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TM001 - Introduction to Raman spectroscopy

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1. s e 5 moa v 94 ri aN SUDAN we dr OY The Navigator Navigator a Single scan measurement 7 a E3 Single scan measurement 8 Single scan measurement 9 E A Measurement 7 a Viewers A Single scan measurement 10 H A Measurement i o 58 Viewers 3 8 Single scan measurement 11 H A Measurement e Enables control of what data is open and where it is viewed e Windows Measurement Viewers and data housed together different viewers for different types of data e Measurement enables identical or modified conditions to be used for new data collection TMO002 02 A Introduction to WIRE and System start up Notification area Q e c c memet emm Amt Pese Phe Tec ee D SE2ZOe aN CHOAGECROREO LASS Eel teI EnEV K ok ie Meg URBE 3 ijn T 4 5 H gt r 0 pi HIHI 1 1 i i verne seos E gt Notification area 4 X 35143 0 Y 3762 0 25 9 x 2442 74 y 51223 86 50T411 HA o e Progress bar indicating data acquisition progress File signing when using 21 CFR pt 11 e Sample location within video XYZ values e XY spectrum co ordinates or XYi Raman image values e Laser interlock status Complete system start up This procedure assumes all electrical components relating to the use of the inVia Raman microscope are switched off initially and that the user has suitable knowledge of Renishaw s WIRE 4 software 1
2. Gain Speed Low 1 Scan type is used when Extended scan is selected in the Range tab to select the type of extended scan to use SynchroScan is recommended for most applications as it does not contain the artefacts present in stitched scans The Step option is included for samples that are very strong Raman scatters and might saturate the detector if the SynchroScan option which requires a minimum 10 second exposure is used TM004 02 A Measurement set up and data acquisition amp 1D apply innovation Camera Gain switches between the sensitivity settings of the detector and should usually be set to high Camera Speed should be set to low Pinhole allows the user to set whether the pinhole is n or Out The pinhole may improve the beam profile The pinhole can be used to convert a line laser into a spot laser This function only operates on systems with a motorised pinhole Its primary uses are in those instruments with True Raman imaging and where the automated alignment functions are set up Binning allows the co addition of adjacent signal from pixels on the detector to improve the signal to noise ratio in extended scans only However excessive binning reduces the spectral resolution Use values of 2 or 3 unless the Raman bands are naturally broad when larger values can be used The default value is 1 Laser focus controls the use of the beam expander 0 indicates the laser is tightly focussed while 100 9o indicates it
3. the baseline i x 50000 40000 Counts 30000 20000 200 300 400 500 600 700 800 900 1000 1100 1200 1300 Raman shift cm 1 25000 20000 Counts 15000 10000 5000 200 300 400 500 600 700 800 900 1000 1100 1200 1300 Raman shift cm 1 TM012 02 A Data processing and simple analysis apply innovation Intelligent fitting can be applied to single spectra and multifiles e g mapping dataset The baseline will automatically fit each spectrum within the multifile Through fixed points Selecting Through fixed points as the fitting mode enables the user to manually specify points in XY to determine the baseline shape The user can choose between polynomial and the order and cubic spline options Cubic spline is only available if 2 points are added 4 total points This method can be applied to single spectra or multifiles but the baseline is fixed and will not fit to different spectra within multifiles It is useful to zoom in by left clicking and dragging in either window and adjusting the points added in the top window by moving them with the mouse Subtract baseline 28000 27000 26000 25000 Counts 24000 23000 22000 21000 20000 Frequency Raman shift cm 1 5000 4000 Counts 3000 2000 1000 Raman shift cm 1 TMO012 02 A Data processing and simple analysis apply innovation Through chosen point
4. 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 nan Figure 10 Map Review LUT Control 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 R Set crosshair position Zoom out Edit images Save to Crosshairs Axes wv Scalebar Copy view Tools Add Remove Docking Properties After collection v None ww RENISH AWS apply innovation Renishaw plc Tel 44 0 1453 524524 RE N IS H AW i Spectroscopy Products Division Fax 44 0 1453 523901 el Old Town Wotton under Edge Email ishaw R ue Goes GP TD i P mamanenieawcom apply innovation United Kingdom www renishaw com TM007 White light image capture montaging and WiRE 4 0 Surface generation This document aims to show the WIRE 4 user how to collect and save white light images and montages from the microscope camera and set up the Surface option This training module covers the collection of 1 Single images Capture of optical video images 2 Manual montages Capture of multiple manual video images to quickly and easily define mapping regions over very large areas 3 Automated montages Capture of multiple automated video images to easily define mapping regions over a variety of areas spatially connected to the white light view of the sample 4 Surfaces Capture of multiple manual video images with XYZ positions used to define a 3D sample surface for subsequent image or data capture 5 Surface and montage Capture of multi
5. 1 Turn on the appropriate laser s 2 Run WIRE 4 there will be no prompt for motor referencing as the current state of the motors will be recognised by the software and referencing is not necessary 3 Wait for the required time period for the optimum laser stability to be reached Having powered up all the system components and waited for stability to be reached the system is now ready to be configured Renishaw plc Tel 44 0 1453 524524 RE N IS H AW Spectroscopy Products Division Fax 44 0 1453 523901 Old Town Wotton under Edge Email raman renishaw com snan Gloucestershire GL12 7DW apply innovation United Kingdom www renishaw com TMO003 Sample viewing and configuration change WiRE 4 0 This module details recommended procedures for 1 Viewing different types of samples using the Renishaw video 2 Selecting different laser grating CCD camera configurations within the inVia Raman microscope Suitable knowledge of the WiRE 4 software is assumed in this document Sample viewing inVia and inVia Reflex Raman microscopes typically consist of direct microscope sample viewing using eye pieces and or a microscope video camera The Sample review is opened from the View menu or short cut button c The Sample review contains aser shutter control within inVia Objective selection not motorised Laser power control for viewing Laser defocusing control for viewing e Selection control of Laser e
6. Turn on the system using the main on off power button situated to the right hand side of the instrument the CCD camera will take 20 minutes to cool to its operating temperature 2 urn on the desired laser s and ensure all keys and switches are correctly set please refer to the laser user manual for individual laser start up procedures TMO002 02 A Introduction to WIRE and System start up 3 The laser interlocks will not be activated until the WiRE 4 software has been opened From point of lasing each laser requires at least 30 minutes to reach optimal pointing and power stability 4 Turn on the PC and run the WiRE 4 programme 5 The software will prompt for a position check of the relevant motors WiRE Motor Reference Options FER a B Sisters Beam Expander parallel un referenced Beampath Mirror 1 parallel un referenced Reference All Motors Beampath Mirror 2 parallel un referenced Grating Motor parallel un referenced Reference Selected Motors Holographic Notch Filter parallel un referenced rerum qui acp rtg E Linefocus Motor serial un reterenced Unreferenced serial motors will still be Podule Lower Selector Wheel serial un referenced referenced Podule Upper Selector Wheel serial un referenced Slit Master serial un referenced l Ignore Referencing Parallel Motors Slit Slave serial un referenced If referencing of Parallel motors is skipped they will be assigned their last known positions Serial motors will always be
7. height a greater number of points need to be added 300 200 100 100 200 300 300 200 100 0 100 200 300 Figure 4 Image view of Surface with points red In addition to the simple image view control the image view can be changed in the following way from the right click menu Show points View options Show data points Show region available for defining the map area Properties Image tab Surface edge Clear images Select points Remove points Double click on the image to move the sample to the selected surface XYZ point when the cross hairs are active TMO007 02 A White light capture montaging and Surface generation amp jl apply innovation Z axis values o um Figure 5 Surface view of Surface rainbow LUT The Surface view can be changed in the following way from the right click menu Reset view LUT colour and contrast View options Show LUT Top down view View options Top down Interpolation options for points beyond the defined Surface Constant default or Linear Double click on the Surface to move the sample to the selected surface XYZ point when the cross hairs are active Using the Surface to collect mapping data The minimum spectral acquisition time which can be used with Surface to ensure correct sample focus is dependent on the rate of XY motion relative to the rate of Z motion The rate of XY motion is dependent on the spectral acquisiti
8. scans between the upper and lower limits entered in Spectrum range and is used when a static scan will not cover the required wavenumber range 2 Configuration allows the user to select the laser grating and detector to be used 3 The Confocality box allows the user to choose between high and standard confocal performance The confocality defines the sample volume that signal is collected from Using the High confocality option reduces this volume increasing the spatial resolution but also reducing the total Raman signal Note the instrument is always confocal due to the optical layout High confocal mode is not available in line focus or StreamLine imaging configurations Acquisition The Acquisition tab allows the user to alter scan conditions such as the exposure time and laser power to be used 2 TMO004 02 A Measurement set up and data acquisition REN ISHAW O gt apply innovation Vap vereren cene TT n Exposure time s 1 00 Objective 100 Cosmic ray removal Accumulations 1 Laser power 100 Response calibration T Restore instrument Live im al ME imaging state on completion Close laser shutter on completion Not using live imaging Jit LU Minimize laser exposure on sample Title Simple mapping measurement 4 Description This is a mapping measurement created by the map setup wizard cox e Gate Gate Figure 4 Acquisition tab 1 Exposure time is the time the
9. define mapping regions over a variety of areas with a complete white light montage When performing imaging experiments over large areas it is often desirable to be able to compare the white light image of the sample area imaged with the Raman data Where the Raman image is greater in size than the field of view of the white light image a montage of these images can be created It is also easier to define the image area from the montage 1 Focus on the sample with the objective to be used for the montaging Note this can be different to the objective to be used for collecting the Raman data but the sample should be flat for both if the image and collected Raman date are to be in focus 2 Zero the co ordinates using the Set origin button SB 3 Setthe correct objective in the Sample Review this is reflected in the scale bar of the Video viewer TM007 02 A White light capture montaging and Surface generation REN ISHAW A apply innovation 4 Ensure the video displays the desired brightness and contrast to enable a uniform joining of the images this will be dependent on sample type A more seamless montage is often collected by opening the aperture stop and or field stop of the microscope This is mounted on the Leica microscope for non Reflex models and is accessed in the Sample Review tool on Reflex models Aperture stop Objective E ma bd s x50 magnification Field stop 5 Select Live Video Snap
