorbance ~5 um thick
Image @ @ Image 0.00 1725 cm 1725
3500 3000 2500 2000 1500 1000 Red: PE Wavenumber
Green: Polycarboxylic acid 0.15 Polyamide
Layer 5, 8 & 10 1 1
polymer (possibly) - ~7um (left) 0.10 Blue: Polyamide ~2um (centre)
White: Polypropylene Absorbance 0.05 ~3um (right) Image @ @ Image Cyan: EVOH cm 1202 0.00 3500 3000 2500 2000 1500 1000
Wavenumber RBGWC composite image 0.08
Layer 6
0.06 Polypropylene 1 1 - 0.04 ~20um 3 5 6 8 10 0.02
4 7 9 11 Absorbance 0.00
Image @ @ Image 2951 cm 2951 3500 3000 2500 2000 1500 1000 Wavenumber 0.06 EVOH
Layer 9
1 1 0.04 - ~5um 0.02 (spectral overlap from
Absorbance Polyamide present) Image @ @ Image 1090 cm 1090 0.00 3500 3000 2500 2000 1500 1000 Wavenumber
June 12, 2015 Confidentiality Label
43 Sausage Packaging – ATR Chemical Images (Spot 3)
15x high mag. obj. vis image ATR Chemical Image PP (possible with an
Layer 11 additive as evident by
0.15 peak at 1060cm-1)
0.10 ~37 um thick
70 um70 0.05
70 um70 Absorbance
0.00
3500 3000 2500 2000 1500 1000 70 um 70 um Wavenumber
0.15 Layer 5, 8 & 10 Polyamide ~2um (left) 0.10 ~3um (right) RBC composite image
Absorbance 0.05
Red: PE 0.00 Blue: Polyamide 3500 3000 2500 2000 1500 1000 Cyan: EVOH Wavenumber 0.06 EVOH 8 10 11 Layer 9 ~5um thick 7 9 0.04 (spectral overlap from Polyamide
0.02 present) Absorbance
0.00
3500 3000 2500 2000 1500 1000 Wavenumber
June 12, 2015 Confidentiality Label
44 Sausage Packaging – ATR Chemical Images
40x obj. vis image (polarized) 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 um 140 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. 180 um
~3 ~3
~11 ~24 ~11 ~5 ~7 ~20 ~5 ~37
~6
)
ester ester Pixel size: 1.1micron
Obj mag: 15x, 0.62NA
PE
PE
PE PE PE PA PP PE PA PA
EVOH FOV: 70x70um
(in microns)(in
with additive with polymer (possibly polymer
FTIR ATR imaging ATR FTIR Total system mag: 36x
Polycarboxylic acid Polycarboxylic determined identity & identity determined approximate thickness approximate 1 2 3 4 5 6 7 8 9 10 11 ~130 um
June 12, 2015 Confidentiality Label
45 Sausage Packaging – 25x Transmission High mag Chemical Images
40x obj. vis image (polarized) - 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
1 micro ATR
- Most books and papers still talk of ~10um 140 um 140 spatial resolution for transmission imaging! - The 25x, 0.81NA is a revolution in objective 2 design 3 EVOH, layer 3
180 um
PA, layer 2 Pixel size: 0.66 micron
Absorbance Obj mag: 25x, 0.81NA PE, layer 1 High mag: ON FOV: 85x85um Total system mag: 61x
3500 3000 2500 2000 1500 1000 Working distance: 12mm Wavenumber
June 12, 2015 Confidentiality Label
46 Sausage Packaging – 15x Transmission High mag Chemical Images
40x obj. vis image (polarized) 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. 1 140 um 140
EVOH, layer 3 2
3
PA, layer 2
180 um Absorbance
PE layer 1 Pixel size: 1.1micron Obj mag: 15x, 0.62NA High mag: ON 3500 3000 2500 2000 1500 1000 FOV: 140x140um Wavenumber Total system mag: 36x Working distance: 21mm
June 12, 2015 Confidentiality Label
