Company Confidential Almega Introduction Almega Visible Raman Microscope Nicolet Instrument Corporation 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) 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% Company Confidential Almega Introduction Dispersive vs. FT-Raman Source: SDI Global Assessment Report (1997) v Breakdown by technique (New Sales) w % Dispersive Raman w % 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 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 % Note: Unit numbers are estimations based on market data Company Confidential Almega Introduction Market Share - Dispersive Raman 1998 RegionEst. UnitsPercentage N. America5032 Europe3422 Japan2516 Pacific Rim1711 Latin America128 R.O.W1711 Total % Note: Unit numbers are estimations based on market data 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. Company Confidential Almega Introduction Almega System Company Confidential Almega Introduction Truly Integrated and Versatile!!! Company Confidential Almega Introduction Macro Sampling and Accessories 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 Company Confidential Almega Introduction Vibrational Spectroscopy Virtual States Electronic level 1 Electronic level 0 IR absorption & emission Rayleigh Vibrational levels Stokes Raman 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) 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 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 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 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 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 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 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 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 Company Confidential Almega Introduction Detector Objective Sample Adjust z-axis with scope focus knob The Confocal Analysis...More on Confocal later 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) Company Confidential Almega Introduction Raman Shift Spectrum 3000 cm cm -1 0 cm cm cm -1 Stokes Anti-Stokes Wavelength (nm) In Raman, 3000 cm -1 is longer absolute wavelength than 100 cm -1 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 Company Confidential Almega Introduction 1064 nm 532 nm Electronic level 1 Electronic level nm785 nm 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) Company Confidential Almega Introduction Laser Power More is not necessarily better!! Laser WavelengthTypical Applied Power (mW) 1064 nm nm nm and 514 nm 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 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 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 Company Confidential Almega Introduction Spectrograph - The heart of the System Torroidal Mirrors Grating Aperture Shutter Detector 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 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 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 Company Confidential Almega Introduction The wavelength dependence can be visualized High cm -1 Low cm -1 Grating Spectral Resolutions increases 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 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 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 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 Company Confidential Almega Introduction CCD Range Why not use them everywhere? v CCD Range => 400 nm 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 Company Confidential Almega Introduction How do they Work? Exposure, Shutter open Registry Row Covered so no photons reach these pixels Company Confidential Almega Introduction Close Shutter and Read out Registry Row Registry Row Read out charge in each pixel across Company Confidential Almega Introduction Close Shutter and Read-out detector Transfer Charge down one row 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 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 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 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 (50 counts): Read noise is insignificant For weak signals (