1. Company Confidential Almega Introduction Almega™ Visible Raman Microscope Nicolet Instrument Corporation

2. Company Confidential Almega Introduction Molecular Spectroscopy Demand by Industry Government 12% Chemicals 12% Polymers 8% Ag/Food 8% Indep. Testing 6% Academia 14% Pharmaceuticals 16% Med/textiles/pulp and paper 21% Petrochem 3% Source: SDI Global Assessment Report (1997)

3. Company Confidential Almega Introduction Total Raman Market Demand by Region 1998 N. America 32% Japan 15% R.O.W. 12% Pacific Rim 10% Europe 23% Latin America 8%

4. Company Confidential Almega Introduction Dispersive vs. FT-Raman Source: SDI Global Assessment Report (1997) v Breakdown by technique (New Sales) w 60 - 65% Dispersive Raman w 35 - 40% FT-Raman v Total Market 1998 w Approx. $ 40 million U.S. Dollars v Projected total Raman Growth through 2002: 8-12% ; resulting in Approx. $60 million market

5. Company Confidential Almega Introduction Market Share - Dispersive Raman 1998 According to Nicolet Marketing Company Est. Units Percentage Renishaw5837 % JY Horiba5737 % (ISA/Jobin-Yvon/Spex/ Dilor) Kaiser2315 % Other Dispersives1711% (Incl Jasco, Chromex, etc) Total 155100 % Note: Unit numbers are estimations based on market data

6. Company Confidential Almega Introduction Market Share - Dispersive Raman 1998 RegionEst. UnitsPercentage N. America5032 Europe3422 Japan2516 Pacific Rim1711 Latin America128 R.O.W1711 Total 155100 % Note: Unit numbers are estimations based on market data

7. Company Confidential Almega Introduction Dispersive is Expanding v More emphasis from dispersive suppliers in traditional FT-IR/FT-Raman areas. w Pharmaceutical »R&D »Identification »Raw materials analysis w Forensics w Polymers w Chemicals, Petrochemicals All assuming NO FLUORESCENCE. It must be evaluated case by case.

8. Company Confidential Almega Introduction Almega™ System

9. Company Confidential Almega Introduction Truly Integrated and Versatile!!!

10. Company Confidential Almega Introduction Macro Sampling and Accessories

11. Company Confidential Almega Introduction Outline of Presentation v Theory w Discussions of Raman - Quick review w Dispersive Raman »Aspects of instrumentation as they apply to specifications »Emphasis on aspects that will be competitive

12. Company Confidential Almega Introduction Vibrational Spectroscopy Virtual States Electronic level 1 Electronic level 0 IR absorption & emission Rayleigh Vibrational levels Stokes Raman

13. Company Confidential Almega Introduction Advantages of Raman v Chemical fingerprint - vibrational spectrum v No sample preparation v Sample through glass, plastic bags, water v Crystal structure information - polymorphs v Backbone structure v Bonding (DLC - diamond like carbon)

14. Company Confidential Almega Introduction Dispersive Raman Spectroscopy v Laser introduction and scatter collection similar for FT-Raman or dispersive v Dispersion is by grating v Detection is by an array detector Sample Grating CCD Detector Laser

15. Company Confidential Almega Introduction Why All the laser Wavelengths? v Dispersive Raman => visible lasers w 785 nm, 633 nm, 532 nm w Other lasers, 514 nm, UV lasers v Raman efficiency proportional to 1/ 4 v This indicates all Raman should be done at the shortest wavelength possible. w Fluorescence proportional to 1/ w Resonance is sample specific

16. Company Confidential Almega Introduction Raman Microscopy v Dispersive Raman is preferred w Higher spatial resolution –Shorter wavelength means smaller diffraction limit –Higher sensitivity allows use of smaller apertures w Confocal microscopy –Also related to diffraction –Allows non-destructive depth analysis, no sample prep ALL w Almega will be ideal addition for ALL FT-IR microscope customers since it allows analysis of smaller samples

17. Company Confidential Almega Introduction Resolution v Spectral Resolution w Not as important for Raman but still a factor w Several instrumental parameters determine this v Spatial Resolution w Better than IR microscopy w Determined by different factors v Confocal Depth Resolution w Dispersive Raman only w Depth “slice” resolution

