Advancing FT-IR Imaging Synchrotron IR Imaging in Your Lab. Dr. Mustafa Kansiz FTIR Microscopy & Imaging Product Manager
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1 Advancing FT-IR Imaging Synchrotron IR Imaging in Your Lab Dr. Mustafa Kansiz FTIR Microscopy & Imaging Product Manager
2 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
3 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.
4 FTIR Microscope Measurement Modes: 3: Linear array Mapping Acquisition of spectra by a row (1x16 or 2x14) of detectors. Faster than single point mapping, but still much slower than FPA imaging 4: FPA Imaging With an FPA detector, up to spectra can be recorded simultaneously in a single measurement.
5 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
6 FTIR Chemical Imaging and its applications Materials/Polymer Polymer laminates (functional layer & adhesive identification) Defect analysis Phase distribution Composites Forensics Car paint layer & structure analysis Trace evidence analysis Pharmaceuticals Ingredient distribution Coating analysis Defect/particle analysis Electronics Defect analysis 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 Art Conservation Painting components & layer identification Geology Study of inclusions Food/Cosmetics Study of emulsions, eg cheese, mayonnaise, cream
7 A New Method of Magnification Enhancement
8 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 Total system magnification - It is important to note that, pixel resolution is therefore NOT ONLY governed by the objective.
9 FTIR Microscope Magnification Schematic FPA 40x40 um pixel Total system magnification = Native detector size/sample pixel size Pixel size = (native detector size/obj mag) / other mag Pixel size = 5.5 microns Stage
10 FTIR Microscope Magnification Schematic 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 Stage 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
11 Microscope objectives: some comparisons Agilent Synchrotrons & Other Systems 15x 0.62 NA 4x 0.2 NA 74x 0.65 NA 36x 0.5 NA 15x 0.4 NA 21 mm 38 mm 1 mm Pixel size: 0.5 m 10 mm Pixel size: 1.1 m 24 mm Pixel size: 5.5 m (normal) 1.1 m (high mag) Agilent s new 15x objective Pixel size: 19 m (normal) 3.8 m (high mag) Agilent s new 4x objective Pixel size: 2.7 m 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.
12 Advancing FTIR Imaging with the Agilent Cary 620 Highest spatial resolution Superior signal-to-noise Largest Field of View Fastest data collection February 3,
13 Synchrotron FTIR microscopy - 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.
14 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.
15 Synchrotron single point FTIR microscopy 10 um synchrotron source Transfer optics (spectrometer & microscope) 1:1 Single point MCT (100x100um) In single point mode, beam does not need to illuminate full area of MCT detector. High brilliance of synchrotron beam is utilised
16 Synchrotron FPA FTIR Imaging FPA (128x128) 10 um synchrotron source Transfer optics (spectrometer & microscope) 1: mm 5.1 mm In imaging mode, beam must illuminate full area of FPA detector, this therefore requires a 260,000x defocussing, losing the synchrotron intensity advantage
17 Agilent s Thermal source FPA FTIR Imaging Globar (double image) FPA (128x128) Transfer optics (spectrometer & microscope) 1:1 5.1 mm 5 mm 5.1 mm 7.5 mm 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
18 High IR Magnification A Synchrotron on your bench! Standard Magnification 5.5 um pixel size 2500 cm-1 Standard Magnification 5.5 um pixel size 2500 cm-1 High Magnification 1.1 um pixel size 2500 cm microns 280 microns 700 microns 280 microns 280 microns AGILENT CONFIDENTAL
19 280 microns 280 microns 3750 cm microns 2500 cm microns High Magnification Transmission Rayleigh Criterion %T Contrast Group 8 30 Group cm-1 Group cm Pixel Modulation Transfer Function (MTF) Lp/mm = 166 Line Width = 3.0 m Lp/mm = 205 Line Width = 2.4 m
20 Achieved Spatial Resolution Summary Pixel Size (obj/mode) Achieved Spatial Resolution 3750 cm-1 Achieved Spatial Resolution 2500 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 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 USAF target imaged (280x280um) at 1.1 um resolution (high mag mode) with 15x objective in 8 minutes. 2x2 tile mosiac with 128FPA
21 ADVANTAGES AND BENEFITS OF THE NEW FEATURES
22 Cary 620 top 4 advantages Highest Spatial Resolution New high mag optics >400% IR energy Synchrotron comparable high resolution data Largest Field of View Proprietary 4x IR objective Measure cm x cm areas in minutes Fastest analysis time >10x - other FPA s > 50x - linear array >100x - single point Live FPA imaging Enhanced chemical contrast software mode Eliminate sample prep Avoid damaging samples
23 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
24 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. 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 USAF target imaged (280x280um) at 1.1 um resolution (high mag mode) with 15x objective in 8 minutes. 2x2 tile mosiac with 128FPA
25 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
26 4. Live FPA Imaging with enhanced chemical contrast 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.
