Characteristics and Applications of Imaging Microscope FT-IR. Teo Wei Boon PerkinElmer Singapore
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1 Characteristics and Applications of Imaging Microscope FT-IR Teo Wei Boon PerkinElmer Singapore
2 Infrared (IR) Spectroscopy. IR Spectroscopy measures the radiation absorbed by materials due to molecular vibrations H O H -C-H cm -1 -C=O cm -1 =C-H cm -1 -C-O cm -1 -C=C cm -1 -O-H cm -1 -C-Cl cm -1 -N-H cm -1 -C-Br cm -1 Vibration frequencies are influenced by Atom Size Environment Bond Strength Page 2.provides a vast amount of chemical information
3 Interactive Interpretation Spectral interpretation is to find out more about the functional groups present in the compound Page 3
4 PerkinElmer Euclidean Search A spectrum from an unknown compound is searched through reference spectra to try to match and identify the unknown compound Page 4
5 What is IR Microscopy?
6 When the sample spot is much smaller than the IR beam IR Beam in spectrometer is around 8mm in diameter Analysis of surrounding areas and not the spot. To analyse small areas, beam spot must be smaller than sample area. Page 6
7 Microsampling : IR microscopy/imaging AutoImage Spectrum Spotlight 400 Microscope schematic Motorized stage Detector MCT Cassegrain Toroid Coupling Optic To Optical Viewer or Video Camera Remote Aperture Position Reflectance Mirror Objective Cassegrain Sample Position Condenser Cassegrain View/IR Mirror Page 7
8 What Can An IR Microscope Study? Small samples down to about 10 x 10 µm : fibers, particles, crystals Small inhomogenieties in a larger matrix: contaminants in paper, polymers, electronic components Surface studies: defects on coatings, painted surfaces Microscopic changes of composition across a sample: additives in polymers, purity of materials And more... The motorised stage helps to collect spectra at a fixed spatial interval. The collection of spectra can be manipulated to produce new images showing spatial changes. Page 8
9 Page 9 FT-IR Microscopy data collection
10 FT-IR Microscopy provides. 130 spectra, 30 minutes IR Microscopy µm single-point measurements - single spectra in seconds Wavenumber 625 spectra, 90 minutes point-by-point accumulation of linescans point-by-point mapping µm µm... fast single-point data, but IR images very slowly Page 10
11 Composition is a key subject in Imaging and Surface Science techniques are a collection of analytical tools which can characterise the following properties Topology The surface features How it looks Morphology Shape and size of the particles Crystallography Structure Composition Elements and molecules making up the material Concentrations Distributions EDX spectra of syringe contaminant IR Microscopy identified the Tin Salt FT-IR Microscopy helps determine component distribution in high performance polymer Electrograph of car-tape deck identified white particles. FT-IR microscopy truly identified the polymer Page 11 understanding materials
12 IR Imaging gives complementary information. Molecular IR Imaging Raman Imaging Chemical Information Content Mass Spec X-Ray None Optical Microscopy SEM/TEM Scanning Probe Low Spatial Resolution High.to a wide range of imaging and surface-science techniques. Page 12
13 FT-IR Imaging enable chemical composition... FT-IR Microscopy single-point measurements - single spectra in seconds point-by-point accumulation of linescans point-by-point mapping generates images Images take may hours to generate FT-IR Imaging A many element detector generates an image Sometimes the sample is moved over the detector to create very large images Images take only minutes to generate Huge success for applications where very large sample areas have prevented practical studies Biomedical research Pharmaceutical tablet Successes in traditional microscopy markets where the organizational culture is one that enables early adopters to investigate imaging Page 13...from large areas to be obtained quickly