18. Company Confidential Almega Introduction Spatial Resolution v Same meaning as in FT-IR microscopy but different influencing factors w FT-IR => spatial resolution is a function of aperture size »Redundant apertures to limit both the illumination and image at detector w Raman => function of both laser beam size and aperture »Laser beam size limits how much of sample is irradiated NO APERTURE! »Spectrograph entrance aperture limits image on detector, located at spectrograph, not in the microscope v Still limited by diffraction; shorter wavelength light gives higher resolution

19. Company Confidential Almega Introduction v A major advantage of dispersive Raman microscopy is higher spatial resolution  FT-IR 10  m  FT-Raman 7  m (realistically 25-30  m) w Dispersive Raman 1  m v This should be good for both Raman AND FT-IR microscopy customers who need higher resolution … More on Spatial Resolution later

20. Company Confidential Almega Introduction Depth Resolution or Confocal Axial Resolution v A new concept for FT-IR users v The ability to analyze different depths in the sample without cross-sectioning w Simply focus on the surface of the sample, take a spectrum w Move the focus knob to focus a little deeper, take a spectrum w Repeat until all layers are analyzed v Analyze up to several hundred microns deep w Sample dependant v Resolution dependant on pin hole aperture size

21. Company Confidential Almega Introduction Detector Objective Sample Adjust z-axis with scope focus knob The Confocal Analysis...More on Confocal later

22. Company Confidential Almega Introduction Laser Wavelengths v 1064 nm w FT-Raman w Diode pumped, external cavity w Powerful (up to 3 Watts) v 785 nm w Most common dispersive Raman laser w Diode laser - external cavity stabilized w Typically offers low output power v 633 nm w HeNe gas tube laser w Low power, gain power by making tube bigger w More power than FT-IR alignment laser (1 mW) v 532 nm w Frequency double Nd:YAG w Smaller, simpler version of 1064 nm laser w Lower power v 514 nm w Argon Ion gas tube laser w lower power w Additional wavelengths v UV w Many available w 488 nm line of Ar Ion often doubled to 244 nm w All are expensive ($30k - $100k)

23. Company Confidential Almega Introduction Raman Shift Spectrum 3000  cm -1 100  cm -1 0  cm -1 -100  cm -1 -3000  cm -1 Stokes Anti-Stokes Wavelength (nm) 10641563107510641053807 7851027791785779635 633781637633629532 532633535532529459  In Raman, 3000  cm -1 is longer absolute wavelength than 100  cm -1

24. Company Confidential Almega Introduction Fluorescence is the main driver in laser wavelength selection Fluorescence NIR Excitation Visible Excitation Electronic level 1 Electronic level 0 UV Excitation

25. Company Confidential Almega Introduction 1064 nm 532 nm Electronic level 1 Electronic level 0 633 nm785 nm

26. Company Confidential Almega Introduction Which Laser should be used? v Longer wavelengths are more universally applicable w Less fluorescence w For greatest versatility, offer 1064 or 785 v Shorter wavelength results in greater efficiency w Raman signal is proportional to 4 v Shorter wavelength for higher spatial resolution v Resonance enhancement w 633 nm often used for DLC due to slight resonance enhancement w 532 also used for silicon to get high spatial resolution v Aqueous samples are better at 532 or 633 v Shorter wavelengths for dark samples (less heating)

27. Company Confidential Almega Introduction Laser Power More is not necessarily better!! Laser WavelengthTypical Applied Power (mW) 1064 nm500 - 1000 785 nm25 - 100 633 nm10 - 20 532 and 514 nm1 - 30 v Factors affecting choice of power w Raman intensity = f(power) w No need for as much power at short wavelengths »Intensity  (1/ ) 4 w Thermal damage and sample heating »Tighter focus means thermal damage is more likely

28. Company Confidential Almega Introduction How Much Power? v Use the highest power that does not damage the sample v Recommend starting with low power and slowly increase v Remember, power density is much greater at the tight focus of a microscope

29. Company Confidential Almega Introduction Other Laser Wavelength related Phenomena v Photoluminescence w Broadband emission, similar to fluorescence but different mechanism. w Wavelength specific w Common for Silicon analyses v Resonance Raman w Virtual state exactly equals an upper electronic state w Preferentially amplifies select Raman peaks by several orders of magnitude w Resonance Raman spectra can be a million times more intense than regular Raman spectrum v SERS ( Surface Enhanced Raman ) w Surface phenomenon; any Raman system can do this w Highly dependant on metal surface or particles and laser wavelength

30. Company Confidential Almega Introduction Spectrograph - The heart of the System Torroidal Mirrors Grating Aperture Shutter Detector

