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Standard Operating Procedures with relevant Renishaw training
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1. 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 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 ij 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
2. TMO004 02 A Measurement set up and data acquisition 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 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 t
3. 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 generally 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
4. 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 Integration 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 TM012 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
5. as they give a higher power density at the sample The box 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 subseque
6. but only covers a limited range e Extended SynchroScan 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
7. Figure 4 Acquisition tab 1 Exposure time is the time the 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
8. 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 566660 polystyrene 500 1000 1500 2000 2500 3000 Raman Shift cm 1 Fig 4 Raman 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 Peak
9. 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 photons 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 Ra
10. and requires a short training do not use the 50x long working distance objective without first getting this training from the facility manager Remember that rotating the coarse and fine focus knobs counterclockwise towards the user moves the microscope stage down 7 For Raman imaging switch the upper mirror selection wheel to 1 and the lower mirror selection wheel to 2 Pull out the camera selection rod on the upper left side of the microscope trinocular to the fully out position which ensures that no light gets to the eyepieces 8 DO NOT LOOK THROUGH THE EYEPIECE FROM THIS POINT ONWARD USE THE CCD CAMERA IMAGE ON THE SOFTWARE 9 The only manual adjustment of hardware you need to make in the spectrometer are the three lenses and the pinhole do not touch the filters The lenses that need changing when you go to a different laser are the A2 B2 C2 for 488 and 532 nm and A1 B1 C1 for 785 nm lenses The pinhole should be in for 755 nm operation and out for 488 and 532 nm operation The Rayleigh filters are on a motorized rotating panel and will be put in place automatically when you choose the laser using the menus along the bottom of the main software panel there is an icon that looks like a laser spot either SM 488 nm Raman for the 488 nm or 785 nm edge for the 785 nm or SM 532 nm ULF Raman for the ultra low frequency Rayleigh filter operating with the 532 nm laser If you want to use the 532 nm laser for high speed l
11. 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 also 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 42
12. don t see a laser beam on the sample just run a measurement and the optics position should correct itself If it looks like the optics on the linefocus motor are staying in a bad position after you run a measurement follow the procedure below 1 From the Tools menu choose Reference Motors 2 Choose Reference selected motors and highlight in the list to the left ONLY Linefocus motor 3 Click on OK 4 Go through the following process below for moving the optics to Good To move the wheel to the GOOD position first choose the laser 785 nm Linefocus if you are using 785 nm or SM 488 nm Raman Linefocus if you are using 488 nm This will move the wheel to close to the WORST position Then choose the laser Multi Line Fiber Raman This should rotate the wheel to the GOOD position Open the spectrometer door and check the wheel position If it is not in the GOOD position close the door and choose the Linefocus laser setting you used before and then try again Multi Line Fiber Raman Repeat until the wheel is in the GOOD position Then pick the laser your normally use If this repeatedly fails then start the entire process with the other linefocus laser setting than you started with VERY BAD 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
13. esac apply innovation Temperature The temperature 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 B
14. on the Waiting for Key message turn the key off wait 20 seconds hit Restart and turn the key back on Wait until the message box shows Completed highlighted in blue Remove safety goggles You can adjust the laser power with the software from that point on If you adjust the power above 100 mW put the laser goggles on whenever the shutter is open Turning off 532 nm laser Turn the power key on the laser off Turn down the power in the software Disconnect the laser from the Cobolt software Wait 2 minutes and turn off the 532 nm laser cooling fan at the terminal strip Exit the laser software Installing Wire 4 1 on your computer You can get Wire 4 1 from the desktop on the new computer the folder is called Wire 4 1 CD it is also available to Cornell users as a zipped file at https cornell box com s gxy4pkeo2odlbdukdx3d Copy this folder to your memory stick and follow the instructions in the file Wire Install Notes that is in the Wire 4 1 CD folder Be especially careful to copy the Wire 4 1 CD to your desktop do not try to install from the memory stick directly You do not need to uninstall Wire 3 4 Help File The user guide is on the desktop as User Guide chm and is also available at http www ccmr cornell edu raman Storing Data ALL data should be stored on the Users folder on the D drive in a folder with your netID as its name there is a shortcut on the desktop to th
