Carol Hirschmugl

1. Frontiers of infrared spectroscopy: Infrared Beamlines and Applications in Biology, Geology and Environmental Remediation Carol Hirschmugl

2. What is Infrared Spectroscopy? Ultraviolet X rays Infrared 1012 1013 1014 1015 1016 1017 1018 1019 1020 103 104 105 106 107 108 109 1010 1011 1012 l: (m) Molecule vibration e near Fermi Level Valence band e Core e f: (Hz) Absorption of Low Energy (IR) Photons causes: Displacement of atoms (Vibrations) Functional groups (CH, OH, etc.) within larger molecules exhibit similar absorption energies

3. What is a Static Dipole Q is charge r is distance between the charges P is dipole -Q r P=Qr +Q Which of the following molecules are dipoles: CONO Which of the following molecules are dipoles: O2, CO, N2,NO?

4. What is a Dynamic Dipole -Q +Q’ Separated charge distribution that is moving

5. Which of the following would be Dynamic Dipoles: Vibrating N2 Vibrating CO Vibrating O2 Vibrating NO Vibrating CO2 Vibrating CO Vibrating NO Vibrating CO2???? Ionic bonds in molecules can have infrared active vibrations, Covalent bonds in molecules are not infrared active

6. CO2 Vibrations: Stretching Modes

7. CO2 Vibrations: Stretching Modes

8. CO2 Vibrations: Net Dipoles

9. CO2 Vibrations: Net Dipoles

10. What are greenhouse gases? Air: N2,O2, CO2, CH4, H2O Which are absorbing the infrared radiation reflecting from the earth?

11. Vibrational Strengths High Frequency Vibrations: Stretching Modes Lower Frequency Vibrations: Rotational Modes (bending and wagging modes)

12. Vibrational Strengths k m1 m2 region = region Atoms in Typical Functional Groups include C, N, O, H, P, S, Si, Al fingerprint region = region X-H Stretching 4000 3000 2000 1000 0 Wavenumbers (cm-1)

13. Signatures in Infrared Spectroscopy? (a) Transmittance 100 IS/IR[%] Frequency, n/c[cm-1] (b) Absorbance A [arb. units] 0 Frequency , n/c [cm-1] The energy for each vibration is dependent upon the mass and spring constant, IR absorption spectroscopy is chemically specific.

14. Sources of Infrared Light Blackbody Radiation Storage Ring Radiation Hot filament or rod (Globar or Nernst Glowar) Flux radiated in all directions Flux  Temperature From relativistically accelerated charged particles Flux radiated in a tangential, well defined cone Flux  Beam current

15. Synchrotron Characteristics: IRSR Flux IR Flux  Beam Current IR lc increasing Wavelength IR Source: wide beam profile Large opening angle Mixed polarization Beam Profile

16. Why use a storage ring source? Storage Ring Spectromicroscopy Grazing incidence reflection-absorption spectroscopy Small source size Small solid angle Very small angular acceptance < 5° Very small sample 1 mm x 1 mm Globar Large source Large solid angle Small angular acceptance > 10° Small sample 30 mm x 30 mm

17. Advanced Light Source Infrared Facility Necessary Beamline Components: Large extraction port Large Water Cooled Flat Mirror Off-Axis Parabolic Mirror with long focal-lengths Wedged Diamond Window at focal point Collimating Optics Spectrometer: FTIR (Vacuum or Nitrogen Purge) w/ LN2 or LHe cooled detectors Optional Beamline Components: (Based on experiment) Infrared Microscope Surface Science Chamber Beam position feedback system

18. NSLS, BNL Vacuum Tank Schematic

19. NSLS, BNL Vacuum Tank IR and UV

20. Advanced Light Source Infrared Facility m1: Large Water Cooled Flat Mirror m2: Off-Axis Parabolic Mirror with long focal-lengths Wedged Diamond Window at focal point Collimating Optics