10. for the tiny fraction that has been Raman scattered often holographic notch or dielectric edge filters e A device such as a diffraction grating for splitting the Raman scattered light into component wavelengths i e a spectrum Alight sensitive device for detecting this light normally a CCD camera e A computer to control the instrument and the motors and analyse and store the data Figure 5 shows the layout of Renishaw s inVia Reflex Raman microscope with all the key components highlighted Auto excitation Auto Research grade Leica wavelength confocality microscope switching control Ultra high precision multiple grating stage Auto view Raman changeover Auto calibration Auto performance verification UV capability Raman imaging capability Near excitation filter capability Wavelength optimized Class 1 laser safe laser beam control enclosure Auto alignment Flexible sample optimization handling Safety interlock control Fig 5 Renishaw s inVia Reflex Raman microscope TM001 02 A Introduction to Raman spectroscopy e amp ID apply innovation Renishaw plc Tel 44 0 1453 524524 RE N ISH AW e Spectroscopy Products Division Fax 44 0 1453 523901 eL Old Town Wotton under Edge Email raman renishaw com ema Gloucestershire GL12 7DW apply innovation United Kingdom www renishaw com TMO002 Introduction to WIRE and System start up WIRE 4 0 The aim of th
11. is completely defocused by the beam expander Defocusing reduces the power density at the sample and so can reduce sample damage in sensitive samples but reduces spectral resolution Values greater than 096 are used for True Raman imaging measurements Input polarisation is used in instruments with polarising filters to select the polarisation of the laser beam The different Raman scattering response of a sample to different laser polarisation can be useful in assigning the symmetry of the vibrational modes involved Image capture allows the user to specify the capture of a white light image of the sample before or after or both for time temperature or mapping measurements by using the Mode drop down menu Delay sets the time the camera is allowed to adjust its settings to the conditions so that a good image is obtained The default time is 2 5 seconds The image capture feature applies to both inVia and inVia Reflex models On the former the user is prompted to switch the optics such that an image can be collected then back again such that a spectrum can be acquired When reviewing one dimensional datasets time and temperature series the acquired video images are displayed in the top right frame of the Map Review window If images were acquired before and or after spectral acquisition these images may be toggled selected from the right click context menu Show video image TMO004 02 A Measurement set up and data acquisition
12. selection Operation type Arithmetic v Auxiliary data bl removed wdf Acquisition 1 Result 3 Data z 1 Auxiliary i not il reaions absen UL I T CUIUI IS au Fill with value 0 ok J ce J It can be useful to multiply either the sample file or the auxiliary file by a factor so that the Y axes are comparable In the example below 1 the sample file has been used and 2 the background correction file used to remove the baseline Accept the change either from the context menu or by closing the window Data arithmetic 90000 bs Ao jh i Counts 60000 50000 ADIT RDM ce neo 40000 Verus VW RM 30000 0 700T00700T0000T0000T1T707077 200 300 400 500 600 700 800 af Data arithmetic Properties Raman shift cm 1 Arithmetic operation selection Operation type Arithmetic 30000 r 4l Auxiliary data bl wdf Acquisition1 idi Result 1 Data umm 2 Auxiliary ounts E 25000 4 o 4 Ez Filling Fill with value 0 Freque 20000 n C ones 15000 I L I LC T C 7 B M M PLT E T mme r 200 300 400 500 600 700 800 900 1000 1100 1200 1300 Frequency Raman shift cm 1 25000 20000 15000 A a N y 200 300 400 500 600 700 800 900 1000 1100 1200 1300 Frequency Counts e e o Frequency Raman shift cm 1 TMO012 02 A Data processin
13. spectra of diamond and polystyrene The bottom axis of the graph represents the energy of the Raman shift measured in cm and may be plotted right to left or vice versa A value of 0 cm would indicate that no energy has been exchanged with the sample and the incident light is scattered with no change in wavenumber Carbon hydrogen bonds give rise to Raman bands around 3000 cm due to the small mass of hydrogen and resulting high frequency vibrations Peaks at lower wavenumber relate to lower energy vibrations such as those of bonds to carbon or oxygen TMO001 02 A Introduction to Raman spectroscopy RENISHAW apply innovation What information can you get from Raman spectroscopy Raman bands can be analysed to obtain chemical and structural information for the material identification investigation of material properties and spatial analysis The table below illustrates the variety of results that can be obtained from point and mapping measurements __ Band parameter Information O O OO Identification material composition Characteristic Raman frequencies Compare characteristic Raman frequencies Intensity variation with changing polarisation Variation in absolute relative intensity Variation in Univariate Raman band width Variation in Raman band position Any of the above parameters applied to multiple spectra e Univariate based on raw data or curve fitting e Multivariate algor
14. to analyse your samples under high pressure Q9 want to be able to perform polarisation measurements Is this possible A polariser and half wave plate set for each wavelength may be purchased from Renishaw These enable you to examine the molecular symmetry of your sample and assist in assigning bands to vibrations within the molecule Motorised laser polarisation control is also available Q10 l ve noticed that placing my sample in different orientations gives a different spectrum Why is this This is caused by the laser being incident on different crystal planes within the sample Using a quarter wave plate can help to remove these orientation effects by scrambling circularly polarising the Raman laser light This method is often of use to confirm relative intensity Raman information is not sample orientation induced e g for highly ordered systems such as polymers or single crystals
15. 2250 9 8 78093 672090 17786 9 667 369 716 036 x 8 734 483 7926 38 8 80111 536554 131449 716 861 761 404 a9 929 515 6242 84 9 71824 429559 103378 908 229 951 947 Note that it may be necessary to maximise the window containing the active spectrum in order to see the peak results table window depending on where it is currently docked TMO012 02 A Data processing and simple analysis ij apply innovation Peak Pick detects peaks for the active spectrum of the active spectrum viewer using the current threshold settings and displays the results It also adds a peak results table to the current window which gives details for the picked peaks which can include some or all of the following information e Centre e Height e Width e Area e Absolute intensity e Low edge e High edge Peaks are automatically labelled on selection of the Peak pick option from the Analysis menu Analysis Processing Procedures Tools Window Help Mapping review Curve fit Integrate v Peak pick v Visible Autoset thresholds 4 Single peak Remove peak label Whole spectrum Copy results Show hide columns Information viewer Spectrum search If the peaks are not suitably labelled the following methods can be used to add or remove the labels 1 Use the Autoset thresholds gt Whole spectrum option This sets thresholds so that a limited number of the best defined peaks will be found and then performs peak picking The maximum number of peaks ca
16. 40000 Counts 30000 20000 da li jw A SN ad cJ 200 300 400 500 600 700 800 Raman shift cm 1 25000 20000 15000 Counts 10000 5000 200 300 400 500 600 700 800 Raman shift cm 1 pie T pd o M J Lu M Aa a du Ure JC AMA Ma Jia U i 1000 1100 1200 1300 1000 1100 1200 1300 Accepting a correction When you are satisfied with the correction either select Accept from the context menu or close the window upon which there will be a prompt asking if you want to keep the correction To save the change to your file use the File Save or Save as option from WiRE TM012 02 A Data processing and simple analysis ij apply innovation Arithmetic functions on data A variety of mathematical operations can be performed on single data files For example you can add files together or subtract one from another It can be an effective method of subtracting a background spectrum or filter ripple profile With a file open select Processing Spectral Arithmetic A new viewer will open split into three separate areas The upper displays the sample spectrum the middle will show the auxiliary data i e the data file you would like to add subtract multiply by etc and the lower region will show the result spectrum Use the Spectral Arithmetic Properties window to browse for the auxiliary data and to select the arithmetic function Data arithmetic Properties Arithmetic operation
17. C ai li o D B e B B e meee inc B amp e B a w va v E romans v NO 219 2090 UD we dr O x C TAE f C 100 B201 635 780 c Hi C v 3X aE Master Renishaw CoD Camera 2 p amp 785nm toptica Y Master Renishaw CCD Camera z e Controls the view of the sample on the video white light and or laser e Aids focussing onto the sample e Opens closes the instrument shutter for laser entry into the instrument e Controls the laser grating detector configuration used for new measurements TMO002 02 A Introduction to WIRE and System start up Video Video e e ie see ee tee o t sa D SEZOee aN CHUVABEDOKRG ASR amp V694230C W E IUE M g 1 annm v w v d e Mn dee eee ee ee ee ee ee ee ee ee ee dl we oo T toeo va t boss moa spaa 239 aN UD we dr OY Video Crosshairs Axes Scalebar Video properties Tools Add Remove 3750 3700 15250 35200 35150 35100 35050 e Turn on off and change the type of crosshair used e View the axes e View the scalebat e Change the properties fo the video display including brightness contrast and exposure time TMO002 02 A Introduction to WIRE and System start up Spectrum viewer Window with spectrum viewer Q tm t c memento Aet Pee Pm Tec ee a m n b bEZz65ham GUCAGWCUOCE
18. ROC wEZS MARI teri Ene K eS Megs c Same i a 0 5M we d e u VA v f v 4 N p v 4 VY va V i wee e 4 I 9 s eee ee ee ee ee ee ee ee ee er ee eee ee ee toes E moa v GA 239 aN SUDAN we dr OY 100000 90000 80000 70000 40000 20000 10000 500 1000 1500 2000 2500 3000 Raman shift cm 1 e View and control the spectrum or spectra e Control the view add labels e View processing and analysis operations TMO002 02 A Introduction to WiRE and System start up Map review Map Review x Pr a White light image n S a 2v Counts L Visible Component ref 4 wxd DCLS Visible BE Component ref 5 wxd DCLS Visible Lack Of Fit ref 5 wxd L Visible Component 532 mix 2 off pigment ref wxd DCLS Visible 18000 16000 14000 12000 10000 8000 6000 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 Raman shift cm 1 e View the individual or combined white light image and Raman images e View spectra from different image locations e Review how spectra have been analysed in conjunction with the Raman image e Access the look up table to control image colour contrast and brightness TMO002 02 A Introduction to WIRE and System start up Navigator BT Q tm t memet emm Aet Pee Pm Tec y 2m n D DELONAan SGaserouic wLE X Sher teI S50 aG b ei x Mea Semen i 7H ve rw 0d
