1. Infrared Microspectroscopy A training guide for using light microscopy and infrared spectroscopy to analyze materials

2. Table of Contents Lectures I. Introduction II. Basic Principles of Microspectroscopy III. Transmission Theory IV. Reflection Theory V. Solving Problems with Infrared Microspectroscopy Laboratories I. Microscope Performance Validation a. Performance Validation of the IR-Plan Research Microscope b. Performance Validation of the IR-Plan Analytical Microscope c. Performance Validation of the Nic-Plan Microscope II. Transmission Experiments III. Reflection Experiments

3. Infrared Microspectroscopy A new technology formed by combining light microscopy and FT-IR spectroscopy Seeing the sample assures analysis of the correct area of interest Understanding microscopy principles is the foundation of infrared microspectroscopy

4. Microscopy Applications Small samples Plastics Packaging materials Pharmaceuticals Fibers Trace evidence Contaminants Multi-phase mixtures Failure analysis Coatings & inks Electronic materials Migration, diffusion and aging studies Reverse engineering Art conservation And much more

5. Rules of Microscopy The ruler in the microscopic world is marked in micrometers  m = 0.001 mm or 0.0004 inches A human hair is 75 to 150  m; the resolving power of the eye is 75 to 100  m

6. Rules of Microscopy Spatial Resolution is the ability to separate objects into distinct parts Resolution limit or resolving power is the smallest distance two objects must be separated so they are seen as separate parts Detection is the ability to sense the presence of a feature with a reasonable degree of certainty

7. Infrared Microspectroscopy Observe SampleDefine Area of InterestCollect Spectrum Basic concept is a simple 1, 2, 3 operation 123

8. Infrared Microspectroscopy Transmission Theory Microscope Training Course

9. Transmission FT-IR Microscopy Fundamental Optical Factors Affecting FT-IR Microscopy –1. Diffraction –2. Refraction –3. Reflection –4. Scatter

10. Diffraction * Aperture * Sample Area * Shaded Area Indicates Diffraction

11. IR Transmission Path Diagram

12. Spurious-Energy Diagrams 50004000300020001000 Frequency (cm-1) 0 20 40 60 80 Spurious Energy for Various Width Specimens % of Total Signal 10um 20um 50um Numerical Aperture = 0.50 Single Aperturing

13. Spurious-Energy Diagrams 50004000300020001000 Frequency (cm-1) 0 20 40 60 80 Spurious Energy for Various Width Specimens % of Total Signal Numerical Aperture = 0.50 Dual Remote Image Masking 50um 20um 10um

14. Effects of Stray Light on Absorbance Value No Stray Light 5% Stray Light 10 % Stray Light Measured Absorbance 0 1 2 12 Predicted Absorbance

15. Hair Sample Using Three Different Aperturing Techniques 0 1 2 3 4 5 6 7 8 9 10 %Transmittance 1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1) 1 2 3 1 - Lower Mask Only 2 - Upper Mask Only 3 - Both Upper & Lower Masks

16. Experiment LAYER 1LAYER 3 2 220 um 25 um Cross-Section of a Laminated Film

17. Outer Layer - Polypropylene 0.2 0.4 0.6 0.8 Absorbance 1000 1500 Wavenumbers 220 um wide layer

18. Middle Layer - PVA (top) 25 MICRON LAYER #2 - single aperture (bottom) 25 MICRON LAYER #2 - dual apertures Absorbance 1000 1500 Wavenumbers

19. Refraction Normal n1n1 n2n2 n 1 < n 2

20. Windows and Their Effects Spherical Aberration is produced when the sample is placed on, or between, infrared transparent windows Spectra-Tech objectives and condensers correct for these aberrations, resulting in improved resolution window Spherical aberration is caused by the rays at high incident angles not coming to the same focal point as the low angle rays

21. Spherical Aberration Correction Images of Multi Layered Polymer Before Reflachromat CorrectionAfter Reflachromat Correction

22. Ray Trace of Reflachromat Objective

23. Internal Reflection in Transmission Measurements 123

24. Nylon 6.6 Fiber, flattened, in air 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 %Transmittance 1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1) Interference fringes 25502010 t(mm) = N/2 x [10/(W h - W l )] t(mm) = 2/2 x (10/540) = 0.0185 mm

25. Interference in a Thin Film AIR (n =1.0) SAMPLE (n =1.5) A B T  REFLECTANCE = (n-1)2 (1.5 -1.0)2 --------- ------------ = (n+1)2 (1.5 +1.0)2 = 0.4

26. Transmission Spectroscopy The ideal sample for transmission measurement –(1 to 15 um) thickness –large and uniform surface –low reflectivity to avoid thin film interference fringe patterns in the spectra Mounting in a micro compression cell between two infrared windows makes an ideal transmission sample

27. Micro Compression Cells Standard Micro Compression Cell with standard 13x2 mm Windows Diamond Micro Compression Cell

28. Micro Compression Cell Sample KBr AB1 B2

29. Sample Preparation Tools Roller KnifeTungsten Probe Kit

30. Preparing a Sample in a Micro Compression Cell

31. Nylon 6.6 Fiber Mounted in a Micro Compression Cell 0 10 20 30 40 50 60 70 80 90 100 %Transmittance 1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1) 4000

32. Scatter

33. Sample Preparation onto an Infrared Window Step #1 Step #2 Step #3

34. Scatter Experiment Photocopier Toner Prepared in a Micro Compression Cell Photocopier Toner -10 0 10 20 30 40 50 60 70 80 90 100 % Transmittance 1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1)

35. Scatter Experiment Photocopier Toner (baseline corrected) 60 65 70 75 80 85 90 95 100 105 %Transmittance 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1) Spectrum after Data Manipulation (Baseline Correction)

36. Transmission Experiments Practical Hands-On Sample Preparation