15. 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 s e E moa v 94 ri aN SUDAN we dr O v The Navigator Navigator a Single scan measurement 7 g 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
16. 07 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 TM012 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
17. 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 12250 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
18. Frequency Raman shift cm 1 5000 4000 Counts 3000 2000 1000 Raman shift cm 1 TM012 02 A Data processing and simple analysis ij apply innovation Through chosen points 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 fitti
19. Renishaw InVia Quick Operation Summary July 2015 This document is frequently updated if you feel information should be added please indicate that to the facility manager currently Prof Kit Umbach ccul Q cornell edu Thurston 114 office and cell 607 216 8872 All procedures are subject to change If you have any questions about the operation of the instrument do not take any risks first contact the facility manager he has his cell phone with him at all times Reservations and Enabling on Coral The Renishaw InVia Raman microscope is on the CCMR Coral equipment reservation and enabling system Users are allowed to reserve the instrument for as much time as they need However users who reserve time and do not cancel it in advance will be charged abuse of the reservation system can result in suspension of CCMR facility privileges You can access Coral either from your laptop or from the Coral computer in Bard B47 to which you will be given access Safety This instrument uses Class IIIb lasers so please be alert to the safety issues you learned in the EH amp S laser safety course Use of laser safety eyewear is not required except in the case of the operation of the 532 nm laser Logbook Record the date your name and hours of usage Comment on problems Keeping lab orderly Please dispose of any glass slides that you use into the red plastic sharps container that 1s kept on the floor near the base of the system Also dispose of
20. arge area imaging see the facility manager about manually replacing the 488 nm Rayleigh filter with a 532 nm Rayleigh edge filter You also need to pick a grating in software For 488 excitation pick the 2400 I mm grating using the pull down menu at the bottom of the main software panel For 785 excitation pick the 1200 I mm grating using the pull down menu For 532 nm ULF filter operation choose the 2400 l mm grating For high speed imaging with the 532 nm laser you can use either the 2400 I mm grating or manually replace that grating with a 600 I mm grating see the facility manager for additional training on changing the filters manually high speed imaging and using the ULF 10 Open the shutter If you get an error box Warning Interlock relays are disabled check that the spectrometer door is locked tight Then hit Retry in the error box At that point it is possible that the 785 nm laser needs to be restarted if that is the laser you are using close the shutter and open it again to restart the 785 nm laser 1 1 Bring the power up to about 50 or 100 make sure you have picked the SLOWEST speed on the trackball control and use the fine focus ring on the microscope track ball to sharpen the image of the laser Adjust the fine focus ring in small steps or you risk crashing the objective into the sample 12 Under the Measurement pull down menu on the top menu bar pick New Spectral Acquisition Take a static spectru
21. cessing 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 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 b
22. croscope 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 Renishaw plc Tel 44 0 1453 524524 RE N ISH AW le 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 this 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 expor
23. e 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 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 ww 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
24. 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 iek 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 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 m
25. evert and just use the initial values If after doing these things the counts are less than 50 of the expected values please make a note in the logbook For the 785 nm laser the spot is defined by a pinhole The pinhole is manual and needs to be left in for 785 and moved to the out position for 488 and 532 nm operation For those of you who want more signal with 785 nm you can leave the pinhole out so that your sample 1s illuminated by the full beam profile of the laser which is a line however you will lose spatial resolution and the facility manager will need to show you how adjust the CCD area to take full advantage of this extra power 13 From the Tools menu pick Calibration Quick Calibration to ensure that the spectral calibration of the instrument is correct 14 Rotate out 100x and place your sample onto the microscope stage Proceed with your sample measurement More information on setting up measurements is available in the photocopied Renishaw documents and via the Help files of the Wire software 15 Shutdown Put the 5x objective into place lower the microscope stage remove your sample put the mirror selection wheels into the position for White Light imaging turn down the intensity of the micrsoscope halogen illuminator with the dial at the lower left of the micrsoscope base exit the Wire software turn off whatever laser you are using exit Coral Operating Tips When you are setting up a new measurement and are i
26. ill 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 selection Operation type Arithmetic v Auxiliary data bl removed wdf Acquisition1 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 shif