19. Selection control of grating Where configured within inVia additional controls for laser polarisation for viewing and CCD detector selection may be available inVia shutter Objective Laser power Grating Sample Review Laser defocus Laser Figure 1 inVia sample review In addition to these the inVia Reflex Sample review contains e Viewing control of eye piece and video white light only e Viewing control of video only white light and laser e Viewing control of internal reference laser focus e Illumination brightness on off and intensity e Aperture stop control e Field stop control For inVia these options are all configured manually using the microscope _4 TM003 02 A Sample viewing and configuration change REN ISHAW e gt apply innovation Sam Review Video only laser Internal reference Aperture stop and white light video only A stop E i d vid Eye place and video _ y trae 2e e 2E I prorrgrgrgrggp gg prr grrrrrrgag a prrrggcgrggprag Illumination Field stop intensity F stop Illumination gt a on off Figure 2 inVia Reflex sample review Whether used manually or through the Sample review the illumination control A stop F stop and camera control are used together to aid focussing and sample viewing and generate high quality white light images of the sample Focussing the sample is aided by F stop white light focus and laser focus Ty
20. TMO012 02 A Data processing and simple analysis ij apply innovation Select Start Fit from the context menu to fit the added curves to the data The algorithm will perform many iterations until the best fit has been achieved You can save the curve fit file as a wxc from the context menu Curve parameters Save curves To reapply this saved curve template perhaps to a similar sample start the curve fit application and use the context menu Curve parameters Load curves and then Start fit You can modify or make changes to the curve fit using the Curve Fit Properties window from the context menu Properties This provides greater control over the fitting process instead of the automatic parameters that are usually used For example you can choose to fix a band centre instead of letting it float during the curve fit or apply limits to parameters This can be useful for complex band shapes Curves can also be named different types of baseline can be used or the curve type can be defined Use the Curve Fit Curves and Baseline tabs to adjust the curve fit Curve fit Properties Curve Fit Curves Baseline Curve 1of6 Curve Name Band at 1636 Curve Type Mixed Float Use Limits Lower Limit Upper Limit Centre 1636 1401 Width 14 883852 Height 117488 51 9 Gaussian 49 376508 The context menu allows the truncation of the fitted region on zooming Fit viewed region It is ge
21. V lasers are sometimes used with no beam expander e Internal laser shutter autochange all e Internal silicon reference re focus Reflex only The flexibility and upgradability of the Renishaw inVia microscope is such that the degree of automation desired can be gained from any previous configuration Manual partial automation full automation and full auto validation options are available For inVia instruments without motorised Rayleigh rejection filter change the user must open the instrument door see instructions below for safe operation of the instrument door and laser interlock and manually swap the Rayleigh filter for the new wavelength Some instruments with motorised filter change may require manual filter change if all four positions on the mount are occupied and the new laser s filter is not currently fitted When the Grating is changed the relevant software changes are implemented but no immediate mechanical change takes place The grating used will affect the spectral range and the spectral resolution The grating change procedure is identical for all inVia Raman microscope models For instruments with more than two gratings the user may have to manually remove and replace a grating to obtain the required configuration Gratings may be mounted back to back and care should be taken in separating pairs of gratings Grating mounts are designed such that any one grating can only be mounted in one of the two positions set durin
22. Wire 4 Training Modules Compilation The following modules are in this compilation TMOO1 TMO002 TMO003 TM004 TM007 TM012 TM026 Introduction to Raman Spectroscopy Introduction to WiRe and System start up Sample viewing and configuration change Data acquisition and measurement set up White light image capture montaging and Surface Data processing and simple analysis FAQ s The modules above as individual documents and other more specialized modules and training videos are available to users with a Cornell NetID in a folder at https cornell box com s gxy4pkeo2od1bdukdx3d The modules not included in the compilation and the training videos are Videos in mp4 format TM007 MO 1 TM007 MO 2 TM007 MO 3 TM007 MO 4 TM011 MO TM012 MO TM013 MO TM013 MO TM017 MO TM018 MO TMO18 MO An introduction to surface Defining a surface Applying a surface to white light images and montages Applying a surface to map data collection 3D viewer Baseline subtraction Cosmic ray removal Noise filtering Image domain analysis Importing and saving Raman data Viewing and controlling Raman images The training modules not included in the compilation but available individually are TMO005 TM006 TM008 TMO009 TM010 TMO11 TMO013 TM014 TMO015 TM016 TMO017 TM018 TMO019 TMO021 Depth profiling FocusTrack Point Imaging StreamLineHR imaging StreamLine imaging 3D imaging Multi file processing Multi file d
23. ad hepar 1000 T T T T 500 1000 1500 2000 2500 3000 500 1000 1500 2000 2500 3000 wavenumber shift cm 1 wavenumber shift cm 1 Fig 1 Fluorescent room lights left monitor phosphorus lines right Fluorescent lights result in spectral contamination from mercury emission lines simply turn off all fluorescent lighting in the room and work under the minimum incandescent light The room should be as dark as practicable Similarly a thorough effort must be made to exclude sunlight from the room since spectral aberrations will results from the numerous emission lines of white light Phosphorous lines are often present due to the phosphour coating in all CRT monitors if such lines are a problem turn the monitor off or reduce the contrast until the screen is darker It is important to remember that emission lines are always present at the same position on an absolute wavenumber scale and will therefore be seen to move on a scale of Raman shift when using different laser wavelength These lines are more prominent when using collection optics of longer working distance TM26 02 A Frequently asked questions FAQ RE N ISHAW e apply innovation Q4 Why do some of my spectra give such an intense background signal that masks the Raman information A high background in a Raman spectrum is the result of sample fluorescence or phosphorescence an intrinsic property of the material of the sample Unfortunat
24. and depth slices e StreamLine image acquisition high speed spectral collection at varying lateral sample positions with a minimised laser power density e StreamLineHR high speed spectral collection at varying lateral sample positions e StreamLineHR 3D acquisition When the appropriate measurement has been selected the set up of that measurement will be automatically displayed This module details the standard set up tabs consistently used throughout the different measurement types These tabs are Range Acquisition File and Advanced TMO004 02 A Measurement set up and data acquisition REN ISHAW O gt apply innovation Range The Range tab covers the basic settings for the scan such as the laser and grating to be used and the type of scan to be performed Grating scan type Static Extended High Confocality Configuration Standard Laser name 514 nm edge a High Grating name 2400 l mm vis M Detector name Master Renishaw CCD Camera Calibration status f Calibrated OK o j cancel Figure 3 Range tab 1 Grating scan type gives the option for two types of scan e Static covers a range of about 200 cm to 500 cm either side of the centre depending on the wavelength and the grating used The desired centre can be entered in the Spectrum range box A static scan is quicker to perform than an extended scan but only covers a limited range e Extended SynchroScan
25. as it degrades the spectral resolution Smoothing is no substitute for performing a better measurement i e using longer acquisition times or more accumulations When using SynchroScan the binning function can be used again with caution to gain a better signal to noise ratio To perform smoothing with the file you wish to smooth open select Processing Smooth A new window will open with the sample spectrum at the top and the result spectrum below This data will be smoothed To increase or change the degree of smoothing select Properties from the context menu to see the Smooth Properties window Smooth Data Properties Savitsky Golay parameters Smooth window Polynomial order 2 The application uses a Savitsky Golay algorithm Use the Smooth Window and Polynomial Order functions to change the degree of smoothing Pressing Apply performs the change and OK completes the operation You can use the zoom function to see more closely the effect of the smoothing You will be asked if you want to accept the resulting smoothed spectrum Zap Stray bands can be removed from the spectrum using the Zap function Generally these will be cosmic ray features or other spurious lines Ideally the measurement would be re performed but you may decide that zapping is acceptable To remove a band on an open spectrum select Processing Zap A new viewer will open with the sample spectrum at the top and the result
26. ata analysis univariate Multi file data analysis multivariate Database searching and creation Image domain analysis Importing viewing and saving spectral and image data Labelling and printing Polarizer analyser accessory Renishaw plc Tel 44 0 1453 524524 RE N ISH AW d Spectroscopy Products Division Fax 44 0 1453 523901 e L Old Town Wotton under Edge Email raman renishaw com nan Gloucestershire GL12 7DW apply innovation United Kingdom www renishaw com TM001 Introduction to Raman spectroscopy WIRE 4 0 What is Raman scattering Raman scattering is named after the Indian scientist C V Raman who discovered the effect in 1928 If light of a single colour wavelength is shone on a material most scatters off with no change in the colour of the light Rayleigh scattered light However a tiny fraction of the light normally about 1 part in 10 million is scattered with a slightly different colour Raman scattered This light changes colour because it exchanges energy with vibrations in the material This makes Raman scattering an excellent tool for probing vibrations in materials The aim of Raman spectroscopy is to analyse the Raman scattered light and infer from it as much as possible about the chemistry and structure of the material More on Raman scattering Scattering occurs when an electromagnetic wave encounters a molecule or passes through a lattice When light encounters a molecule the vast majority of pho
27. captured Right click in the Still Image viewer to add remove crosshairs axes and scale bar Also right click to save the image as a bmp or jpg 50 40 20 30 60 50 40 30 20 10 0 10 20 30 40 50 60 70 Figure 1 Example single white light image 50x objective 2 Multiple manual white light image capture XY montaging Quickly and easily define mapping regions over very large areas Repeat the process described in 1 moving the sample on the motorised stage in XY to produce an area partially filled with white light images The sample can be either moved freely using the track ball or in a more grid like manner using the XYZ stage control typing spatial co ordinates to move the sample known distances one axis at a time As many or as few images as desired can be added by the user This process is useful where a large area is required to define the mapping area without the need to collect a large number of images over the entire area e g a large area on a sample with no meaningful white light viewable features The resulting montage will have black regions where no image capture has occurred Figure 2 TMO007 02 A White light capture montaging and Surface generation amp ID apply innovation 300 200 100 100 200 300 300 200 100 0 100 200 300 Figure 2 Example manual white light montage multiple 50x objective images 3 Multiple automated white light image capture XY montaging Easily
