FTIR Microscopy and Imaging

Transcription

1 FTIR Microscopy and Imaging A world of applications and solutions Dr. Mustafa Kansiz FTIR Imaging and Microscopy Product Manager Agilent Technologies mustafa.kansiz@agilent.com

2 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

3 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

4 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.

5 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 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

6 70 um = What do the Coloured Images Mean? 1724 cm-1 PET ATR Chemical Image O O C 70 um

7 - 70 um = What do the Coloured Images Mean? 1724 cm-1 PET ATR Chemical Image O O C 3295 cm-1 Nylon 70 um H N

8 um = What do the Coloured Images Mean? 1724 cm-1 PET ATR Chemical Image O O C 3295 cm-1 Nylon 2915cm-1 PP 70 um H N H C

9 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

10 Infrared Microscopy Sampling Techniques Transmission Reflectance Absorption/ Reflectance Micro - ATR Objective Sample Stage θ θ θ θ ATR - IRE Kevley Slide TM Condenser Sample thickness: mm Sample thickness: NA Sample thickness: 5-10 mm Sample thickness: NA

11 Large Sample Microscopy Sampling Techniques Measure small areas on large samples Objective Objective GAO Large Sample Reflectance Large Sample Micro ATR Large Sample Grazing Angle large sample large sample large sample External sample stage External sample stage External sample stage Sample thickness: NA Sample thickness: NA Sample thickness: NA

12 Abs. Large Sample Objective Agilent s patented 1 Large Sample Microscope Objective allows you to measure unlimited sized samples in reflection or ATR single point or imaging mode It s Examples: as simple as: 1. Taking Investigation your sample of new to coating the microscope materials on helmets (military / aerospace / etc.) 2. Placing Large vehicle it against component the 90 degree analysis, objective ex: car door/panel 3. Press collect and get your results! cm -1 Large sample 15x Objective Mirror assembly LS

13 Single point analysis (Single-element detector analysis)

14 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 µm.

15 Transmission IR Analysis of Microtomed 3 layer Laminate (cm -1 ) (cm -1 ) (cm -1 )

16 500 mm 2.0 mm 500 mm Plastic Tubing Defect analysis 4x glass visible objective 15x reflective vis/ir objective ATR 2.5 mm 15x reflective vis/ir objective mm Tube spectrum # ATR Tube spectrum #2 660 mm

17 500 mm 2.0 mm Plastic Tubing Defect analysis 4x glass visible objective 0.10 Glue spectrum # mm 15x reflective vis/ir objective ATR 660 mm

18 Infrared Mapping - Single Point - Linear Arrays

19 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

20 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.

21 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

22 Infrared Mapping in Material Analysis: Multi-layer Paint Sample 1.5 Layer Layer (cm -1 ) (cm -1 ) 0.9 Layer Layer (cm -1 ) (cm -1 ) 1.0 Polyester resin (cm -1 )

23 Infrared Mapping in Material Analysis: Multi-layer Paint Sample Feature image based on absorbance at 3692 cm -1. Abs. at 3692 cm -1

24 Infrared 2-D Focal Plane Array Imaging

25 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) n 2 spatially resolved spectra are collected simultaneously (e.g., array 256 spectra) One pixel (~5.5µm) is an entire spectrum

26 Achieved Spatial Resolution Summary 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 25x obj 0.98um 1.4um 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

27 Advancing FTIR Imaging with the Agilent Cary 620 Highest spatial resolution Superior signal-to-noise Largest Field of View Fastest data collection June 12,

28 MARKETS AND APPLICATIONS

29 Polymer Film Laminate FTIR Imaging

30 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

31 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

32 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)

33 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

34 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

35 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

36 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

37 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

38 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

39 Sample Presentation 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 June 12, 2015 Confidentiality Label 39

40 Sausage Packaging (Red): Visible images ATR Imaging Sampling Location 500 um 15x obj. vis image ATR spot 1 ATR spot 3 ATR spot 2 Collection Conditions: 670 um Resolution: Scans (time): Spectral Range: Collection Mode: Pixel Size & FOV: 4 cm-1 32 scans (~30 sec) per spot cm-1 Micro ATR & transmission (5 micron microtomed slices) 1.1 microns/pixel, 70x70 microns

41 Different collection mode comparison 1. Micro ATR (1.1 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

42 70 um 70 um 70 um 70 um 70 um Sausage Packaging ATR Chemical Images (Spot 1) ATR 2850cm Layer 1&3 PE ~46 um thick ATR 1607cm PE (with additive) ~23 um thick Layer 2 70 um x obj. vis image 70 um ATR Chemical RGBY Composite Image Red: PE Yellow: PE (with additive) Green: Polycarboxylic acid ester (possible) Blue: Polyamide ATR 1725 cm um Polycarboxylic acid ester Layer 4 polymer (possibly) ~5 um thick ATR 1202cm Layer 5 polyamide um 70 um June 12, 2015 Confidentiality Label 42