27. inning Polarization Laser focus Figure 9 Advanced tab 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 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 indica
28. inutes 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 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 i
29. is Users folder I have transferred all user folders dating from 2013 and later from the old User folder to the new User folder If you need to get to your data or measurement settings from the old computer let me know No longer will any data files on the desktop or on the system drive be tolerated they will be deleted You must put your data onto the Users folder on the D drive Data Formats Renishaw now uses a new data format wdf instead of wxd This format cannot be read by Wire 3 4 However all the data that you took with Wire 3 4 as wxd files can be read by Wire 4 1 So if you want to view the data you take with Wire 4 1 you can either save your data as txt files in which case you can use Wire 3 4 or Wire 4 or other programs to view the data or you need to install Wire 4 1 on a Windows 7 machine to view the wdf files According to Renishaw Wire 4 1 is not going to function with XP or with Windows 8 but some users have been able to install Wire 4 1 on Windows 8 machines so you can give it a try Raman Users Occasionally the Raman system has problems with very low counts on the Si or you are seeing the laser as a line instead of a spot This might occur because of a system crash after which a dialog box comes up and you are asked to re reference the motors Then the optics might start out in a bad position where some of the light is blocked as it passes toward the Rayleigh filter So after re reterencing even if you
30. m at the wavelength you want to use At 785 nm that you should take the alignment spectrum at 100 power at 100 you should see 10 000 counts for a 1 second acquisition time At 100 power for 488 nm counts are at 7 000 for a 1 second acquisition time If you are not getting these counts you will need to first manually align the laser beam position to be at the center of the crosshair box in the video image This is done with the Manual Beamsteer option in the pull down menu on the top menu bar under Tools When using Manual Beamsteer Only use the left set of arrows when steering the beam manually After centering the beam use AutoAlign goto Tools gt AutoAlign gt Align for the CCD and the slits Do no use AutoAlign for the laser position use it only for the CCD and slits after you have manually aligned the laser position As indicated above before using Manual Beamsteer set up and run a measurement Anytime you use Manual Beamsteer you should then do Align CCD Area followed by Auto Align Slit Optimise Important Warning on AutoAlign CCD the AutoAlign CCD will give you Initial and Final values of the pixels on the CCD where the system will collect the Raman scattered light These pixels should be in the range from 15 to 30 for 488 nm and 1 to 15 for 755 nm Sometimes the routine makes an error and gives as a final value a range of pixels that 1s far from the initial If this happens do not accept the results r
31. man 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 accompanying 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
32. n 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 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 mi
33. n the Acquisition tab there is an option Restore instrument state on completion Check this option This will bring the power to whatever level you have set it to manually after the measurement is done You can also choose the option of closing the shutter after the measurement to avoid overexposing light sensitive samples The origin of the mapping stage can be reset to zero by picking Choose Origin under the Live Video pull down menu along the top menu bar Be careful about making sure that confocality is set to Standard and not to High in the Range tab in the Measurement Setup window We have changed the sign convention for the z stage position more negative now means deeper into sample same as moving stage up Turning the 532 nm laser on Make sure the 532 nm laser cooling fan is turned on at the terminal strip you should be able to hear the fan but do not reach in to check the fan at the laser because you may disturb the optics Put on laser safety goggles Start the laser software on computer the software is called Cobolt Monitor Pull down the File menu and choose Connect Click on the radio button Set Power Set laser power level in software to 40 mW Click on the Restart button in the software Turn on the power key to laser The message box on the right hand side of the Cobolt Monitor software window should quickly highlight in blue the Warming Up message If it is stuck
34. ng 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 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 w
35. nitial 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 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 e L Old Town Wot