28. ccompanying Raman scattering left Rayleigh scattering right anti Stokes lines Stokes lines y n a m R Rayleigh scattering Raman shift em 14000 500 500 1000 wavelength nm 514 absolute i i wavenumbers tem 20400 19900 19400 18900 18400 Fig 3 Raman spectrum of silicon 514 nm excitation showing the Rayleigh scattering at the laser wavelength and the Stokes and anti Stokes line of the Raman scattering TMO001 02 A Introduction to Raman spectroscopy REN ISH AW O K apply innovation What does a Raman spectrum look like Figure 4 shows the Raman spectra of two carbon based species diamond and polystyrene In Raman spectroscopy we are interested in how much the scattered light differs from the incident light so the spectrum is normally plotted against the difference between the two the Raman shift Diamond has one main Raman band only because the tetrahedral lattice is symmetrical and all the carbon atoms and connecting bonds are equivalent Polystyrene has different functional groups consisting of differing atoms and bond strengths Each Raman band represents either a discrete function group e g C H from benzyl group at 3200 cm 1 or a combining of small groups into a larger group e g CsHsR breathing mode from benzyl group at 1000 cm Diamond CHe CHa CHa CHa CHa CH polystyrene 500 1000 1500 2000 2500 3000 Raman Shift cm 1 Fig 4 Raman
29. ced sample damage To prevent damage it is prudent to start initial analysis with low laser powers especially when using NIR and UV systems from here the power can be increased balancing sample damage prevention with the needs for a strong signal If reducing the laser power to very low levels lt 1 still results in sample damage use of a line focus accessory may help The line focus reduces the laser power density by spreading the laser power out over a greater area This increases the number of Raman scatterers and the resulting Raman signal is therefore significantly higher than conventional methods Conventional methods include Using a lower magnification objective to reduce the power density at the sample this produces a larger spot size but also produces less Raman singal as the numerical aperture is significantly lower e Defocusing the laser spot using the beam expander Line focus is a superior option for faster data collection as it not only reduces the power density but also optimises the throughput in the spectrometer unlike the beam expander spot defocus method TM26 02 A Frequently asked questions FAQ RE N ISHAW e apply innovation Q7 I can t fit my sample on the stage because its a liquid powder very large What can do While Renishaw Raman instruments provide an excellent way of analysing samples with very little preparation some samples can t simply be placed on a microscope slide It is p
30. cks unless the instrument laser shutter accessed from the sample review is closed If the interlock circuit is broken close and re lock the instrument door and reapply the laser interlock Tools Interlock Reset 5 Change the Grating Note that this should not prompt a lens set change unless multiple gratings are configured for the same Laser x Sample Review B gra cree 100 ie mm ivi 1200 l mm 633 780 1800 Irom vis 2400 Irom vis 300 lmm Of course if the configuration already set is the same as that desired then no configuration change is needed and the above protocol can be skipped Configuration change as part of a measurement The configuration can also be changed by editing the current measurement This is useful when the user requires analysis from the same region of the sample but with different excitation Use the Setup measurement button Pd to edit the laser and or grating in the Acquisition tab the laser power exposure time and accumulations may also need to be adjusted for the new laser TMO003 02 A Sample viewing and configuration change AN apply innovation Running this edited measurement may prompt the user for a lens change if motorised lenses are not avialable Renishaw plc Tel 44 0 1453 524524 RE N IS H AW Spectroscopy Products Division Fax 44 0 1453 523901 e L Old Town Wotton under Edge Email raman renishaw com snan Glouceste
31. cus e g 10 um means 5 um from the optimum focus point e Dis not the main objective diameter but is calculated as the lens radius added to the DoF mm This ensures gradients of up to 45 degrees can be safely analysed 15 Renishaw plc Tel 44 0 1453 524524 RE N IS H AW i Spectroscopy Products Division Fax 44 0 1453 523901 ew Old Town Wotton under Edge Email raman renishaw com Gloucestershire GL12 7DW apply innovation United Kingdom www renishaw com TMO012 Data processing and simple analysis WiRE 4 0 This document aims to show the WiIRE 4 0 user how to process single spectra and perform basic analysis The following methods are discussed Baseline subtraction processing e Arithmetic functions on data processing e Smoothing processing Zapping processing e Peak Pick analysis e Curve fitting analysis e Integration analysis Baseline subtraction Samples may exhibit Raman spectra with varying degrees of fluorescence or thermal background Providing that there is sufficient Raman signal on top of the sloping background the baseline may be subtracted to yield a spectrum with a flat baseline In some cases the measurement can be re performed with an alternative excitation wavelength to more effectively remove the effects of fluorescence The following methods are available Intelligent fitting default intelligent automated option e Through fixed points user control
32. d noise and improving the signal to noise ratio Careful adjustment of these two parameters allows the maximum possible exposure without saturation and will improve the signal to noise ratio It is worth bearing in mind that the signal to noise ratio is proportional to the square root of the number of accumulations 4 accumulations provides a two fold improvement in the signal to noise ratio TM26 02 A Frequently asked questions FAQ RE N ISHAW e a apply innovation Another factor as important as the signal to noise ratio is the signal to background ratio these two ratios are intimately linked If the background component is high it will mask the Raman signal and contribute noise to the system See also Why do some of my spectra give such an intense background signal which masks the Raman information for further details Q6 How can I stop my sample from being damaged by the laser The laser spot incident on the sample has a high power density This is especially true of UV systems and those with high laser powers Unfortunately some samples are susceptible to thermal or photo degradation The resulting spectrum will contain features caused by modification not natively present in the sample for example broad amorphous carbon bands around 1500 cm Often viewing the white light image before and after acquisition will indicate a clearly altered region of sample Figure 2 where the laser was incident Figure 2 Laser indu
33. detector is exposed to the Raman signal Longer exposure times give a better signal to noise ratio in the spectra The minimum exposure time for a static grating scan is 0 02 s If the Extended option is selected in the Range tab the exposure defaults to the minimum required 10 s There is no maximum exposure in either case 2 Accumulations is the number of repetitions of the scan The accumulations are automatically co added to produce spectra with better signal to noise ratios Using several accumulations of a short scan can be preferable to performing one long scan For example e If the sample has a high fluorescence background a long scan will saturate the detector whereas several short scans will not This allows an improvement in the single to noise ratio e If cosmic ray removal is used two extra accumulations are performed So if the scan consists of 10 accumulations of 10 seconds then two extra 10 second accumulations are performed If the scan consists of one 100 second accumulation then two extra 100 second accumulations will be performed which is clearly more time consuming Generally it is preferable to conduct longer exposures when possible as each accumulation adds readout noise from the CCD to the collected spectrum 3 Objective indicates the magnification of the objective being used Better signal to noise is usually obtained from higher magnification objectives as they give a higher power density at the sample The box
34. e series measurement tab is only available when suitable heating freezing temperature stages have been installed with the appropriate WIRE feature permission By default the use check box is unchecked To activate the temperature series parameters check the box Range Acquisition File Timing Temperature FocusTrack Advanced V Use temperature parameters gam to 25 0 C 10 0 C min Cancel Apply Help Figure 7 Temperature tab See module TM22 for instructions on the set up of temperature series measurements FocusTrack To maintain the laser focus for spectral acquisition for example during time temperature and mapping measurements you may use the FocusTrack function Refer to module TM6 for guidance notes The Focustrack tab allows the user to enable this function and specify how often it is used during the measurement TMO004 02 A Measurement set up and data acquisition Spectral acquisition Range Acguisition Eie Timing Temperature FocusTrack Perform FocusTrack after how many scans Figure 8 FocusTrack tab Advanced RENISHAW apply innovation a Properties The advanced tab covers more specialised options for the measurement SynchroScan Step Minimum pixels in overlap pixel 9 Match Overlap Discard overlap pixels Input polarization Binning Polarization Laser focus Figure 9 Advanced tab
35. ely this is an unavoidable consequence of laser irradiation and in many cases the fluorescence is stronger than the Raman signal Despite fluorescence being an unavoidable side effect steps can be taken to minimise or irradicate the problem e Change laser wavelength the approach that will have a most significant effect for highly fluorescent samples In general fluorescence is worse with visible lasers and moving to a laser in the UV or NIR is likely to cure or reduce the problem Renishaw manufacture and can supply a wide range of lasers from UV through to NIR e Quenching possible with some samples By leaving laser light incident on the sample for a period of time before acquiring a Raman spectrum it is sometimes possible to quench reduce the fluorescent background enhancing the Raman features The period of time required is sample dependent but normally some effect is observed in seconds to minutes It is worth noting however that quenching is exponential and therefore the greatest effect will be seen initially Cycle the spectrum to see this affect occur with a live update e Confocal mode by acquiring data from the small sample volume that is strongly irradiated by the laser the fluorescence may be greatly reduced This approach may also be beneficial where the sample being investigated is contained within a substrate that is strongly fluorescent for example a sample confined within a fluorescent matrix If there is too much amb
