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1. k oO LabSpec 5 user manual Page 119 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA malian Scientific the baseline point from the baseline curve The entire baseline can be cleared by Clear clicking on Clear When the baseline curve is completed click Sub Sub to subtract it from the spectrum 4 6 2 3 Setting a Baseline for Baseline Correction of a Multidimensional Spectral Array The procedure as outlined above section 4 6 2 2 page 118 should be followed but the baseline curve should be created on the Splm spectral image window In general it is useful to have the Attach mode set to Yes when applying a baseline to the Splm window since this ensures the baseline points can adapt to the varying intensities typical in a multidimensional spectral array Individual spectra within a multidimensional spectral array can be baseline corrected by applying the correction to the individual spectrum displayed in the Point window When the baseline has been subtracted ensure the corrected spectrum is re inserted into the spectral array by clicking on the blue arrow icon displayed in the point window or click Correct in the Map Analysis dialog window see section 4 6 9 page 147 This must be done before the map profile cursor is moved 4 6 3 Spectral Normalization and Correction oP h
2. In general the higher the degree the more adaptable the baseline curve will be to strangely shaped non uniform backgrounds Page 117 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA Tews Scientific Attach Select the Attach mode for manual definition of baseline points used to define the baseline curve o No baseline points are set at the exact intensity and spectral position cm or nm specified by the user o Yes baseline points are forced to sit on the spectrum at the spectral position cm or nm specified by the user The Attach mode is particularly important when defining a baseline to be used for correction of multidimensional spectral array data In this case Attach should be set to Yes to ensure that the baseline points will adapt to the varying intensities of the spectra within the multidimensional spectral array Style Set the display style of the baseline curve The colour width and line style of the baseline curve can be set using the Style drop down box 4 6 2 2 Setting a Baseline for Baseline Correction of a Single Spectrum Select the spectrum which is to be baseline corrected Click on the Baseline icon to open the Baseline MA Apt dialog window Page 118 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific H
3. drop down box The apodization reaches zero at the Limit position Four modes are available No no apodization Line linear apodization function Sart parabolic apodization function Cos cosine apodization function O O O O Filter Select whether a filter will be used for the Fourier fite Na transformation using the Filter drop down box Two modes are available o No no filter o Traffic traffic filter OK Click OK to permanently apply the smoothing to Ie the active spectrum and close the Fourier Transform dialog window Cancel Click Cancel to close the Fourier Transform Cancel without applying the smoothing The original spectrum will be left unchanged 4 6 5 2 Using the Fourier Tranform Function to Smooth a Spectrum Select a spectrum to be smoothed Open the Fourier Transform dialog window by PA pr clicking on the Fourier Transform icon in the Icon Bar Page 128 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA Explore the future HORIBA Scientific The real and imaginary Fourier functions of the active spectrum are displayed in the Fourier Data window Select the Apodization and Filter functions to be used from the Apod and Filter drop down boxes Set the limit for high frequency contribution which will be removed from the spectrum The spectrum is continuously updated allowing
4. Loren Lorentzian GaussLoren mixed Gaussian Lorentzian o Area the area of the peak in area units based on the units displayed on the spectrum s X and Y axes The Area column is only displayed if the Use area box is ticked in the main Peaks dialog window see section 4 6 7 1 page 134 o Fix the Fix tick boxes to the right of each of the p a w and g parameters allows a parameter to be fixed A fixed parameter will not be varied during the peak fitting routine When a box is ticked the parameter is fixed When a box is unticked the parameter will be varied during the fitting routine o Map the Map tick boxes which are only displayed for the Splm window of a multidimensional spectral array to the right of each of the p a w g and Area parameters allows a profile or map image to be generated based on the parameter For example it is possible to create an image based on peak position illustrating how the peak position varies across the map area To display a profile image based on a peak parameter tick the appropriate box and click Apply A new map profile image will be created To close the map profile image untick the box and click Apply Copy Click on Copy to copy the parameters displayed L in the Peak Parameters dialog window Cw Parameters which have been copied can be pasted into other programmes such as Microsoft Office or into the Peak Parameters dialog window This fu