36. not opened the video camera follow the instructions below If you do need to change the settings then you MUST restore them to their original settings before you end your session on the microscope The original settings are on the desktop in a file Video Settings Failure to restore to the original settings will result in a usage charge repeated failures could result in restrictions on your access to the Raman microscope If Wire software does not open the Video Camera Under View choose Live Video If you cannot see the sample well then you can turn on AutoGain right click when the mouse is in the field of view of the video camera then choose Video Properties then VideoSource tab then CaptureFilterProperties then in the Image Controls window check on Full Auto As mentioned above please restore the video settings back to where they were originally when you are through with your session 6 Place the Si reference sample underneath the objective and focus on the Si using white light Rotate in increasingly higher power objectives until you have the 100x in place You can use the regular 50x objective but because of some absorption due to coatings on the objective the signal strength with the 50x will be lower than with the 100x most noticeably with the 488 nm laser If you are imaging powders liquids foils or fibers you should use the 50x long working distance objective which is a different objective from the regular 50x objective
37. ntly viewed during data collection This feature is used in conjunction 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 be
38. o more effectively remove the effects of fluorescence The following methods are available Intelligent fitting default intelligent automated option e Through fixed points user controls 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
39. r mirror selection wheel is set to 1 The power on the white light illuminator can be turned up to whatever level 1s appropriate for you sample this 1s the wheel at the bottom left of the microscope body The illuminator is always left on and should be turned down to its lowest setting at the end of your session Because of the different lighting in the room light from the overhead fluorescents can enter through the eyepieces and be seen in the imaging camera when you are not looking through the eyepieces To eliminate this problem there is a rod on the left hand side of the trinocular with three positions when it 1s pulled all the way out then the eyepieces are blocked and the light from the sample only goes to the camera It should be left in the middle position for use of both the eyepieces and camera When using white light imaging pulling the rod out is optional However you should pull the rod out when aligning the system with the lasers and taking Raman spectra You should also turn off the room lights for sensitive measurements Video Camera The video camera should be set to not have auto gain This means that in white light you may need to adjust the white light intensity using the dial on the lower left of the microscope base to get the best white light image In normal operation you should not need to change the settings for the microscope camera If for some reason you need to increase the gain on the camera or if the computer has
40. s 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 algorithm 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 Informatio
41. t cm 1 25000 20000 15000 A a ah dal 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 processing 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 TM012 02 A Data pro
42. ted 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 ta L2 8 Li alien AA EE 1e 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 CHOCABKWOLHO RASS Eer tesvstSG0 ef N v MV 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 gra
43. tes the laser is tightly focussed while 100 indicates it 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 0 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
44. 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 can 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 TM012 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
45. ting 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 Beet ter SG0 2F E ue M g 1 annm v w v d e Mn dee eee ee ee ee ee ee ee ee ee ee dl we 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 GUCAGWCUOCEROC wEZS MARI teri Ene K ZEE 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 O 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 TM002 02 A Introduction to WIRE and System start
46. tion spectral collection at varying lateral sample positions 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 TM004 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
47. ton under Edge Email raman renishaw com snan Gloucestershire 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 acquisi
48. tween each one This function may be useful to monitor the lifetime 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
49. wipes tape etc when you are done with them Steps to run system 1 Enable the instrument on Coral 2 Verify that main power is on and that the computer is on Normally these are left on If the main power is off check with facility manager before turning the system on The computer can be rebooted if needed no password is currently needed If the Windows logon dialog comes up log on as CCMR User with password ccmruser Start the Wire 4 1 software Pick Reference un referenced motors only if the Motor Reference Options dialog box comes up 3 Verify that the shutter is closed 4 Turn on the lasers you wish to use if they are not already on You should wait 10 minutes if you need very stable power or frequency for a quick measurement you can begin right away There is a switch labeled L1 for the 785 nm laser on the side of the InVia spectrometer The 488 nm laser which has the blue fiber optic connected to the laser output has a key on the power supply It must be turned to Start and held there momentarily for the laser to be started Do not adjust the power knob on the 488 nm laser power supply There is a manual shutter on the 488 but it is left open so as not to disturb the fiber optics The procedure for turning on the 532 nm laser is at the end of this document 5 Set the Leica microscope up for white light imaging Put in the 5x objective Verify that the upper mirror selection wheel is set to 2 and the lowe
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