47 Sausage Packaging – 25x Transmission std mag Chemical Images
40x obj. vis image (polarized) With the larger pixel size, now at 3.3um, even with the high NA 25x objective, the ~3um PA layers cannot
be spatially resolved
140 um 140
180 um
Pixel size: 3.3micron Obj mag: 25x, 0.81NA High mag: OFF FOV: 420x420um Total system mag: 12x
June 12, 2015 Confidentiality Label
48 Sausage Packaging – 15x Transmission std mag Chemical Images
40x obj. vis image (polarized) As the pixel size gets larger, now at 5.5um, layers that are ~5 um now start to blur out
140 um 140
180 um
Pixel size: 5.5 micron Obj mag: 15x, 0.62NA High mag: OFF FOV: 700x700 um Total system mag: 7.3x
June 12, 2015 Confidentiality Label
49 Principal Component Noise Reduction (PCNR)
1720 cm-1
0.25 - Removes ONLY noise 0.20 1 scan
0.15 - Typical S:N 0.10 improvement of Absorbance 1 scan with PCNR ~5-10x 0.05 128 scans 0.00 - Corresponding time 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 saving (for equivalent Wavenumber
S:N) is 25-100x ! 0.01
0.00 Absorbance - Only FTIR imaging -0.01 Residual spectrum system to include
PCNR 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber
Agilent Confidential June 12, 2015 50 Polymer Film Defects - Visible Images & ATR Imaging Sampling Location 15x obj. vis image – cross-section view ATR Chemical Image
defect, image @ 1402 cm-1
0.15
0.10
m m
ATR 0.05
70 70 Absorbance
0.00 480 um 480
3500 3000 2500 2000 1500 1000 Wavenumber 70 mm
0.06 polymer, image @ 1402 cm-1
640 um 0.04
Composite (Green/Red) image 0.02 Absorbance 0.00
GREEN – DEFECT
RED - POLYMER 3500 3000 2500 2000 1500 1000 Wavenumber At initial analysis, it appears that the defect is likely to be an Inorganic material, most probably a carbonate, or a carbonate containing mixture Micro ATR (FPA) imaging of defects in black rubber sample Vis image IR image
0.15
1 0.10
-
ATR Absorbance 0.05 PE 480 um 480
0.00
at 2848 cm 2848at Image created Image
3500 3000 2500 2000 1500 1000 10 um Wavenumber
640 um 0.10
0.08 Polyamide
1
0.06
-
m 0.04 m
Absorbance 0.02 70 70
0.00
at 1644 cm 1644at Image createdImage -0.02
3500 3000 2500 2000 1500 1000 70 m Wavenumber m 0.18
0.16
0.14
0.12
0.10
Absorbance 0.08 0.06 Polyisoprene (natural rubber)
3500 3000 2500 2000 1500 1000 Wavenumber 25x, 0.81 NA Objective in “high mag” mode for Transmission Polymer bead analysis 2915 cm-1 1442 cm-1 1118 cm-1
40x vis image (3.4 microns) (6.9 microns) (8.9 microns)
m
m 85 85
85mm
5 micron polystyrene beads on BaF2 substrate are clearly chemically distinguished.
Pixel size: 0.66 micron Even at relatively long wavelengths (low Obj mag: 25x, 0.81 NA wavenumbers), 5 micron beads are clearly resolved High mag: ON FOV: 85x85 um Total system mag: 61x Working Distance: 12 mm
Agilent Confidential June 12, 2015 53 350um Exposed green plastic plastic green Exposed 15x obj 470um . vis ATR
image
Sampling Location Sampling –
Visible & images ATR Imaging
70 um 70 70 um
Sample Placement & Measurement
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.