14 What is FT-IR Imaging?
15 FT-IR Images show.. FT-IR Imaging is an analytical tool which provides a complete view of the spatial distribution of a molecule or functional group across an area of a sample. This provides a series of molecular 'pictures' of the sample showing the locations of different components. Visible Image IR Image showing carbonyl distribution.. the distribution of molecular species Page 15
16 Spectrum Spotlight 400 Imager Spectrum One LED AutoIMAGE automation & throughput Breakthrough array detector technology Integrated System Control Spectrum Spotlight 400 Page 16
17 16X1 Staggered Linear Array Elements are 6.25µ apart in horizontal and vertical plane At the sample! High Definition Mode Stage is moved in 6.25µ steps Achieves close to 100% fill factor Uses more of the available photons Improves sensitivity Page 17 Is The Foundation For Superb Signal Processing
18 The Gaps Are Filled As The Stage Is Stepped... First Step Second Step Third Step Page % Fill Factor Delivers Great Sensitivity
19 Keep On Stepping Imaged area highlighted in yellow This equates to the sample area chosen by the user Data from first two columns and last two columns not used This is outside the area selected by the user Page 19 8 steps To Collect Any Size Image
20 Stepping The Stage Direction Of Stage 16 element array views sample in blocks of 100µ x6.25µ or 400µ x25µ Precision automated stage is stepped left at up to 5 steps per second At the end of a row the stage sweeps back to start the next row p Up to 5 steps per second and 80 scans per second at 16cm -1 Allows Users to Define Any Image Size Page 20
21 Small Linear Array design any image size Crane Fly s Wing 18.5mm x 4.5mm, 132,000 spectra Fingerprint 20mm x 13.5mm IC 0.3mm x 0.3mm Page 21 without mosaicing or wasted data collection time
22 IR Imaging provides.. IR Imaging some systems > 5000 high-quality full-range spectra in seconds! Pixel size 6.25 x 6.25 µm Gives a fast, COMPLETE, high resolution picture of molecular distribution in a sample the complete picture, RAPIDLY. Page 22
23 IR Images show.. Color temperature shows molecular distribution e.g a carbonyl impurity in a polypropylene matrix Some systems provide 2-D surface or 3-D projection.. the distribution of molecular species Page 23
24 Each pixel of an IR Image contains. Full-range spectra. Generate further images Identify unknowns Page 24 a full-range IR Spectrum.
25 Functional groups (including user defined) are selectable to. Page 25 create various functional group images
26 In Summary, IR Imaging provides.. Very fast - some systems are up to 1000 times faster than pointby-point mapping On the latest PerkinElmer Spectrum 400 model it can create an image of 1 cm X 1 cm in 8.4 minutes Ideal for needle in a haystack problems Provides chemical information about ALL of the sample, not just areas which look different in visible light the COMPLETE picture of molecular distribution Page 26
27 The Spotlight 400 Imaging System
28 Announcing the innovative Spotlight 400 The Spotlight 400. The most powerful laboratory infrared imaging system in the world Page 28 more detail, faster, easier the results will inspire
29 The innovative Spotlight 400 The Spotlight 400. The most powerful infrared imaging system in the world Large sample stage option for more and bigger samples ATR Imaging Accessory. The highest quality ATR spectra over the largest sample area. Environment enclosure for the most challenging applications Liquid Nitrogen Reservoir for increased unattended analysis times 160 spectra for second enabling extremely fast image collection Multiple image collection increases efficiencies and productivity 1.56µ to 50µ pixel sizes the widest range of options proving speed and detail on the same system Page 29 more detail, faster, easier
30 Infrared Sampling Modes Transmission Reflection-Absorption Transmission ATR Reflection-absorption Diffuse Reflectance c Internal reflection Diffuse reflection Page 30
31 The Spotlight 400 ATR Accessory
32 Why Use ATR Imaging? Extends the range of samples which can be measured Too thick for transmission imaging Typically need 5-10µ Where preparation into thin slices may alter or damage the sample Where reflection spectra are of poor quality or complicated Where the only the top surface of the sample is of interest Potential to increase spatial resolution! Page 32