36. es and possessing a very small FWHM 1 5 To confirm the presence of a cosmic ray immediately re capture the data and you will notice a distinct absence of that feature If however the line still exists it is most likely a result of spectral contamination from room lights etc For further information see keep getting repeatable sharp peaks in my spectra Cosmic rays become increasingly common with increasing exposure time For long scans where the presence of cosmic rays must be avoided consider using the cosmic ray removal feature This is an option in the experiment set up window when activated the spectrum is collected in triplicate equivalent to 3 accumulations The software uses the median value at each wavenumber value to ensure no cosmic ray features are seen Q3 I keep getting repeatable sharp peaks in my spectra What are they If you have repeated the scan and the spurious lines are still present in exactly the same place the possibility of them being cosmic rays has been ruled out Such sharp repeatable lines are usually due to emissions from fluorescent room lights or phosphorous in CRT monitors figure 6 1 Using long working distance objectives worsens the problem Mercury emission lines from fluorescent room lighting at 514 nm excitation Phosphorous lines from CRT monitor at 633 nm excitation 7000 6000 50004 40004 30004 counts arbitrary units counts arbitrary units 2000 AL n
37. exposure from this dialogue The size of the video window can be changed to balance the desired image resolution and image size Select the Video capture pin button and choose a display resolution Ensure the aspect ratio of the video is kept the same as this will otherwise affect the calibration of the video Figure 5 Latest Renishaw video option Configuration selection The sample review shows the current instrument configuration laser grating and also allows the configuration to be changed TM003 02 A Sample viewing and configuration change REN ISHAW e gt apply innovation x a Seb lil 1800 lmm eEsew xx Figure 6 inVia Reflex sample review A Current Laser and grating configuration Changing configuration To change configuration The user must know the desired laser grating and detector they intend ot use for their analysis Some combinations are not appropriate and when attempting to collect data a message will appear to inform the user that this configuration is not calibrated and therefore should not be used When the Laser is changed several or all of the following will occur immediately on selection dependent on the system type e Motorised beamsteer autochange only if the motorised beamsteer mirrors are installed e Motorised Rayleigh filter change only if the motorised Rayleigh filter change is installed e Beam expander autochange all although U
38. f view to significantly reduce the number of images and make the montaging much faster e Ensure the Window containing the Surface is selected e Change the objective to a lower magnification if desired e Select the correct microscope objective if changed Select Live Video Snap montage or the toolbar button IJ e Enter the X and Y values over which the montage will be collected typically the same as the Surface e Select Run The depth of field information for the objective is used to ensure any single video image is in focus over the entire field of view This is automatically determined and where the focus changes over the field of view multiple images are collected at different Z positions The in focus regions of the combined Z stack are then used together in the final montage The result will be a montage collected using one objective but the Surface generated with another Remember to collect the Surface first then the montage Example using Surface to generate an in focus montage using the same objective TMO007 02 A White light capture montaging and Surface generation REN ISHAW e ce apply innovation 50x montage over non level sample 50x montage over non level sample using Surface Figure 11 Original montage compared with in focus montage Note how the original montage goes out of focus on the right hand side whereas the Surface montage is in focus over the entire area orange box Example using S
39. ferent montages to be queued TMO007 02 A White light capture montaging and Surface generation apply innovation 4 Multiple manual white light image capture XYZ Surface Used to ensure data collection or image capture occurs with the sample in focus The Surface option enables the user to manually define in focus positions for samples which are not level or flat This process can enable Large in focus white light montages to be generated based on the defined surface e Mapping data to be collected with automatic Z change based on the defined surface Initially a surface must be generated Generating a Surface opecific objective properties working distance WD depth of field DoF and diameter D are automatically added to WIRE on installation for standard objectives 5x 10x 20x 50x 50xL 100x Other objectives need to have this information added manually if they are to be used with the Surface option see Appendix 1 for this procedure 1 Position the sample on the microscope stage The sample should be appropriately constrained so it is unable to move during Surface generation 2 Select Surface New uy 3 Setthe correct objective in the sample review The surface should be generated using the same objective that will be used for data collection Lower magnification objectives whilst having a larger field of view will not enable the focus to be accurately set for higher magnification objectives nor
40. fetime of a biological sample for example by its Raman spectrum Set the total number of spectra to be acquired in the first box Number of acquisitions and the interval in the second Time series measurement settings A profile can be created at the end of the sequence from the data For example the intensity at one frequency in the spectrum with acquisition number time Vap measer eae UNE ERR M 800 Range Acguision Ele Timing Number of acquisitions Time series measurement settings 0 00 Sample bleaching Software triggered series measurement settings Bleaching time seconds D 00 E Wait on a software trigger before each scan Send a software trigger after each scan Lc j caei jJ ay jJ Figure 6 Timing tab Sample bleaching also called photobleaching or photoquenching is a phenomenon whereby fluorescence is observed to decrease simply by the having the laser incident on the sample There are various mechanisms that part contribute all or in part to this effect Setting a value in the box exposes the sample with the laser for a set time before the spectrum is acquired The period of time may range from seconds to tens of minutes and will be sample and laser dependent Software triggering is only required in special cases using external hardware 5 TM004 02 A Measurement set up and data acquisition RE N ISH AW e esac apply innovation Temperature The temperatur
41. g and simple analysis REN ISHAW A apply innovation Image arithmetic A special case of arithmetic is performing functions on Raman image data i e images created from mapping measurements Ratio images can be generated form the Map generation option see TM014 More complex image arithmetic is performed using the data arithmetic option The initial image is loaded into the viewer data tab of the Navigator derived data right click load dataset Under Processing Data arithmetic select auxiliary data image by browsing for the mapping measurement wxd file then selecting the image from the drop down in Use derived data Use the value boxes to adjust the Data and Auxiliary scaling The format image or surface and LUTs of each image initial auxiliary and result can be adjusted from the context menu View View mode and LUT control Accept or reject the result image 14434 1160 MSS Image 1 14432 A 1150 14431 14430 1140 1730 1740 1750 176 Xi um 20 1160 bs 16 Auxiliary z i E i image gt 1150 12 10 1140 1730 1740 1750 176 1600 Result 1160 i 1400 Image E x Image 1 i divided by 1000 auxillary 1140 1730 1740 1750 1766 X um TMO012 02 A Data processing and simple analysis ij apply innovation Smoothing It can be useful to smooth data This operation has the effect of improving the signal to noise ratio but must be used with caution
42. g the system build phase To manually add replace a grating remove the spectrograph cover plate attach the grating dust Covers remove the gratings separate fit the new grating remove dust cover and replace the spectrograph cover plate Configuration change protocol 1 Ensure all files and windows are closed checking that no unsaved files are still required TM003 02 A Sample viewing and configuration change REN ISHAW e uu apply innovation 2 Decide on the desired configuration 3 Change the Laser Note the laser change does not only necessarily change the laser wavelength but is also used to select for example different Rayleigh filter types of the same wavelength and the use of line focus with the same wavelength Sample Review x B4B ey Mio z Renishaw 532 8nm 514 5nm low cut off Laser Physics 514 5nm Renishaw 532 8nm Renishaw 785nm Renishaw 830 nm Instrument laser shutter 4 On changing the laser a dialogue will appear prompting the user to change the relevant spectrometer lenses if change is required If this dialogue appears open the instrument the instrument laser shutter will be automatically closed and carefully remove the appropriate lenses from their kinematic mounts Replace with the new lenses ensuring that each is correctly seated Close and re lock the instrument door Note under standard use opening the instrument door will trip all laser interlo
43. gration The integration option provides a method where the total area under the spectrum can be determined The left and right vertical bars determine the region which is being analysed within the spectrum The properties are selected by using a right click on the spectrum These enable the exact start and end position to be defined and the type of integration Trapezoid or cubic spline to be selected Integrate Properties jm eS Integration Properties Integration Type Trapezoid Area Cubic Spline Edit Regions Regions Start End Regiond 663 561751 1725 236500 v Allow Overlapped Regions Came TMO012 02 A Data processing and simple analysis AR apply innovation Trapezoid calculates the area between adjacent points using a trapezium drawn between the points and the x axis Cubic spline approximates the spectrum with local cubic polynomial models and uses integration to get an estimate for the area between adjacent points In each case the result is the sum of areas across all pairs of points in the region TM26 02 A Frequently asked questions FAQ RE N ISHAW e i apply innovation Ua i ASKE AUESUONS ITAN WIRE TA Introduction This module offers a list of common questions and problems offering possible solutions to them without having to delve into the manual or contact Renishaw for technical support Q1 Help It s not working Whether new or experienced to their operatio
44. ient light in the room either fluorescent or incandescent it is possible this may cause unnecessary background signal in your spectra It is best to work with lighting at a minimum however if this is not possible for example if the instrument room is used by other people consider the use of a Renishaw enclosure This prevents stray light from entering the instrument and further minimises exposure to the laser beam The enclosure is available in either Class l or Class 3b laser safety forms Q5 Why is my signal so weak and or why do I get such a poor signal to noise ratio If the signal is weak first check that the sample is correctly placed under the microscope and sharply in focus you could also try moving to a different sample point Check that the instrument is set up for Regular mode and check the laser power setting if the power is less than 100 try increasing it to improve the signal If the spectrum is very noisy this may be improved by increasing the scan time or number of accumulations e Increasing the scan time allows the CCD to acquire more Raman signal enhancing the features over the extraneous noise This method is ideal if both the background and Raman signal are low however if either of these is intense then increasing the scan time increases the chance of saturating the CCD e Accumulating the data takes a number of identical scans and co adds them together enhancing weak Raman features from the random backgroun