27 MARKETS AND APPLICATIONS
28 A world of 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 Polymer laminates - Total elapsed time to measure sample < 5min - PE Layer ~3um thick Live cell in water - IR image measured in water in ~6 minutes - Data collected using high mag mode with a 128x128 FPA
29 Live Cell Imaging IN WATER 100 microns 100 microns 100 microns 100 microns 100 microns Visible image C-H image Lipid image Protein image Composite (RGB) image Lipid Red Conditions: Micrasteris Species, 8 micron pathlength, CaF2 liquid cell. 8 cm-1 resolution 128x128 FPA, 1.1micron pixel size (21mm WD), 6 mins total collection time C-H Green Protein - Blue Traditionally, FTIR is thought to be incompatible with measurements through water Agilent s new high throughput high magnification system allows for live cell imaging at biologically relevant time frames with spatial resolution now exposing organelles
30 Breast Carcinoma Tissue Total IR FOV = 1400x1400 um (2x2 mosaic 128FPA), pixel size = 5.5um, total data collect time ~ 6 mins, Visible image CH image ( cm-1) Amide I image 1233 cm-1 image 1.2 Absorbance Absorbance Wavenumber Note: red box, indicates area for single tile high mag collect on next slide Wavenumber
31 Breast Carcinoma Tissue Total IR FOV = 280x280 um (2x2 mosaic 128FPA), pixel size = 1.1um, total data collect time ~ 12 mins, 40x obj vis image CH image ( cm-1) Amide I image 1233 cm-1 image Absorbance Absorbance Wavenumber Wavenumber
32 Normal Mag (5.5um) High Mag (1.1um) comparison, CH image Normal Mag (5.5um) High Mag (1.1um) 280 um 280 um um um 0.5 Absorbance Absorbance Wavenumber Wavenumber
33 Normal Mag (5.5um) High Mag (1.1um) comparison, Amide I image Normal Mag (5.5um) High Mag (1.1um) 280 um 280 um 280 um 280 um
34 Normal Mag (5.5um) High Mag (1.1um) comparison, 1233 cm-1 image Normal Mag (5.5um) High Mag (1.1um) 280 um 280 um 280 um 280 um
35 Polymer Film Laminate FTIR Imaging
36 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 razor Step 4. Place micro-vice (with sample) on microscope stage & touch ATR
37 ATR Contact with sample IR light out IR light in IR light out IR light in micro ATR micro ATR micro-vice Sample 100 micron wide (thick) STEP 5. raise stage to make contact & collect data micro-vice Microscope Stage Sample 100 micron wide (thick) Microscope Stage
38 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)
39 Live/Real-Time ATR contact monitoring Standard Live ATR direct FPA IR Image without correction Stage is raised No Pressure (before contact) First Contact Live ATR direct FPA IR Image with Enhanced Chemical Contrast Stage is raised No Pressure (before contact) First Contact
40 Live/Real-Time ATR contact monitoring Standard Live ATR direct FPA IR Image without correction Stage is raised No Pressure (before contact) First Contact Increasing Pressure Live ATR direct FPA IR Image with Enhanced Chemical Contrast Stage is raised No Pressure (before contact) First Contact Increasing Pressure Complete Contact
41 Live/Real-Time ATR contact monitoring Standard Live ATR direct FPA IR Image without correction Stage is raised No Pressure (before contact) First Contact Increasing Pressure Live ATR direct FPA IR Image with Enhanced Chemical Contrast Stage is raised No Pressure (before contact) First Contact Increasing Pressure Complete Contact
42 Live/Real-Time ATR contact monitoring Standard Live ATR direct FPA IR Image without correction Stage is raised No Pressure (before contact) First Contact Increasing Pressure Live ATR direct FPA IR Image with Enhanced Chemical Contrast Stage is raised No Pressure (before contact) First Contact Increasing Pressure Complete Contact
43 Live/Real-Time ATR contact monitoring Standard Live ATR direct FPA IR Image without correction Stage is raised No Pressure (before contact) First Contact Increasing Pressure Live ATR direct FPA IR Image with Enhanced Chemical Contrast Stage is raised No Pressure (before contact) First Contact Increasing Pressure Complete Contact