33 Sampling flexibility provides the most information Accommodates samples up to 10mm thick for the maximum applications flexibility 600 µ diameter crystal tip of which 500 µ guaranteed defect free Enables 400 µ areas to be collected 100 x bigger than currently available More information from your samples A number of rectangular or square areas can be selected Reducing redundancy and increasing the analysis time Page 33 the most efficiently
34 Spotlight 400 Lightening Speed! With no compromise on our world class performance
35 Our EXCLUSIVE detector design (and patents) Patented Dual Mode MCT Detector Exclusive to PerkinElmer 16X1 NBMCT array and single point MBMCT on the same substrate in the same dewar cm -1 Guaranteed 5%T at 720cm -1 but can measure below this Superb linearity 100µ Spectacular signal-to-noise Far superior than the competitions Page 35 gives better imaging performance
36 High signal-to-noise, means fewer scans, faster collection 25µ pixel size sensitivity comparable to aperturing single point to 25µ Guaranteed 600:1 for 4 scans Typically >800:1 6.25µ sensitivity the highest, high resolution sensitivity Guaranteed 200:1 for 4 scans Typically >400:1 16cm-1, through IR polarizer Crane Fly Wing Page 36 but is not the only reason Spotlight is fast
37 So we get often get great data with 1 scan Spotlight 300 scans (full range spectra): 80 per second 4,800 a minute Spotlight per second 10,000 per minute Page 37 so the Spotlight 400 now collects spectra even quicker
38 The Spotlight 400 can provide Spotlight µ, 80 spectra/sec Spotlight µ, 170 spectra/sec 30 minutes 8 minutes!!! Same quality images, same quality spectra but much, much faster!!! Page 38 the same quality information, faster
39 Spotlight 400 combined speed and intelligent software Spotlight 400 provides the fastest IR images available on the market Large image areas can be collected 4 times faster than today! But what do customer s do with the data once they have collected the image? Show Structure capability in Spotlight 400 Page 39 provides information faster
40 One button press is all it takes Page 40 to reveal the true chemical variations in a sample
41 Spotlight 400 Unmatched Flexibility
42 Spotlight 400 exclusive detector design enables Spectral range <700cm -1 Guaranteed 5%T at 720cm -1 Measure material characteristics that competitors cannot do! Inorganics Polymorphs Superb detector linearity Highly absorbing bands more accurately measured Relative strengths weak/strong bands more accurate Page 42 applications others just cannot do!
43 The Spotlight 400 Environment Enclosure Purge input for removal of atmospheric spectral interferences For demanding applications where these small spectral changes can affect analyses Biomedical Hinged lids for easy access to dewar and sample Page 43 addresses the most challenging applications
44 Spotlight 400 provides a large stage option Standard Stage travel = 75x50mm Remember Spotlight 400 is can be changed quickly between Reflectance, Transmission and ATR and also has a single element microscope detector for the most difficult single point analyses Large stage option Stage travel = 160x60mm Page 44 for the bigger and more samples at a time
45 Spotlight 400 Purposeful Productivity
46 Image All Markers Image 5.2 x 3.6mm 208 x 144 spectra Page 46 speeds data collection for samples with large non-sample areas
47 Spotlight 400s easy setup Can Add Image Markers to visible image Set up shape, size, label and focus position at each marker point (Auto focus all markers also available) No limit on the number of markers Scan and save all marker images in a single operation Can save and retrieve image marker position files Page 47 enables this to be done quickly
48 Liquid Nitrogen filler Multiple image acquisition means extended unattended Spotlight operation times are likely New dewer reservoir extension can extend unattended operation to ~18 hours Spotlight can work when everyone else is asleep!! Page 48 provides long periods of unattended operation
49 The NEW Spotlight 400 No other IR imaging system can match the Spotlight 400 for the number of applications, performance and productivity Page 49 powerful, flexible and productive!
50 The NEW Spotlight 400 Highest performance Sensitivity Speed Spatial resolution Pixel size of 1.56µ Environment enclosure Unsurpassed imaging flexibility 50, 25, 6.25, 1.56µ ATR, transmission, reflectance Any image size Large stage option Most productive Multiple images Auto-sampling Fast survey mode Page 50 powerful, flexible and productive!