45. ine imaging example Figure 9 Raman image of angled grid with no surface correction 5 2 um step size 50x objective The bright image regions are in focus darker regions are out of focus The left side of the image is in focus and the sample is going further out of focus as X increases 11 TM007 02 A White light capture montaging and Surface generation REN ISHAW e ain apply innovation Im M 200 um Figure 10 Raman image of angled grid with surface correction 5 2 um step size 50x objective The image regions are all in focus producing no contrast resulting from changes in signal level The right side of the image is now as sharp as the right side of the image and less blurry than before Controlling the sample height prior to data collection The Z position of the sample can be manually defined separately in the area setup tab of the measurement Use fixed Z This option is defaulted to off meaning data collection will commence at the current Z position unless Surface has been used Tick the Use fixed Z box to force a defined Z sample position for map data collection On map data completion the sample is moved to the first data collection point in XY and Z if a Surface has been used regardless of whether the Restore instrument state on completion box has been ticked on the Acquisition tab of the measurement setup Therefore thought needs to be given to the Z position of the sample which will be poten
46. is greyed out The value reflects the value set in the Sample Review window TM004 02 A Measurement set up and data acquisition REN ISHAW O gt 10 apply innovation Laser Power is the percentage of maximum laser power that will be used for the scan Higher power will give a better signal to noise ratio but can damage some samples depending on the laser used Cosmic ray removal removes random sharp peaks due to cosmic background radiation The cosmic rays are eliminated by automatically obtaining three spectra and taking the median average of the three Restore instrument state on completion is used to automatically restore the instrument to the state it was in prior to collection as defined in the Sample Review This function applies largely to inVia Reflex Raman microscopes where there is a greater degree of motorisation Close laser shutter on completion forces the laser shutter to be closed after data collection and will override the Restore instrument state on completion checkbox recommended when performing imaging experiments Minimise laser exposure on sample will close the laser shutter when data is not being collected during a single measurement e g temperature ramp measurement Response calibration will collect data using a pre defined transmission profile normalising the instrument response Live imaging allows Raman images to be defined and subsequently viewed during data collection This feature is used in c
47. is module is to provide a general overview of the WiRE software and detail the correct procedure for Raman microscope laser PC and software start up Please note that this module is a guide and not a complete protocol WIRE software WIRE software is designed to e Control Renishaw Raman instruments e View the sample e Control data collection parameters e View data e Provide processing functions to improve data e Provide analysis options to determine information from Raman data e Enable data and results to be printed and exported for analysis reporting Architecture of the WIRE software Menu and toolbar Menu and toolbar TMO002 02 A Introduction to WIRE and System start up n File Edit View Measurement Livevideo Analysis Processing Procedures Tools Window Help n DRYAS ig d VU ES a isla M dd L2 8 Li alien AA EE Ba i ee it tent JH uu E te 2 Standard Renishaw XYZ X 35143 0 Y 37619 zZ 5 9 v Measurement v Analysis v Video v View e Toolbar buttons offer shortcuts to menu items al ean e Right click on the toolbar to control which groups 7 an bad are shown E Customize e Right click on a group to control the individual buttons which are shown e The toolbar contents is configurable for different users who log on to the PC Sample review Sample review Q tm c memet emm Ate Pom Pm b ee 9 aen D SE2ZEe aN DGUCAMRWUNELERD RASS Beer tesvstSG0 ef N v MV
48. ithm based e g DCLS PCA MCR ALS or EmptyModelling Differentiation Crystallographic orientation Absolute relative concentration Crystallinity Temperature Stress state Any of the above information in conjunction with different dimensions such as time temperature distance area and volume e g e Thickness Intensity with depth 1D Domain size and distribution Intensity with area 2D 3D Table 1 Information obtainable from analysis of different Raman band parameters TM001 02 A Introduction to Raman spectroscopy REN ISH AW e esac apply innovation Raman spectroscopy obtains such information by probing the vibrational states of materials Renishaw s inVia can also be used for photoluminescence PL measurements which is a competing effect to Raman PL is typically much stronger in intensity and is a function of the electronic states of the material The PL effect can sometimes provide an unwanted broad background that can mask the Raman bands However PL measurements can also provide useful complementary information on material properties such as conjugation structural vacancy and atomic substitutions What does a micro Raman instrument usually consist of It usually consists of e A monochromatic light source normally a laser e Ameans of shining the light on the sample and collecting the scattered light often this is a microscope e A means of filtering out all the light except
49. lso for complex band systems where there may be two three or more bands that overlap Curve fitting can produce a wxC file that can be saved and applied later to a spectrum or set of mapped data To fit a curve to a band or series of bands select Analysis Curve fit to open the Curve fit window zoom in to a region that contains the band and some baseline data either side A baseline may be added automatically between the end points of the spectrum This can be used or removed via the context menu Use the mouse to position the approximate centre of the band Click to add the band and repeat for the centres of other bands if part of a system of bands You may need to use the context menu and select Add Curves if the curve symbol does not appear with the cursor Pressing Remove curve from the context menu will remove the last node you added I Single scan measurement 1 o Curve fit x 130000 120000 110000 100000 w 90000 80000 70000 60000 ntensity Counts un eo e 40000 30000 20000 10000 1550 1600 1650 1700 1750 Frequency Raman shift cm 1 Curve Name Centre Width Height Gaussian Type Area ChiSq Curvel 1636 14 14 8839 117489 49 3765 Mixed 2 30964 e 006 1 01113 Curve 2 1607 54 12 4207 47762 28 5127 Mixed 846212 Curve 3 1594 75 15 0094 715974 50 5112 Mixed 1 53162e 006 Curve 4 1579 87 9 28587 21895 30 9211 Mixed 287534 Curve 5 15501 14 4396 9596 66 0 Mixed 217669 m
50. mally used for data collection 4 Determine the area the surface will be collected over and set the XYZ origin if desired Note Setting the origin on a discrete and easily recognisable sample point can enable any generated Surfaces to be saved and re used even if the sample is removed from the stage 5 Navigate around your sample modifying the focus and select add new point A Move the sample in XY using either e the XYZ stage control by typing values and selecting Go to e the high speed encoded stage HSES trackball Adjust the focus using the HSES trackball DO NOT USE THE FOCUS WHEELS ON THE MICROSCOPE 6 Adding points images will build up the Surface The images are shown in the Image mode the Surface is shown in the Surface mode These modes are accessed from the right click menu You can also show the points defining the surface from this menu A double left click on either the Image or Surface view when the cross hairs are active will move the sample to that point in XYZ If this point is not in focus focus the sample and add a new point T TM007 02 A White light capture montaging and Surface generation amp FS apply innovation Image points form the 3D surface using triangulation linear interpolation Producing a Surface for samples which are inherently flat but not level is therefore fast and easy A large number of Raman samples consist of such a form Where samples have more complex variations in sample
51. montage or the toolbar button EA Surface montage le 1491 9 To 1310 0 822 5 To 705 3 Set From Set To Reset Montage with manual Z Status Number of snaps X 2 Number of snaps Y 2 Image start 1547 5 861 2 Image end 1254 2 666 5 Video area 70 33 685 438 No surface available using current height Several options are available in this dialogue X and Y start values are in micrometers taken from the current motorised stage position The user specifies the area in micrometers to collect the montage from The X and Y distance of a single video image can be determined using the axes of the video viewer TM007 02 A White light capture montaging and Surface generation amp D apply innovation he Set From and Set To options enable the user to move the sample stage to the extreme end points they require the montage to be collected from top left and bottom right points e Montage with manual Z enables the user to focus each montage image in Z using the trackball to also generate a Surface e The dialogue also confirms if the montage will be collected using a pre defined Surface or fixed Z height Several options are available within Advanced Advanced Montage Options Raster Overlap 96 Snake Settling time ms Spiral Min dearance 95 Apply background removal Force max number of images per snap e The background removal option applies a post pr
52. n the cure is nearly always very simple Below are summarised a few of the common reasons why you may not be getting a spectrum If you are having trouble making the instrument or laser operate check all of the following to ensure that the instrument and all accessories are powered up correctly e With the WiRE software closed check that the instrument and accessories are plugged in and switched on Ensure that the laser and if necessary its power supply is plugged in and switched on Since there are many different types of laser refer to its individual manual if you require further help with this e Check that the door of the instrument is securely closed and locked and that the interlock is operational e Check that the Class 1 enclosure door if present is securely closed e Start WIRE If all these operations have been checked you are ready to capture your spectrum With your sample under the microscope and the WiRE software loaded and ready check the following if you are still not getting a spectrum e Use a standard sample such as a silicon wafer This is strong and sharp with the 1 order band located at 520 cm e Ensure that your sample is loaded correctly under the microscope that it is sharply in focus and that you are looking at the correct portion of the sample It is often worth trying a different region within the sample because of the possibility of impurities giving unexpected results e Check all the set
53. n be set on the Automatic Thresholding tab 2 Use Autoset thresholds gt Single peak option Zoom in on a single peak including some baseline either side of the peak and then select this option This function sets thresholds to locate all peaks in the spectrum that are as well defined or better defined than the displayed peak Peak picking is then performed TMO012 02 A Data processing and simple analysis ij apply innovation 3 Manual peak addition is performed by using a double left mouse click close to the peak to be labelled The software will locate the closest peak with 3 falling point either side of the maximum and label this peak Complete manual control can be achieved by reducing the number of falling points to 1 To reduce this value to 1 right click on the peak pick table and select properties In the find peak tab reduce the number of falling points option to 1 and select OK A double click on the spectrum will now add a peak label exactly where mouse cursor is located 4 Manual peak removal is performed by right clicking on the relevant peak label in the peak pick table and selecting remove peak label The peak result table may be copied to the Windows clipboard by selecting the Copy results option from its context menu shown by right clicking it From here it may be pasted into e g spreadsheet or word processing programs Curve fitting Curve fitting calculates highly accurate values for simple single bands but a