31. Company Confidential Almega Introduction Gratings v Spectral resolution dependant on blaze of grating w Blaze or ruling in units of lines/mm w More lines/mm, higher resolution at given wavelength w Shorter wavelengths require more lines/mm to attain same resolution as at longer wavelength v Typical Gratings (rulings) w 360, 500, 672, 1200, 1800, 2400 lines/mm v There are limits to the spectral range of gratings with high rulings due to the maximum angle that can be diffracted. v Grating throughput is wavelength dependant

32. Company Confidential Almega Introduction High vs. Low Resolution CCD Detector Low Resolution Grating High Resolution Grating CCD Detector High Resolution distinguishes closely spaced peaks provides only a small portion of the spectrum at a time Low Resolution provides full spectrum in preview mode Is sufficient for many applications. Exp 1

33. Company Confidential Almega Introduction Resolution is affected by several factors Resolution  w n n = grating pitch (lines/mm) w = CCD pixel size (usually 25 um) Resolution is inversely proportional to grating pitch and proportional to wavelength Spectral Resolution

34. Company Confidential Almega Introduction The wavelength dependence can be visualized High  cm -1 Low  cm -1 Grating Spectral Resolutions increases

35. Company Confidential Almega Introduction Too many lines/mm - long wavelengths dispersed too much. High  cm -1 Low  cm -1 Grating Cannot collect Angle is too steep However There are limits to lines/mm

36. Company Confidential Almega Introduction 785 Raman shift region 633 Raman shift region 532 Raman shift region Gratings are optimized for Throughput Grating for 785 nm low Resolution

37. Company Confidential Almega Introduction Grating Summary v Gratings are chosen for resolution w pitch (lines/mm) goes up => resolution gets better v Gratings also must be chosen for wavelength throughput v Cannot simply rely on higher and higher grating pitch to get better and better resolution

38. Company Confidential Almega Introduction Charge Coupled Device (CCD) v Silicon based detector composed of an array of light sensitive elements called pixels. v Each pixel is on the order of 25  m v Arrays of 256 x 1024 pixels v CCD offers exceptional sensitivity w Several orders of magnitude more sensitive than Ge v CCD has large spectral range

39. Company Confidential Almega Introduction CCD Range Why not use them everywhere? v CCD Range => 400 nm - 1000 nm –Can be extended to 1050 nm by proper selection of device –Can also be extended to less than 400 nm by selection of device or additional manufacturing steps w Using 1064 nm lasers »100  cm-1 to 3000  cm-1 = 1075 nm to 1563 nm w Using 785 nm lasers »3000  cm-1 is equivalent to 1027 nm, very close to cut-off

40. Company Confidential Almega Introduction How do they Work? Exposure, Shutter open Registry Row Covered so no photons reach these pixels

41. Company Confidential Almega Introduction Close Shutter and Read out Registry Row Registry Row Read out charge in each pixel across

42. Company Confidential Almega Introduction Close Shutter and Read-out detector Transfer Charge down one row

43. Company Confidential Almega Introduction And the read out moves down the rows until the entire array is read Registry Row Read out charge in each pixel across Now it is ready for another exposure

44. Company Confidential Almega Introduction Binning v Bin-On-Chip w Combine multiple rows before read-out w Single read cycles w Image data (individual rows) is not preserved v Bin-In-Memory w Read each row w Combine row data in computer memory w Maintains individual row data v Binning mode chosen in OMNIC

45. Company Confidential Almega Introduction Why Bother Binning? In this example, only 3 rows are illuminated. By summing those three only, get increased signal and avoid background from other dark pixels Wavenumber scale (  cm-1) Small  cm-1 Large  cm-1

46. Company Confidential Almega Introduction Bin-on-Chip vs. Bin-in-Memory v Bin-In-Memory w Saves information from each row w Allows software manipulation of data (cosmic ray detection, etc.) w Should be used except when signal is very low (<300 electrons) w Get multiple read operations; more read noise v Bin-On-Chip w Used mainly when signal is very low w Lowest noise; avoids additional read noise w Individual row information is lost

47. Company Confidential Almega Introduction Three CCD Types All devices have pixels fabricated on front surface with gate material on top (electronic metal connections) v Front Illuminated (FI) w Photons strike the front surface and must go through the gating material »Smaller wavelength range »Lower QE in portions of region of interest

48. Company Confidential Almega Introduction v Back Illuminated (BI) w Back is etched (thinned) and illumination is from back side w Higher QE, no gate material to pass through w Higher noise (5x to 20x more than OE or FI) w More defects because more fabrication steps w Fringing v Open Electrode (OE) w Same as FI, but part of gating material is remove on each pixel w Better QE over entire range w lower noise w no fringing