51 Spotlight Applications
52 Infrared Sampling Modes Transmission Reflection-Absorption Transmission ATR Reflection-absorption Diffuse Reflectance c Internal reflection Diffuse reflection Page 52
53 Applications: Fillers in polymers Transmission measurement
54 Application 1- Distribution of clay filler The distribution of various components used in polymer making are of great interest to polymer chemists as they affect the physical and chemical properties of the polymer considerably. In this example the distribution of a clay filler can be imaged by Spotlight using the transmission mode. The filler added was 10 percent and the distribution studied Page 54..in polymer film
55 Application 1- Distribution of clay filler The total absorbance map of the polymer film with 10 % clay filler Page 55..in polymer film
56 Application 1- Distribution of clay filler %T 40 Clay peak cm-1 Page 56..in polymer film
57 Application 1- Distribution of clay filler Area under the curve from cm-1 Page 57..in polymer film
58 Application 1- Distribution of clay filler Clay distribution image of the polymer film with 10% clay filler Page 58..in polymer film
59 Applications: Pharmaceutical ingredients distribution Diffuse reflectance measurement
60 Application 2- diffuse reflectance analysis An example of a diffuse reflectance sample is shown in the next slide.in this case, the distributions of an active ingredient, an unknown excipient, and sucrose within a tablet are clearly distinguished especially well suited to measurement in reflectance mode. Page 60..provides results without sample preparation
61 Application 2- diffuse reflectance analysis Active Ingredient Unknown Excepient Sucrose Page 61..provides results without sample preparation
62 Applications: IR Imaging of a multilayer tubing
63 Application 3 - IR Imaging of a multilayer tubing Multilayer tubing Car fuel pipe layer Nylon 12 Melting 175 Adhesive layer modified ETFE Melting point layer ETFE Melting point 218 Temp 80 80,, 160,, 240 Cooled Sample heated,cooled and tubing microtomed to 5 m 5 m thickness Page 63
64 Application 3 - IR Imaging of a multilayer tubing IR Imaging Parameters Method IR Transmission Spectral resolution 4cm -1 No of Scans 8 Spectral range cm -1 Pixel size 6.25 m m Imaging Area 498 m m No of pixels Sample visible image Red square is imaging area Outer layer Adhesi Inner layer Page 64
65 Application 3 - IR Imaging of a multilayer tubing Original material spectrum with 1 scan using ATR technique 2974cm - 1 area Absorbance CH3 vibration of t-buty Inner Adhesive cm Page 65
66 Application 3 - IR Imaging of a multilayer tubing CH 3 of adhesive layer band area image Room temp Adhesive layer material diffuse into inner layer band area image With heating the adhesive layer diffuses into the inner layer Page 66
67 Contaminant on washer Specular Reflectance Measurement
68 Application 4 - specular reflectance analysis The sample is a spring washer, a component of a small motor, which was sticking and causing failure. Visible image, measures 15mm 2. Page 68..provides results without sample preparation
69 Application 4- specular reflectance analysis The infrared image was recorded for an area approximately 6.5 x 4mm, at 25 m pixel resolution. This represents spectra The spectrum extracted from the area of contamination is shown in the lower figure. Page 69 Unknown Excepient..provides results without sample preparation
70 The Spotlight 400 ATR Accessory
71 Why Use ATR Imaging? Extends the range of samples which can be measured Too thick for transmission imaging Typically need 5-10µ Where preparation into thin slices may alter or damage the sample Where reflection spectra are of poor quality or complicated Where the only the top surface of the sample is of interest Potential to increase spatial resolution! Page 71
72 A carefully considered optical design Accommodates samples up to 10mm thick for the maximum applications flexibility 600 µ diameter crystal tip of which 500 µ guaranteed defect free Enables 400 µ areas to be collected High quality standards and advanced calibration of crystal ensures that each crystal delivers the optimum spatial resolution (3µ) Page 72 ensures high quality results fast