43 cm cm cm cm-1 70 um 2850 cm-1 70 um Sausage Packaging ATR Chemical Images (Spot 2) 15x obj. vis image ATR Chemical Image Layer 3, 7 & 11 PE (with additives) ~6 um thick (centre layer) 70 um 70 um Layer 4 Polycarboxylic acid ester polymer (possibly) ~5 um thick 0.00 Red: PE Green: Polycarboxylic acid polymer (possibly) Blue: Polyamide White: Polypropylene Cyan: EVOH RBGWC composite image Layer 5, 8 & 10 Layer 6 Layer 9 Polyamide ~7um (left) ~2um (centre) ~3um (right) Polypropylene ~20um EVOH ~5um (spectral overlap from Polyamide present) June 12, 2015 Confidentiality Label 43

44 Sausage Packaging ATR Chemical Images (Spot 3) 70 um 70 um 15x high mag. obj. vis image ATR Chemical Image Layer 11 PP (possible with an additive as evident by peak at 1060cm-1) ~37 um thick um 70 um RBC composite image Layer 5, 8 & 10 Polyamide ~2um (left) ~3um (right) Red: PE Blue: Polyamide Cyan: EVOH Layer 9 EVOH ~5um thick (spectral overlap from Polyamide present) 0.00 June 12, 2015 Confidentiality Label 44

45 Sausage Packaging ATR Chemical Images FTIR ATR imaging determined identity & approximate thickness (in microns) PE PE with additive PE Polycarboxylic acid ester polymer (possibly) PA PP PE PA EVOH PA PE 140 um The sample was measured across its entire width with three slightly overlapping ATR measurements, with a total collection time of ~ 1.5 mins. 40x obj. vis image (polarized) 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. 180 um ~11 ~3 ~3 ~24 ~11 ~5 ~7 ~20 ~6 ~5 ~ ~130 um Pixel size: 1.1micron Obj mag: 15x, 0.62NA FOV: 70x70um Total system mag: 36x June 12, 2015 Confidentiality Label 45

46 Sausage Packaging 25x Transmission High mag Chemical Images 140 um - 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 um PA, layer 2 PE, layer 1 Pixel size: Obj mag: High mag: FOV: Total system mag: Working distance: 0.66 micron 25x, 0.81NA ON 85x85um 61x 12mm June 12, 2015 Confidentiality Label 46

47 Sausage Packaging 15x Transmission High mag Chemical Images 140 um 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) 1 EVOH, layer PA, layer um PE layer 1 Pixel size: Obj mag: High mag: FOV: Total system mag: Working distance: 1.1micron 15x, 0.62NA ON 140x140um 36x 21mm June 12, 2015 Confidentiality Label 47

48 Sausage Packaging 25x Transmission std mag Chemical Images 140 um 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) 180 um Pixel size: Obj mag: High mag: FOV: Total system mag: 3.3micron 25x, 0.81NA OFF 420x420um 12x June 12, 2015 Confidentiality Label 48

49 Sausage Packaging 15x Transmission std mag Chemical Images 140 um As the pixel size gets larger, now at 5.5um, layers that are ~5 um now start to blur out 40x obj. vis image (polarized) 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

50 Principal Component Noise Reduction (PCNR) 1720 cm-1 - Removes ONLY noise scan - Typical S:N improvement of ~5-10x - Corresponding time saving (for equivalent S:N) is x! scan with PCNR 128 scans Only FTIR imaging system to include PCNR Residual spectrum Agilent Confidential June 12,

51 480 um 70 mm Polymer Film Defects - Visible Images & ATR Imaging Sampling Location 15x obj. vis image cross-section view ATR Chemical Image 0.15 defect, 1402 cm ATR mm 0.06 polymer, 1402 cm um 0.04 Composite (Green/Red) image 0.02 GREEN DEFECT RED - POLYMER 0.00 At initial analysis, it appears that the defect is likely to be an Inorganic material, most probably a carbonate, or a carbonate containing mixture

52 70 mm Image created at 1644 cm um Image created at 2848 cm-1 Micro ATR (FPA) imaging of defects in black rubber sample Vis image IR image ATR 0.05 PE um 640 um Polyamide mm Polyisoprene (natural rubber)

53 85 mm 25x, 0.81 NA Objective in high mag mode for Transmission Polymer bead analysis 40x vis image 2915 cm cm cm-1 (3.4 microns) (6.9 microns) (8.9 microns) 85mm 5 micron polystyrene beads on BaF2 substrate are clearly chemically distinguished. Even at relatively long wavelengths (low wavenumbers), 5 micron beads are clearly resolved Pixel size: Obj mag: High mag: FOV: Total system mag: Working Distance: 0.66 micron 25x, 0.81 NA ON 85x85 um 61x 12 mm Agilent Confidential June 12,