21. Water Cooled Plane Mirror and Off Axis Paraboloid for ALS IR Beamline Off Axis Paraboloid Plane Mirror

22. NSLS U4IR and U2B Infrared Beamlines Off axis Paraboloid

23. UHV Chamber Detector (Bolometer) NSLS Source (Reflected From Below) Michelson Interferometer Diamond Window Elliptical Mirror Infrared Beamline Overview: NSLS Beamline U4IR

24. NSLS Beamline U4IR Michelson Interferometer Diamond Window UHV Chamber Detector

25. FTIR Michelson Interferometer polychromatic light monochromatic light volts Data Points

26. Rapid Scan Michelson Interferometer Broadband IR from Storage Ring (1 cm-1 - 40,000 cm-1) Moving mirror velocity = 1 cm/s + “Noise” from Electron beam motion ( e.g. 100 Hz) Examples include: RF Side bands (700-3000 Hz) Ripple on Power Supplies (multiples of 60 hz) etc. Broadband IR Signal (1 cm-1 - 40,000 cm-1) + Signal Frequency “Noise” (100 cm-1 spike)

27. Thermo Nicolet Infrared Microscope at ALS Collimated beam from Storage Ring Infrared Microscope FTIR Spectrometer

28. Infrared Microscope Schwarzchild Objectives Sample x-y motorized stage Synchrotron Radiation Michelson Interferometer Detector

29. Infrared Beamlines U12, U10A and U10B at the NSLS

30. How Much Signal? 1000 100 10 Wavelength [mm] Signal to Noise Ratio: is 100-1000 times larger from a synchrotron source than from a globar through a limited aperture Liquid Helium Cooled detectors to reduce background noise

31. Interstellar micron size dust analysis Molecular water Aliphatic CH Cristallised Olivine OH- silicates 0,5 2 Synchrotron- 3x3 m m 0,0 Absorbance 2 Black Body- 12x12 m m Black Body 6x6 m m2 -0,5 4000 3500 3000 2500 2000 1500 1000 Wavenumber ( cm-1) Particles collected from the MIR shuttle comparison SRS-Black body: 3 micron Particles from asteroid “Orgueil” From: G.Quitte J.P.Bibring, L.D’Hendecourt ( IAS), P.Dumas ( LURE), G.L.Carr, G.P. Williams ( NSLS)

32. Chemical Fingerprint of Ink Wilkinson et al., Physics World, March 2002, 43

33. Infrared Microscopy: IR Imaging of Living Cells Euglena gracilis Carol Hirschmugl, Maria Bunta, Justin Holt Physics Department, University of Wisconsin-Milwaukee Andrej Skylarov Advanced Analysis Facility, University of Wisconsin Milwaukee J Rudi Strickler WATER INSTITUTE, University of Wisconsin Milwaukee and Mario Giordano Marine Biology, University of Ancona, ITALY

34. Algae Biology : Euglena gracilisapprox 15 x 15 microns

35. 5 mm 5 mm Euglena gracilis under the microscope Euglenagracilis encysted Euglena gracilis swimming

36. Experimental Conditions Drying algae Gold plated slide Measured in reflection geometry, Infrared light is absorbed by the algae twice

37. Chemical Mapping and Chemical Image: Y X Frequency (cm-1) X,Y Position

38. Reflected and Absorbed Intensities From Euglena gracilis Reflectivity from A) Gold B) Gold + Alga Absorbance from C) Alga #1 D) Alga #2

39. Infrared Spectrum from Alga and Chemical Standards Giordano, et al., J. of Phycology, 37, 271 (2001)

40. Euglena gracilis and IR spectra Spectrum d Spectrum a Spectrum c Spectrum b

41. Lipids a. C=O functional group C C C C=O Stretch vibration at 1742 cm-1 H H H H O b. CH2 functional group Asymmetric Stretch Symmetric Stretch symmetric CH2 stretch vibration at 2852 cm-1