73 Embedding Process A B Resin mold Fixation clips C D Sample + resin in place Cured block removed Page 73
74 Applications: Multilayer polymer film ATR measurement
75 Example 5 Packaging Material Embedded sample Visible image IR reconstructed image after principal components Page 75
76 Application 5 -Spectra from Layers 10 microns %T cm-1 Page 76
77 Applications: Pharmaceutical ingredients distribution ATR measurement
78 Application 6 -Example (1) Low concentration of active ingredient Sample 5 components present Active (<1%) Microcrystalline cellulose Starch Calcium phosphate Magnesium stearate Experimental Spotlight Imaging System with ATR accessory 16 cm-1 resolution 1.56u image pixel size 400x400u image area 1-16 scans/pixel Page 78
79 Application 6 - Suspected Raw Ingredients Spectra Active TBC Microcrystalline cellulose Calcium phosphate Magnesium stearate Starch Povidone (PVP (not present!) ATR spectra 1 st derivative Page 79
80 Application 6 - Composite Image from 4 of the Major Components Avicel Starch Calcium phosphate Magnesium stearate Page 80
81 Application 6 - Least Squares Fitted Image for Microcrystalline Cellulose Raw Ingredient spectrum Spectrum from Image Page 81
82 Application 6 -Distribution for Starch Raw Ingredient spectrum Spectrum from Image Page 82
83 Application 6 -Distribution for Active (< 1%) Raw Ingredient spectrum Spectrum from Image Page 83
84 Application 6 -Active Ingredient Particles <10 microns ~10 microns Page 84
85 Cockroach Wings in Beer Analysis using IR Imaging Spotlight System Transmission Measurement
86 Application 7 - Cockroach in Beer Chemical changes in cockcoach wings with time it has been in beer How long has the cockroach been in the beer? From the shop? From the factory There seems to be some chemical change with the cockroach wings depending on the length of time it has been in the beer Can IR Imaging be used to determine the length of time the cockroach has been in the beer? 0-13 days- from the shop or by the consumer More than 14 days from the factory Page 86
87 Application 7 - Cockroach in Beer IR Transmission Images of chemical changes in cockroach wings with time it has been in beer Visible Image Band Ratio IR Image Page 87
88 Application 7 - Cockroach in Beer Chemical changes with soaking time of cockroach wings in beer Band Ratio Image(Methyl / Amide bands 2970cm - 1 / 1530cm - 1 ) 0 days 7 days 14 days Page 88
89 Application 7 - Cockroach in Beer Chemical changes with soaking time of cockroach wings in beer Band ratio image (Methyl / Amide bands 2970cm - 1 / 1530cm - 1 ) Amount of wax shown by the Methyl/Amide II Band Ratio IR image decreases with time 0 days 7 days 14 days With longer soaking time the the peak around 3300cm - 1 shows changes Page 89
90 Application 7 - Cockroach in Beer Chemical change with soaking time of cockroach wings in beer Band Ratio Image (OH/ NH bands 3380cm - 1 / 3300cm - 1 ) 0 days 7 days 14 days Page 90
91 Application 7 - Cockroach in Beer Chemical change with soaking time of cockroach wings in beer Band ratio Image(OH / NH bands 3380cm - 1 / 3300cm - 1 ) OH /NH band ratio image does not show much differences with 0 day and 7 days of soaking time. However the OH group shows an increase with 14 days of soaking time. 0 days 7 days 14 days Page 91
92 Application 7 - Cockroach in Beer Chemical change with soaking time of cockroach wings in beer Conclusion Amount of wax shown by the methyl/amide II Band Ratio IR image decreases with time OH /NH Band Ratio Image does not show much differences with 0 day and 7 days of soaking time. However the OH band shows an increase with 14 days of soaking time. The could be due to changes in the protein present in the cockroach wings From these changes,we can use IR Imaging to estimate how long a cockroach has been present in the beer Page 92
93 END
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