Confidentiality Label
55 June 12, 2015 Results - Exposed Green Plastic
40x high mag. obj. vis image ATR Chemical Image Image @ 1736cm-1
0.1
0.0
70 um 70 70 um70
Absorbance -0.1
70 um 70 um 3500 3000 2500 2000 1500 1000 Wavenumber
A clear edge containing higher carbonyl content is clearer visible along the expose side edge.
Confidentiality Label
56 June 12, 2015 Pharmaceutical FTIR Imaging
57 Tablet – Contamination Location 15x Vis Image 0.20 Row = 29 Col = 19
0.15
m Active
m m
m 0.10
Absorbance
70 70 480 480 0.05
0.4 3500 3000 2500 2000 1500 1000 Wavenumber 640 mm 70 mm Row = 12 Col = 10 0.3
Lactose Clear and distinct domains from 0.2
4 constituents were detected: Absorbance 0.1 - Active, compared to provided standard 0.0 - Lactose, identified from library search 3500 3000 2500 2000 1500 1000 - Starch, identified from library search Wavenumber - Cellulose identified from library search 0.3 Row = 43 Col = 59
0.2 Starch
Absorbance 0.1
0.0
3500 3000 2500 2000 1500 1000 Wavenumber Defect 0.3 Row = 51 Col = 28
(next slide) 0.2 Cellulose
Absorbance 0.1
0.0 3500 3000 2500 2000 1500 1000 Wavenumber Tablet – Contamination Location 15x Vis Image
0.20 Row = 10 Col = 26
Polyester
0.15
m
m
m m
Absorbance 0.10
70 70 480 480
0.05 3500 3000 2500 2000 1500 1000 Wavenumber 640 mm 70 mm 0.15 Row = 30 Col = 28
Polyamide Clear and distinct domains from 0.10 4 constituents were detected: - Contaminant #1 – Possibly a polyester Absorbance 0.05 - Contaminant #2, - Possibly a polyamide 3500 3000 2500 2000 1500 1000 Wavenumber Contaminant Composite (RG), part 2 From a single FTIR ATR imaging measurement and without damaging the sample, 4 known constituents
and 2 unknown contaminants were imaged in 1 min
m
m 70 70
70 mm Electronics/Semicon FTIR Imaging Spacer contamination on LCD filter
Total analysis time = 2 mins
5 mm Defects identified as dislodged
Spacers 350 um 350 70 um 70 No sample prep and no sample damage
350 um 70 um
0.03
0.02 Absorbance 0.01
0.00
3500 3000 2500 2000 1500 1000 Wavenumber LCD Defect – Protein image (1647 cm-1)
Visible Image 70 microns
480 um 480 70 microns 70
640 um IR image at 1647cm-1 Total analysis time = 1 mins 0.3 Row = 6 Col = 30 1647.960 12.936 Defects identified as protein, most 0.2 probably flake of dead skin Absorbance 0.1
No sample prep and no sample 0.0 damage 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber Contaminated Circuit Board – FTIR ATR Imaging
Visible Image Total analysis time = 2 mins
Spectra library search
reveals contaminant to be 350 um 350 70 um 70 polyetherimide
70 um 0.20 ATR contact damage
caused by other vendor 0.15
0.10 Absorbance 0.05
0.00
3500 3000 2500 2000 1500 1000 Wavenumber Forensics FTIR Imaging Vehicle paint cross sectional analysis 3696 cm-1
3038 cm-1
0.8 Row = 75 Col = 160 1602.985 1.303 1723.527 15.150
Row = 164 Col = 123 2950 1055 0.6
3696 1602.985 3.889 0.6 3399
1723.527 21.695 3696
1695 0.5
1695
3038
1510 1133
0.4 2950
3399 1465
0.4 1055
Absorbance
1133 1465
Absorbance 1561.467 0.031
3038 1375
0.2 1561.4671510 0.115 0.3 1375
0.2 0.0