44 Live/Real-Time ATR contact monitoring Standard Live ATR direct FPA IR Image without correction Stage is raised No Pressure (before contact) First Contact Increasing Pressure Live ATR direct FPA IR Image with Enhanced Chemical Contrast Stage is raised No Pressure (before contact) First Contact Increasing Pressure Complete Contact
45 Polymer Laminate - Visible Images & ATR Imaging Sampling Location 15x obj. vis image cross-section view 350um 70 um ATR 70 um 470um
46 Polymer Laminate : Chemical Images & Extracted Spectra Absorbance cm-1 PE Left side ~11 microns Right side ~20 microns Absorbance cm-1 Nylon ~ 16 microns thick Wavenumber 15x obj. vis image Wavenumber ATR Chemical Image 70 um Absorbance 70 um um 1735cm-1 Polyurethane ~ 2-3 microns thick Absorbance 70 um cm-1 Polyurethane (possibly pyrolyzate based) ~ 5-6 microns thick Wavenumber Wavenumber 46
47 Polymer Film 1 - Visible Images & ATR Imaging Sampling Location 15x obj. vis image cross-section view ATR Chemical Image 0.15 defect, 1402 cm um ATR 70 m Absorbance m Wavenumber 0.06 polymer, 1402 cm um 0.04 Composite (Green/Red) image Absorbance GREEN DEFECT RED - POLYMER 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
48 Micro ATR (FPA) imaging of defects in black rubber sample IR image 0.15 Image created at 2848 cm-1 Absorbance PE 10 um Wavenumber vis image 70 m Image created at 1644 cm-1 Absorbance Polyamide 70 m Wavenumber Absorbance Polyisoprene (natural rubber) Wavenumber
49 Art Conservation FTIR Imaging
50 Cross sections of paint layers for Art Conservation A typical object of studies for specialists of conservation chemistry, Chemically heterogenous, Taken usually during conservation process or examination of a work of art, FT-IR imaging used for nondestructive analysis of a paint cross section m
51 Oil painting from the Netherlands XIX century ATR FTIR imaging Selected IR images for identified substances: 100 m 70um lithopone - white pigment (BaSO 4 + ZnS) oil binding medium zinc stearate/palmitate ageing product cadmium oxalate ageing product barium sulphate - filler ATR FT-IR imaging enables identification of painting materials of various functions as well as ageing products in paint films with a thickness of a few m calcite - filler proteinaceous material impregnant of the canvas Z. Kaszowska, K. Malek, E. Panczyk, A. Mikolajska, Vib. Spec, (2013), 65, 1-11
52 52 Pharmaceutical FTIR Imaging
53 Tablet Contamination Location 15x Vis Image 480 m 70 m Absorbance Row = 29 Col = 19 Aripiprazole (mixed with lactose), 1673 cm-1 (from 2 nd derivative) Active 640 m 70 m 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 Absorbance Absorbance Wavenumber Row = 12 Col = 10 Lactose Cellulose (Avicel), 1058 cm-1 (from 2 nd derivative) Wavenumber Row = 43 Col = 59 lactose, 1168 cm-1 Starch (from 2 nd derivative) 0.0 Defect (next slide) Absorbance Wavenumber Row = 51 Col = 28 Starch(?), 1147 cm-1 Cellulose (from 2 nd derivative) Wavenumber
54 Tablet Contamination Location 15x Vis Image 480 m 70 m Absorbance Row = 10 Col = 26 Contaminant # cm-1 (from Polyester 2 nd derivative) 640 m 70 m Clear and distinct domains from 4 constituents were detected: - Contaminant #1 Possibly a polyester - Contaminant #2, - Possibly a polyamide Contaminant Composite (RG), part 2 Absorbance Wavenumber Row = 30 Col = 28 Contaminant # cm-1 (from 2Polyamide nd derivative) Wavenumber From a single FTIR ATR imaging measurement and without damaging the sample, 4 known constituents and 2 unknown contaminants were imaged in 1 min 70 m 70 m
55 Electronics/Semicon FTIR Imaging
56 Spacer contamination on LCD filter Analysis time = 2 mins Absorbance 350 um um 70 um 5 m 70 um Defects identified as dislodged Spacers No sample prep and no sample damage Wavenumber
57 Contaminated Circuit Board FTIR ATR Imaging Analysis time = 2 mins 350 um 70 um Spectra library search reveals contaminant to be polyetherimide Absorbance ATR contact damage caused by other vendor 70 um Wavenumber
58 SUMMARY Highest Spatial Resolution Largest Field of View Fastest analysis time Live FPA Imaging
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