54 350um Exposed green plastic Visible images & ATR Imaging Sampling Location 70 um 15x obj. vis image ATR 70 um 470um

55 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

56 70 um 70 um Results - Exposed Green Plastic 40x high mag. obj. vis image ATR Chemical Image cm um 70 um A clear edge containing higher carbonyl content is clearer visible along the expose side edge. Confidentiality Label 56 June 12, 2015

57 57 Pharmaceutical FTIR Imaging

58 480 mm 70 mm Tablet Contamination Location 15x Vis Image 0.20 Row = 29 Col = Active mm 70 mm Row = 12 Col = 10 Lactose 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 Row = 43 Col = Starch Defect (next slide) Row = 51 Col = 28 Cellulose

59 70 mm 480 mm 70 mm Tablet Contamination Location 15x Vis Image Row = 10 Col = 26 Polyester mm 70 mm Clear and distinct domains from 4 constituents were detected: - Contaminant #1 Possibly a polyester - Contaminant #2, - Possibly a polyamide Contaminant Composite (RG), part Row = 30 Col = 28 Polyamide 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 mm

60 Electronics/Semicon FTIR Imaging

61 Spacer contamination on LCD filter 70 um Total analysis time = 2 mins 350 um um 5 mm 70 um Defects identified as dislodged Spacers No sample prep and no sample damage 0.00

62 LCD Defect Protein image (1647 cm-1) 480 um 70 microns Visible Image 70 microns 640 um Total analysis time = 1 mins Defects identified as protein, most probably flake of dead skin No sample prep and no sample damage Row = 6 Col = 30 IR image at 1647cm

63 350 um 70 um Contaminated Circuit Board FTIR ATR Imaging Visible Image Total analysis time = 2 mins Spectra library search reveals contaminant to be polyetherimide ATR contact damage caused by other vendor 70 um

64 Forensics FTIR Imaging

65 Vehicle paint cross sectional analysis 3696 cm cm Row = 75 Col = Row = 164 Col = cm cm cm cm Row = 94 Col = Row = 24 Col = Row = 90 Col = Row = 149 Col =

66 MIXED POWDER MICRO ATR ANALYSIS CALCIUM CARBONATE 0.8 Row = 37 Col = CaCO3( ) STPP( ) pyrophosphate( ) disodium phosphate(94

67 MIXED POWDER MICRO ATR ANALYSIS DISODIUM DIPHOSPHATE ANHYDRIDE 0.20 Row = 38 Col = STPP( ) disodium phosphate( ) CaCO3( ) pyrophosphate( )

68 MIXED POWDER MICRO ATR ANALYSIS SODIUM TRIPOLYPHOSPHATE 0.2 Row = 61 Col = 56 STPP( ) pyrophosphate( ) disodium phosphate( ) CaCO3( )

69 MIXED POWDER MICRO ATR ANALYSIS SODIUM PYROPHOSPHATE Row = 17 Col = 17 STPP( ) pyrophosphate( ) CaCO3( ) disodium phosphate( )

70 Food/Cosmetics FTIR Imaging

71 500 um Imaging of mayonnaise heterogeneity: measured in transmission on BaF2 slide 1.5 Row = 69 Col = 63 Visible CCD image 1.0 TmpltPk1( ) water( ) starch( ) oil( ) Row = 28 Col = 106 oil( ) water( ) TmpltPk1( ) starch( ) 650 um Measured on FTS7000+UMA600, using a 128x128 FPA. IR measurement area = 700x700 microns Time of measurement = ~ 2min Resolution = 8 cm Row = 70 Col = 66 water( ) starch( TmpltPk1( ) oil( )

72 Biological & Biomedical FTIR Imaging

73 Breast Tissue section CH image ( cm-1) Amide I image 1233 cm-1 image 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

74 Breast Tissue section (high mag mode) 40x obj vis image CH image ( cm-1) Amide I image 1233 cm-1 image 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

75 280 um 280 um Normal Mag (5.5um) High Mag (1.1um) comparison, CH image Normal Mag (5.5um) High Mag (1.1um) um 280 um The 3 features within this oval shape are each about ~3 um in diameter! June 12, 2015 Confidentiality Label 75

76 Wheat stem cross section FTIR Image See next set of data for med. res. close up 0.8 Protein 1654 cm-1 Row = 26 Col = See following set of data for high. res. close up See next set of data for med. res. close up Lipid 1733 cm-1 Row = 68 Col = See following set of data for high. res. close up See next set of data for med. res. close up 1.0 Carbohydrate cm Row = 57 Col = See following set of data for high. res. close up

77 70 microns 70 microns Visible Image (red box show field of view ATR) 2-D IR image at 1043 cm-1 3-D IR image at 1043 cm Row = 12 Col = 21 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

78 Live cell FTIR imaging in water! 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 Liquid transmission cell pathlength = 7microns Total measurement time ~ 10mins. Pixel size : 1.1 microns June 12,

79 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,