54. nerally beneficial to have this ticked If this is not active then the baseline form and height will be somewhat dependent upon other bands that are present throughout the whole spectral range It may be necessary to re apply the baseline on zooming as its original position will be persisted A curve fitting procedure produces a table of data the columns are selected from the context menu of the table Show hide columns The table lists the various parameters for each of the curves The data in the table can be copied and pasted into a spreadsheet package for example context menu Copy results TM012 02 A Data processing and simple analysis AN apply innovation Curve Fit Results Table Properties Show hide result table columns V Gaussian Type v Area Chisq The fitted curves baseline and result curve sum of the fitted curves and baseline if used can all be saved and reloaded as spectra Once the curve fit has completed select Save curve data from the context menu and save to a location This saves a multifile that can be opened like a spectrum in WIRE 3 Use the Data tab in the Navigator and expand the branches to show the Collected data Highlight each acquisition and right click to show Load dataset To save the curves result or baseline as a separate spectrum or trace highlight the trace in the View tab of the navigator and select Save spectrum as from the context menu Inte
55. ocessing operation to flat field the completed montage This can help remove any effects resulting from uneven illumination on the sample This process is applied immediately on completion of the montage it is also available to be applied anytime after the montage has completed from the Image view right click menu Overlap Settling time and minimum clearance can all be adjusted 6 Select Run and the system will start the collection of the montage automatically moving the stage and adding new frames to the image that appears in the Still Image viewer The image will auto scale to fill the Image viewer size as new frames are added TM007 02 A White light capture montaging and Surface generation REN ISHAW e Saat apply innovation The still white light image can be saved from the context menu Save to as a bmp of jpg Saving as a jpg enables the image to be reloaded into WIRE and used to define Raman data collection in the same way as a montage provided the sample remains on the HSES motorised stage and the co ordinate system has not been reset this cannot be done with bmp files 300 200 100 100 200 300 300 200 100 0 100 200 300 Figure 3 Automated white light montage forty five 50x objective images Multiple montages can be generated by adding a new Window New Window after completing the first montage The co ordinates should not be reset between montages This allows data collection from the dif
56. on change RE N ISHAW e in apply innovation To change the video properties right click on the video image and select Video properties Video Viewer Video Source Video Renderer RR Properties mvBlueFOX Settinc Video Source Properties Video Capture Pin Format Display Properties Frame averaging E Find Cameras Use averaging Number of frames to average Number of frames to discard Figure 3 Video properties Under the main property page the user can apply image averaging to the video This can reduce noise for dark low contrast samples but may affect video responsiveness The Capture filter properties contain the different camera settings For the latest camera Figure 4 set the gain to high no auto and adjust the exposure as necessary TMO003 02 A Sample viewing and configuration change REN ISHAW O E apply innovation Capture Filter Properti e Video General Exposure a Me Brightness Contrast Gamma Color balance Red Green Blue Flip Auto settings Horizontal Full auto Vertical Auto exposure Auto gain Conca Figure 4 Video property settings Using auto exposure adapts the exposure based on the image contrast and brightness but may not be appropriate for all sample types where the F stop is closed or where auto mode continually hunts does not produce a signle stable exposure value In these scenarios turn off auto
57. on time and X and Y step size This relationship also varies with the mapping method If the Z surface position cannot be reached in the available time during mapping data collection the time is not delayed and the appropriate focus position may not be reached This is most likely to occur where the acquisition time is very short or the rate of Z change is very high The following example demonstrates map data collection on an X angled sample which is inherently flat TM007 02 A White light capture montaging and Surface generation REN ISHAW e eae apply innovation e StreamLineHR example Figure 6 Raman image of angled grid with no surface correction 5 um step size 50x objective The bright image regions are in focus darker regions are out of focus The centre of the sample is in focus moving away from the centre in X causes the sample to go out of focus Figure 7 Raman image of angled grid with surface correction 5 um step size 50x objective The image regions are all in focus producing no contrast resulting from changes in signal level 10 TM007 02 A White light capture montaging and Surface generation REN ISHAW e cilm apply innovation Figure 8 Raman image of angled grid with surface correction 1 um step size 50x objective The image regions are all in focus producing no contrast resulting from changes in signal level The image is sharper as the spatial resolution is higher e StreamL
58. onjunction with Map image acquisition and StreamLine image acquisition measurements only This option requires the user to know the expected changes within the Raman data or have pre collected reference spectra of specific components PCA and MCR ALS options are not possible with Live imaging File The File tab covers options for automatically saving the data Either insert a filename or browse to a folder Range Acquisition File Timing Advanced File name C Data renFOD5 wdf Auto increment Figure 5 File tab TMO004 02 A Measurement set up and data acquisition REN ISHAW O uu apply innovation 1 Autosave file saves the file to the file specified in File name directly after collection Its use is recommended as it removes the risk of losing data by forgetting to save it The next dataset will overwrite the first unless the Auto increment checkbox is selected Checking the Auto increment function will force the data to be saved each time this measurement is performed The format will be filename filenameO filename1 filename2 unless the original filename is appended numerically e g filename Timin The Timing tab consists of two main functions time series and sample bleaching measurements The Time series measurement allows multiple spectra with same instrument conditions to be acquired with an identical period of time elapsing between each one This function may be useful to monitor the li
59. ossible to place samples with heights of up to 50 mm directly onto the stage Renishaw s macro sampling kit provides an excellent way of dealing with problem samples such as powders liquid or samples which are large and can t be easily placed on the stage while still requiring no sample preparation Large samples which maynot fit under the microscope can be analysed using a flexible sampling arm This enables Raman analysis external to the microscope and enclosure As the arm is direct coupled it has all the resolution and throughput benefits of the inVia unlike a fibre probe coupling Fibre probes are available and are ideal for distant Raman analysis and integration within other instruments Q8 How can I stop my sample from moving around on the microscope stage It is important that samples can be constrained so they do not move during analysis This is even more important when using Raman mapping methods Flat samples such as polymer films need to be held flat so the laser focus does not change during analysis or depth profiling Other samples need to be held in XY so they do not slide or shift during fast mapping experiments The high speed encoded stage accessory kit HSES enables samples of all types to be firmly held in place to prevent experiments having to be repeated Q8 I d like do be able to examine my samples under different pressures is there a way can do this The diamond anvil cell available from Renishaw enables you
60. pically the sample is initially viewed using a low magnification e g 5x objective If the sample has features these can be focussed using white light Closing the F stop will reduce the field of view and produce an octagonal ring When the edge of this ring is sharp the sample is nominally in focus This is of particular use for featureless samples The laser spot line and white light are co focal for most visible and near infra red laser wavelengths Therefore when the laser spot is in focus the sample is in focus with the white light The sample focus can be checked by moving sample position and seeing an equivalent change on the video Progress through higher magnification objectives refocusing each time until the objective to be used for data collection is reached Achieving the best video image requires appropriate control of the illumination control and video settings for different types of sample different colours and reflectivities The closed aperture stop produces high contrast images at the expense of reduced illumination For most samples the A stop should be closed to achieve the best image quality The properties of the video can be controlled by the user to optimise exposure and gain Depending on the sample reflectivity it may be appropriate to use auto settings to achieve the best quality note that under these settings the frame rate may reduce the responsiveness of the video TM003 02 A Sample viewing and configurati
61. ple manual video images with XYZ positions used to define a 3D sample surface together with multiple automated video images for subsequent data capture i e options 3 and 4 together Defining the field of view used for image capture The captured area can be reduced by selecting Live Video Snap Set up This new area is then used for single or montage image capture only Live Video Setup The black region represents the entire video M area Use the mouse to draw a region within this The region within the orange box will be used for single or montage video image collection Use Select All to collect from the entirety of the video area Reducing the area can help to reduce any montage combining features resulting from Define the dimensions of the sub image to be collected below f n or draw a selection on the image above illumination uniformity this is somewhat dependant on the sample surface reflectivity to white light 524 Left 60 OK TM007 02 A White light capture montaging and Surface generation REN ISHAW e ain apply innovation When capturing video images the objective selected in the sample review must matches the physical objective focussed on the sample 1 White light optical image capture XY A single image can be captured using the Live Video Snap Single option or the Snap video toolbar button A Still Image Viewer will appear with the video camera view