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber Wavenumber 3399 cm-1 1695 cm-1 1561 cm-1 1086 cm-1
0.8 1695 1723.527 20.244 1602.985 -0.465 0.7 1561.467 0.463 1086.493 7.960 Row = 94 Col = 92 1723.527 37.245 1.0 Row = 24 Col = 146 1465 Row = 90 Col = 98 1723.527 13.569 Row = 149 Col = 135 0.6 0.6 0.6 1602.985 -1.255
1133 0.8
1602.985 -2.144 1133 1465
0.5 1055
1375
1375 1055
1133 1723.527 21.636 1561.467 0.301 2950 0.4 0.6 1465 0.4 0.4 2950
2950 1602.985 1.044
1510
2950
1055
3399
Absorbance
3399
1375
1695
1695
Absorbance Absorbance
3399 0.4
Absorbance 1695 1055 0.3
0.2 1561.4671510 0.100
3696 1465
3696 1561.467 0.080
1510
3038
3696
1375
3038
3038
3038 3399
0.2 0.2 0.2 3696 0.0
0.1 0.0 1510 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber Wavenumber Wavenumber Wavenumber MIXED POWDER MICRO ATR ANALYSIS – CALCIUM CARBONATE
0.8 Row = 37 Col = 32
0.6 CaCO3(1420.000)
0.4 Absorbance 0.2 STPP(1140.010)pyrophosphate(987.127)disodium phosphate(945.393) 0.0
3500 3000 2500 2000 1500 1000 Wavenumber MIXED POWDER MICRO ATR ANALYSIS – DISODIUM DIPHOSPHATE ANHYDRIDE
0.20 Row = 38 Col = 48
0.15 STPP(1140.010) disodium phosphate(945.393) 0.10
CaCO3(1420.000) Absorbance 0.05 pyrophosphate(987.127) 0.00
3500 3000 2500 2000 1500 1000 Wavenumber MIXED POWDER MICRO ATR ANALYSIS – SODIUM TRIPOLYPHOSPHATE
Row = 61 Col = 56 STPP(1140.010) 0.2
0.1 Absorbance
0.0 pyrophosphate(987.127)disodium phosphate(945.393) CaCO3(1420.000) 3500 3000 2500 2000 1500 1000 Wavenumber MIXED POWDER MICRO ATR ANALYSIS – SODIUM PYROPHOSPHATE
0.3
Row = 17 Col = 17 STPP(1140.010) 0.2
pyrophosphate(987.127)
0.1 CaCO3(1420.000) Absorbance disodium phosphate(945.393) 0.0
3500 3000 2500 2000 1500 1000 Wavenumber Food/Cosmetics FTIR Imaging Imaging of mayonnaise heterogeneity: measured in transmission on BaF2 slide sugar @1046cm-1 1.5 Row = 69 Col = 63
2999.749 -0.594 water(1650 40.7) 1.0 starch(1046 48.9) 2925.612 0.119 1648.271 24.136 TmpltPk1(3614 0.5) Absorbance 0.5 oil(1745 0.0) Visible CCD image 0.0 2113.148 4.845
2925.612 22.490 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber
1.4 oil @1745cm-1
1.2 Row = 28 Col = 106 oil(1745 9.6)
1.0 2999.749 1.613
500 um 500 0.8 water(1650 2.3)
0.6 starch(1046 0.0) Absorbance 0.4
TmpltPk1(3614 0.0) 1648.271 1.474 0.2 2113.148 0.086
650 um 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber
1.5 water(1650 39.8) Row = 70 Col = 66 water @1650cm-1
Measured on FTS7000+UMA600, using 2999.749 -0.264 1.0 starch(1046 2.0) a 128x128 FPA. 2925.612 0.210 1648.271 24.505 IR measurement area = 700x700 microns TmpltPk1(3614 0.5) Time of measurement = ~ 2min Absorbance 0.5 oil(1745 0.1) Resolution = 8 cm-1 2113.148 4.503 0.0
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber Biological & Biomedical FTIR Imaging Breast Tissue section
CH image (2943-2893 cm-1) Amide I image 1233 cm-1 image
1.2
1.0 1.0 0.9
0.8 0.8
0.7
0.6 0.6
0.5 Absorbance
0.4 Absorbance 0.4
0.3 0.2 0.2
0.1
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber
Total IR FOV = 1400x1400 um (2x2 mosaic), pixel size = 5.5um, total data collect time ~ 6 mins,
