advancing ftir imaging-synchrotron ir imaging in your lab · advancing ft-ir imaging –...

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

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

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


• >10x - other FPA’s• > 50x - linear array• >100x - single point

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

0.8

0.6

0.4

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

0.4

0.3

0.2

0.1

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

0.8

0.6

0.4

0.2

0.0

Wavenumber

Abso

rban

ce

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

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

0.4

0.2

0.0

Wavenumber

Abs

orba

nce

3500 3000 2500 2000 1500 1000

0.7

0.6

0.5

0.4

0.3

0.2

0.1

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) comparison, 1233 cm-1 image

280

um280 um

280

um

280 um


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




First ContactNo Pressure(before contact)

Stage is raised

Stage is raised

First Contact



No Pressure(before contact) Increasing Pressure

Increasing Pressure


First Contact Complete ContactNo Pressure(before contact)

Stage is raised

Stage is raised

First Contact



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

0.3

0.2

0.1

0.0

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

0.2

0.1

Wavenumber

Abso

rban

ce

3500 3000 2500 2000 1500 1000

0.15

0.10

0.05

Wavenumber

Abso

rban

ce

Image @ 1735cm-1Polyurethane (possibly

pyrolyzate based)~ 5-6 microns thick

3500 3000 2500 2000 1500 1000

0.06

0.04

0.02

0.00

Wavenumber

Abs

orba

nce

3500 3000 2500 2000 1500 1000

0.15

0.10

0.05

0.00

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

0.10

0.08

0.06

0.04

0.02

0.00

-0.02

Wavenumber

Abs

orba

nce

3500 3000 2500 2000 1500 1000

0.15

0.10

0.05

0.00

Wavenumber

Abs

orba

nce

3500 3000 2500 2000 1500 1000

0.18

0.16

0.14

0.12

0.10

0.08

0.06

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

3500 3000 2500 2000 1500 1000

0.4

0.3

0.2

0.1

0.0

Wavenumber

Abs

orba

nce

Row = 29 Col = 19

3500 3000 2500 2000 1500 1000

0.20

0.15

0.10

0.05

Wavenumber

Abs

orba

nce

Row = 43 Col = 59

3500 3000 2500 2000 1500 1000

0.3

0.2

0.1

0.0

Wavenumber

Abs

orba

nce

Row = 51 Col = 28

3500 3000 2500 2000 1500 1000

0.3

0.2

0.1

0.0

Wavenumber

Abs

orba

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

3500 3000 2500 2000 1500 1000

0.20

0.15

0.10

0.05

Wavenumber

Abs

orba

nce

Row = 30 Col = 28

3500 3000 2500 2000 1500 1000

0.15

0.10

0.05

Wavenumber

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

0.01

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

0.20

0.15

0.10

0.05

0.00

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





Live FPA Imaging

advancing ftir imaging-synchrotron ir imaging in your lab · advancing ft-ir imaging –...

Documents