42. Proteins CH3 H C CH3 CH2 CH2 N H N H O N N N H H H H H H Symmetric Stretch Scissors Mode Asymmetric Stretch Amides II Amides I a. NH2 functional group • Amides I peak at 1650 cm-1 represents 80% C=O stretching vibration, 10% C-N stretching vibration • and 10% N-H bending • Amides II peak at 1545 cm-1 represents 60% N-H bending and 40% C-N stretching vibration

43. Euglena gracilis and Chemical Map: Proteins 0 10 20 30 40 m 0 10 20 30 40 m 0 10 20 30 m 0 10 20 30 m AMIDES I + Water Peak 1650cm-1 CH3CH2-NH2 (a) AMIDES II Peak 1545 cm-1 CH3CH2-NH (b)

44. Chlorophyll Spectrum 0 10 20 30 40 m 0 10 20 30 m 650 cm-1 4000 cm-1 • Vernon LP, Seely GR, The Chlorophylls, Academic Press, New York, London, 1966, 193.

45. Summary of Findings (a)Protein (b)Lipid (c)Chlorophyll (d) Paramylon (c) (d) 6 mm x 6 mm spectrum at chlorophyll maximum Hirschmugl et. al. (a) (b) Algae 1

46. the protein to carbohydrates ratio : • - sample # 1 alga is 1:0.2 • - sample # 2 alga is 1:10 • the relative strength of the lipid to protein band dramatically different • Sample #2 is nutrient starved • Conclude: sample # 2 is nitrogen starved Sample # 1 Sample # 2

47. Summary for 2 Samples: Euglena Gracilis Alga #1 Alga #2 (a)Protein (b)Lipid (a)Protein (b)Lipid (c)Chlorophyll (d) Paramylon (c)Chlorophyll (d) Paramylon (c) (d) 6 mm x 6 mm spectra at chlorophyll maxima Hirschmugl et. al. (a) (b) Alga 1 Alga 2

48. Summary: Algae IR absorption contour maps of individual alga obtained Contour maps of individual spectral features show similar structure as single cell Chemical Features for two separate alga agree with Nitrogen Starvation Model Future Identify the origin of other ir features Measure different algae species under controlled nitrogen environments

49. Chemical Nature: Inclusions in Geological Specimen (Guilhaumou et al.) ABS CO2 vibrational band Peak OH at 3697 cm-1 4000 3500 3000 2500 2000 1500 1000 Wavenumbers ( cm-1) Distance ( in mm) Distance ( in mm) Aliphatic CH stretch Maximum 0 0 -10 -10 Guilhaumou N, Dumas P, Carr GL, and Williams GP, (1998), Appl. Spectroscopy 52 :1029-1034 -20 -20 -30 -30 -40 -40 -50 -50 -60 -60 Zero -70 -70 0 -80 -60 -40 -20 -80 -60 -40 -20 0 Distance ( in mm) 0 -10 -20 -30 -40 -50 -60 -70 -80 -60 -40 -20 0 High Pressure Gas Phase CO2 Aliphatic Hydrocarbon (Chains) Liquid Phase Terminated with Hydroxyl Groups

50. Interplanetary Dust Particles (IDPS) (Bradley et al.) Si-O Stretch Region at 10 mm Enstatite 9.6 mm + Fosterite 11.2mm + glassy silicates (broad) Amorphous silicates Absorption Spectrum A: GEMS Emission Spectra B: Elias Molecular Cloud C: Trapezium Molecular Cloud D: T Tauri Young stellar object DI Celphi E: M type super gienat m-Cephei Absorption Spectrum A: GEMS rich IDP Emission Spectra B: Comet Halley C: Comet Hale-Bopp D: Late Stage Herbig star Bradley JP, Keller LP, Snow TP, Hanner MS, Flynn GJ, Gezo JC, Clemett SJ, Brownlee DE, Bowey JE, (1999) Science 285: 1716-1718