Note: red box, indicates area for single tile high mag collect on next slide Breast Tissue section (high mag mode)
CH image (2943-2893 cm-1) 1233 cm-1 image 40x obj vis image Amide I image
0.7 1.0 0.6
0.8
0.5
0.4 0.6
0.3 Absorbance 0.4 Absorbance 0.2
0.2 0.1
0.0 0.0
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 Wavenumber Wavenumber
Total IR FOV = 280x280 um (2x2 mosaic), pixel size = 1.1um, total data collect time ~ 12 mins,
Note: red box, indicates area for single tile high mag collect on next slide Normal Mag (5.5um) – High Mag (1.1um) comparison, CH image
Normal Mag (5.5um) High Mag (1.1um)
280 um 280 280 um 280
280 um 280 um 0.7
0.6 0.6
0.5 The 3 features
0.4 within this oval 0.4 shape are each
0.3 about ~3 um Absorbance Absorbance 0.2 in diameter! 0.2
0.1 0.0
3500 3000 2500 2000 1500 1000 3500 3000 2500 2000 1500 1000 Wavenumber Wavenumber
June 12, 2015 Confidentiality Label
75 Wheat stem cross section FTIR Image
See next set of data for med. Protein – 1654 cm-1
res. close up 1651.180 28.040 1654.450 22.965 0.8 Row = 26 Col = 89
3341.250 71.728 0.6 1539.600 7.408 1733.180 -0.889 1044.970 27.715
Absorbance 0.4
See following set of 0.2
data for high. 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 res. close up Wavenumber
See next set of Lipid – 1733 cm-1 data for med. res. close up 1.2 Row = 68 Col = 104 3341.250 162.961 1044.970 74.983 1.0 1651.180 8.118 1654.450 6.133
0.8
1733.180 7.765 1539.600 -0.261 Absorbance 0.6
See following set of 0.4 data for high. 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 res. close up Wavenumber
See next set of Carbohydrate – 1200-950 cm-1
data for med. 1044.970 54.253 1.0 Row = 57 Col = 100 res. close up 3341.250 104.920 1651.180 14.684 1654.450 9.842 0.8
1539.600 2.721
0.6 Absorbance 1733.180 2.899 0.4
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 Wavenumber
See following set of data for high. res. close up
70 microns
70 microns 70
Visible Image 2-D IR image 3-D IR image (red box show field of at 1043 cm-1 at 1043 cm-1 view ATR)
ATR Imaging has provided for a 5 fold increase in 0.16 Row = 12 Col = 21 magnification and REAL 0.14 1043.570 11.216 spatial resolution
0.12 IR spectrum from enhancement. ~5 micron cells wall have 0.10 cross-hairs above been CLEARLY resolved 0.08
Absorbance with 10 micron light (~1000 0.06 cm-1) and have produced 0.04 high quality spectra free of 0.02 artefacts due to diffraction
0.00 and scattering.
3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 Wavenumber Live cell FTIR imaging in water!
Composite (RGB) image Lipid – Red C-H – Green Visible image C-H image Lipid image Protein image Protein - Blue
100 microns 100 microns 100 microns 100 microns 100 microns
Liquid transmission cell pathlength = 7microns Total measurement time ~ 10mins. Pixel size : 1.1 microns
June 12, 2015 78 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
Live FPA Imaging Removes need for complex, time consuming sample prep & allows for delicate analysis
June 12, 2015 79