49. Company Confidential Almega Introduction Quantum Efficiency Curves

50. Company Confidential Almega Introduction Quantum Efficiency doesn’t tell ALL Must think about noise v Read Noise: Noise introduced with each read operation v Dark Current:Baseline noise that will occur at each pixel even in the absence of light. Minimized by cooling detector v Dark Current Shot Noise:Random statistical noise associated with dark current, proportional to dark current intensity v Signal Shot Noise: Random statistical noise proportional to the signal at the pixel during exposure. Major source of noise; much greater than read noise. Minimized by taking multiple, shorter exposures For strong signals (>50 counts): Read noise is insignificant For weak signals (<50 counts): Read noise becomes significant

51. Company Confidential Almega Introduction White Light Correction v Every pixel is not the same v Measure the response of a calibrated white light source to correct for instrument response

52. Company Confidential Almega Introduction Dispersive Raman Microscopy v Spatial resolution dependant on laser spot size and aperture size. w FT-IR microscopy »Beam size is larger than area measured »Targeting aperture limits IR beam size w Raman microscopy »Laser is highly focused »Spot size is dependant on objective magnification Objectives785 nm laser633 nm laser 532 nm laser 20 x10 um8.6 um7 um 50 x4 um3.4 um2.9 um 100 x2 um1.7 um1.4um

53. Company Confidential Almega Introduction Dependence on Aperture v FT-IR microscopy w Redundant aperture limits the stray radiation coming from the sample. v Raman microscopy w Spectrograph entrance aperture limits the stray radiation coming from the sample The big difference is the size of the aperture and the diffraction limit of the radiation being measured

54. Company Confidential Almega Introduction Spatial Resolution Affected by Apertures Objective Confocal Aperture Sample Detector

55. Company Confidential Almega Introduction Microscope Sampling Stage v Automated sampling stage w Mapping is still advocated as the best way to collect wide fields of data w Due to the increased spatial resolution, stage must have smaller steps and better repeatability. »Typically 0.1 um step size, 1 um repeatability v All dispersive Raman microscopes offer automated z-axis and autofocus

56. Company Confidential Almega Introduction Confocal Microscopy Objective Confocal Aperture Sample Detector Confocality = The ability to selectively filter light rays from different depths in the sample

57. Company Confidential Almega Introduction Confocal Microscopy v Pin hole apertures control the spatial resolution v Higher magnification objectives give higher spatial resolution because of tighter focus v Typical aperture sizes: 25 or 10 um v Typical depth resolution limit: 2 um

58. Company Confidential Almega Introduction Depth Resolution Illustrated 2 um Note: The same aperture is also used to enhance spatial resolution

59. Company Confidential Almega Introduction Laser Safety v All lasers for dispersive Raman are Class III or worse. v Design prevents laser from being dangerous without interlocks, but still must comply with laser safety v Class III laser compliance requires w Isolated room w Safety goggles, skin coverings w Laser power interlocks triggered by room door w Safety warning signs and lights outside room w and more….. v If the instrument is not Class I or Ia, then user must do all of this !!!!

60. Company Confidential Almega Introduction View Sample During Data Collect and Remain Laser Safe v Remove eyepieces and use video only w Many microscopists will complain, since the visual image through eyepieces is better than most video images. v Use dichroic filter w Only works in IR and NIR (down to 785 nm) w At shorter visible wavelengths, the dichroic filter blocks the visual image

61. Company Confidential Almega Introduction Fiber Optics v All Dispersive Raman systems offer fiber optics, several use them exclusively for sampling or to connect components v Fiber optic advantages for Raman w Cheap fibers, can be very long w Remote sampling, large or hazardous samples w Process v Special requirements w Low OH silica fibers w Filters at the tip w Specific for each laser wavelength w Universal connectors (FC or SMA)

62. Company Confidential Almega Introduction Almega Fiber probes

63. Company Confidential Almega Introduction Dispersive Applications v Silicon v DLC v Micro-analysis w Below 500 um v Catalysts v Pharmaceuticals v Polymers v Dark Samples v Aqueous Samples v Geology v Gemology v Biological

64. Company Confidential Almega Introduction FT-Raman Applications v Pharmaceuticals w Polymorphs w Combinatorial w Product ID v Incoming materials analysis v Forensics v Polymers v Pulp/paper v Petrochemicals