62. referenced Always Reference don t show this dialog again On running WIRE 2 all motors will be referenced and this dialog will not shown 6 Choose the Reference un referenced motors only option and click on OK Partial system start up Typically some of the components will already be on when the system is to be used and therefore the start up procedure should be modified accordingly The following is an example of how the system might usually be found and the correct procedure in this case to complete the initial start up Example 1 The inVia Raman microscope is on and the PC is on and the WIRE 4 programme is open All lasers are off 1 Clear all data and windows from WiRE 4 checking that no unsaved data is further required 2 urn on the appropriate laser s 3 Wait for the required time period for the optimum laser stability to be reached Example 2 TM002 02 A Introduction to WiRE and System start up The inVia Raman microscope is on and the PC is on and the WiRE 4 programme is closed All lasers are on 1 Open WiRE 4 there will be no prompt for motor referencing as the current state of the motors will be recognised by the software and referencing is not necessary 2 The laser state will not change and all lasers will remain on 3 The system can be used immediately Example 3 The inVia Raman microscope is on and the PC is on and the WiRE 4 programme is closed All lasers are off
63. rshire GL12 7DW apply innovation United Kingdom www renishaw com TM004 Measurement set up and data acquistion WIRE 4 0 The aim of this module is to detail the correct use of WIRE 4 to enable spectral data collection using the different measurement parameters available in conjunction with the inVia Raman microscope Defining the type of measurement Measurements are used within the WiRE software to define the type of data collection Several different types of measurement may be available to the user depending on the exact configuration of the inVia Raman microscope Measurements which are unavailable are greyed out New measurements are accessed using either the menu Measurement New or the toolbar arrow ns Live video Analysis Processing Procedures Tools Window Help W New measurement Spectral acquisition Pa Depth series acquistion 9 amp Depth series acquisition StreamLineHR image acquisition StreamLineHR image acquisition ll Measurement queue Ctrl Q Figure 1 Toolbar new measurement access Figure 2 Menu new measurement access The different types of measurements which may be available are e Spectral acquisition standard spectral collection Filter image acquisition collection of filter spectra and filter images e Depth series acquisition spectral collection at varying sample depths Z only e Map image acquisition spectral collection at varying lateral sample positions
64. s on each spectrum Selecting Through chosen points on each spectrum as the fitting mode enables the user to manually add points vertical lines to the spectrum which are fixed to the data The user can choose between polynomial and the order and cubic spline options Cubic spline is only available if 2 points are added 4 total points This method can be applied to single spectra or multifiles When applying to a multifile common X positions where no Raman bands are present should be found The baseline will optimise based on the X position for each spectrum within the dataset Subtract baseline x 50000 Counts 40000 30000 MI Jh MALA A Nu Nl aes AAT 200 300 400 500 600 700 800 900 1000 1100 1200 1300 Raman shift cm 1 25000 20000 15000 Counts 10000 5000 AMAA as JU 200 300 400 500 600 700 800 900 1000 1100 1200 1300 Raman shift cm 1 TM012 02 A Data processing and simple analysis Through whole spectrum RENISHAW amp apply innovation Selecting Through whole spectrum automatically fits a defined polynomial order through the entire spectrum The context menu enables exclude regions to be added to the spectrum Excluded regions do not contribute to the fitting of the baseline This method can be applied to single spectra or multifiles and is a less intelligent equivalent to the recommended intelligent fitting option Subtract baseline 50000
65. s point positions XY and baseline type e Through chosen points on each spectrum user controls X point which the baseline travels through for each spectrum within the dataset e Through whole spectrum Automatic fitting with no in built intelligence With the spectrum open in a Viewer select Processing Subtract Baseline Intelligent fitting A new Viewer opens with the spectrum in the top half and the result of the automatically applied baseline subtraction in the lower half By default the Intelligent fitting baseline is applied with a polynomial value of 11 This method is Renishaw patented and enables simple or complex backgrounds to be removed automatically Using a right click and selecting properties brings up the property page TM012 02 A Data processing and simple analysis REN ISHAW e iin apply innovation Subtract baseline Properties Subtract baseline etin mode After automatically excuding regions with peaks the baseline will be fitted to all remaining points in each spectrum You can add exclude regions in the upper spectrum viewer to exdude points in these regions from the fit Intelligent polynomial Polynomial order Noise tolerance 1 50 Anchor end points of intelligent polynomial Here the polynomial order can be adjusted if a better fit is needed The context menu also enables exclude regions to be added to the spectrum Excluded regions do not contribute to the fitting of
66. spectrum below The upper spectrum has a zap region between two vertical black lines Grab each vertical bounding line in turn and adjust the position of the zap region so it just encloses the band to remove Then use the zoom function to isolate the band to zap out Notice that the result spectrum updates to show the effect of the zap Additional zap regions can be added from the context menu TM012 02 A Data processing and simple analysis REN ISHAW e cilm apply innovation Add Regions Remove Region Accept Reject View d Tools r rr a vj 1500 Add d Remove Docking Peak Pick Peak pick is a quick and simple method to label band positions on a spectrum and enable these to be printed out together To initiate peak picking select Analysis gt Peak pick or click the Peak pick button on the Analysis toolbar 1661 5 D 77 E E 556 2 694 8 05 7 1606 2320 406 5610 245 1319 3 Jio 0 929 5 12344 ine 1452 5 EN i 500 1000 1500 2000 2500 3000 Raman Shift cm 1 Peak no Centre Height Width Area Absolute inten Low edge High edge 1 155 638 10250 2 15 6173 1 77017 e 006 21800 1 134 509 202 972 2 232 033 4266 84 13 1133 991115 15815 9 203 797 244 215 3 406 69 6914 77 7 37158 901089 15758 5 386 916 436 408 4 505 722 9853 63 7 19457 769548 16845 479 301 527 968 5 560 986 6768 61 9 40512 1 32467e 006 13499 528 793 622 002 6 579 968 4724 68 18 9718 207027 9156 73 623 652 642 624 7 694 777 1
67. tially used for any future queued data collection This is particularly the case if queued data collection consists of a mixture of Surface and non Surface measurements Extracting Surface information from collected map data From collected data the Surface can be extracted so that it can be viewed and manipulated in the same way as when originally produced This also allows any associated white light montages to be back ground corrected post data collection To extract the Surface Select the data tab of the navigator then expand the Measurement configuration node Right click on the map information Surface map type and select Extract surface The Surface will then be loaded into a separate Window 12 TM007 02 A White light capture montaging and Surface generation REN ISHAW e ain apply innovation 5 Using the Surface to generate in focus montages Useful where mapping data can be spatially connected to the variable focus white light image The collected Surface can be used to collect in focus white light montages The Surface is generated using the objective to be used for data collection This is usually of high magnification and produces a small field of view within the video Where the total area for the montage is also relatively small this does not pose a problem As the total area increases the number of images also increases Therefore it is often desirable to use a lower magnification which has a larger field o
68. tings in the measurement setup dialogue If you have cosmic ray removal engaged remember that this takes two additional undisplayed spectra this process may take some time depending on the scan time chosen so don t be concerned with the delay in spectral display e Check the laser spot is on the crosshair of the video If not use the manual adjust for the bottom left beamsteer Tools Manual beamsteer or perform a Laser autoalign e Check the correct lens set is within the instrument The lens set is clearly labelled and the user is prompted on configuration change where a change is necessary e Perform a CCD area auto align inserting a silicon sample under the microscope for non Reflex systems e Perform a slit auto align search and optimise TM26 02 A Frequently asked questions FAQ RE N ISHAW e uH apply innovation Now that this has helped you get a signal from your sample you may find it is particularly noisy if this is the case try the suggestions below on how to improve signal to noise ratio and signal to background ratio Q2 Why do I keep getting random sharp peaks in my spectra These are the result of cosmic rays High energy particles passing through the CCD detector resulting in the generation of electrons which are in turn interpreted as signal by the camera They are completely random in their time of occurrence and the position where they strike Cosmic rays are very intense resembling emission lin
69. tons gt 99 999 are elastically scattered this Rayleigh scattering has the same wavelength as the incident light However a small proportion 0 00196 will undergo inelastic or Raman scattering where the scattered light undergoes a shift in energy this shift is characteristic of the species present in the sample This process is shown in in Figure 1 Before Raman scattering After Raman scattering Molecule vibrating Molecule Laser e Raman excitation scattering Fig 1 Schematic diagram of the Raman effect Figure 2 illustrates the transitions accompanying Rayleigh and Raman scattering The electric field of the incident light distorts the molecule s electron cloud causing it to undergo electronic transitions to a higher energy virtual state not a true quantum mechanical state of the molecule Raman scattering results in the release of a scattered photon with different energy to the incident photon the difference in energy is equal to the vibrational transition AE The relative intensity of stoeks ans anti Stokes lines at room temperature is shown in Figure 3 TM001 02 A Introduction to Raman spectroscopy REN ISH AW e amm apply innovation V v y v v v v V Virtual energy level Virtual energy levels AAT PDL AN ANG A hv hy hv AE hy hv hv AE v hv hv hv y y V V d AE y V Fig 2 The electronic transitions a
70. urface to generate an in focus montage using different objectives For very large areas a lower magnification will provide a faster montage whilst using the accuracy of the Surface defined using the data collection objective The lower magnification is less sensitive to focus changes but will still benefit from the Surface information Figure 12 5x montage over non level sample using 50x Surface Now only 2 images are used to enable white light visualisation over a large area whilst ensuring data collection occurs at the accuracy of the 50x Surface Red points show the location of the 50x Surface points The clear images option can be used to remove white light images added to the Surface during generation to then be replaced by either of the methods above To collect a montage separate to the Surface but on the same sample or over the same area add a new Window first before selecting new montage 14 TMO007 02 A White light capture montaging and Surface generation AN apply innovation Appendix 1 Adding objectives to enable their use with Surface New objectives are added using System configuration System configuration Podule tab objectives Additional to the new objective name and magnification the working distance WD depth of field DoF and diameter D values need to be entered e WD is a value provided by the objective supplier mm e DoF is the distance the sample can travel before it becomes out of fo

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