5. Options section and subtract it Fit Click on Fit to automatically fit a baseline to the Fit active spectrum based on the parameters set in the Options section The best fit baseline curve will be displayed on the spectrum It can be subtracted by clicking on Sub Copy Click on Copy to copy the displayed baseline Copy curve to the baseline clipboard Paste Click on Paste to paste a baseline curve from the Paste baseline clipboard onto the active spectrum Page 116 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific 4 6 2 1 1 Baseline Correction Options The Baseline options control the parameters of the baseline that will be used for baseline correction Baseline M Options Type Palan Clear Degree x Convert E Attach Yes Auta Style 7 Copy Paste Help Fe EEE Close g Type Select the algorithm used to define the baseline curve o Polynom fits a polynomial curve through the baseline points set on the spectrum The degree of the polynomial is set in the Degree drop down box o Lines fits a straight line between baseline points set on the spectrum Degree Select the degree of polynomial equation used to create the baseline curve this option is only used when the baseline Type is set to Polynom
6. Use data style is ticked the display color of the peak label text and arrow marker will be set to match the spectrum color If Use data style is unticked the color will be set according to the selection made in the respective Style drop down box Shape multicolor If Shape multicolor is ticked the individual peak shape display colour will be automatically selected from a default palette in this case when multiple shapes are displayed on a single spectrum each shape will be a different color If Shape multicolor is unticked the colour will be set according to the selection made in the Style drop down box in this case when multiple shapes are displayed on a single spectrum each shape will be the same colour Attach arrow If Attach arrow is ticked the peak arrow marker will be positioned immediately above the peak If Attach arrow is unticked the peak arrow marker will be positioned at the top of the spectrum window 4 6 7 5 Peak Functions Dialog Window The Peak Functions dialog window allows custom peak fitting formulae to be defined so that shapes other than the default Gaussian Lorentzian and mixed Gaussian Lorentzian can be used For example with the Peak Functions dialog window peak shapes such as Voigt or asymmetric Gaussian can be used Page 144 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user ma
7. Graphical Manipulation toolbar see section 5 10 page 172 Init Click on Init to restore peak parameters in the Init Splm window of a multidimensional spectral array to the initial values before the peak approximation Approx or fitting Fit routines were run Clear Click on Clear to clear all peaks from the active Clear spectrum Convert Click on Convert to convert the active spectrum Eanes to the sum of the displayed peaks This function is useful to save a theoretical peak fit solution in a standard spectrum file format Note that the active spectrum will be overwritten by the sum of the displayed peaks Make sure that the file is saved with a different name to ensure the Original spectrum data is not permanently overwritten and lost Peaks Click on Peaks to open the Peak Parameters Peaks dialog window to view and manually set peak position amplitude full width at half maximum height FWHM Gaussian contribution and area values for all peaks labelled on a spectrum Page 137 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductar Scientific HORIBA HORIBA ws Scientific See section 4 6 7 2 page 138 for more information Variables Click on Variables to open the Peak Variables vajahles dialog window to view and set initial values and maximum minimum values for variables within the peak fitting routine See sect
8. Sum C Area Max Page 123 HORIBA HORIBA niai Scientific 4 6 4 Smoothing ray am Y Opens the Filtration dialog window The smoothing or filtration functions allow spectra to be smoothed converted to first and second derivative functions or despiked Typically these functions allow spectral quality to be improved after acquisition 4 6 4 1 Filtration Dialog Window The Filtration dialog window allows the smoothing and processing functions to be configured and performed on both single spectra and multidimensional spectral arrays Filtration m Denoise Standard Apply Matris operations 4 6 4 1 1 Denoise The Denoise function is a unique noise reduction algorithm which can be used to significantly enhance spectrum quality without losing subtle spectral information Standard smoothing functions can result in loss of peak shape and position and subtle features such as weak shoulders on a strong band can be lost The Denoise function ensures that all this important information is retained whilst still reducing noise in the spectrum The spectra shown right illustrate the effect of the Denoise function Page 124 Explore the future Automotiva Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific Two main Denoise algorithms are available from the Denoise drop down box o Standard recomme
9. as follows where a represents the spectral axis a gt 1 2 3 4 5 6 7 8 x gt 10 12 15 25 22 13 10 7 y gt 2 3 2 4 3 Page 132 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific applying the function x y to this data will result in the following values a gt 2 3 4 5 6 7 8 x gt 11 14 18 27 23 14 14 10 y gt 2 3 2 4 3 As another example if Func 1 x 2 a x refers to the intensity value of each pixel of the active spectrum and a refers to the first axis i e the spectral axis with units Raman shift cm value of each pixel Assume the data has the following values a gt 1 2 3 4 5 6 7 8 x gt 10 12 15 25 22 13 10 7 applying the function x 2 a to this data will result in the following values a gt 1 2 3 4 5 6 7 x gt 12 16 21 33 32 25 24 Func 1 or Func 2 select the appropriate active spectrum corresponding to x in the function and click on the adjacent Go button 8 23 Type in the desired arithmetic function for either ppe fo If there are multiple options for the spectrum y a Select Y Select Y message box will ask for the desired 11800_532_300 010 l spectrum to be selected Click on the desired 800_532_300_010 Cancel spectrum and then click OK to complete the arithmetic procedure Combine The Combine function al
10. information about Spectral ID and how it can be used and configured 4 6 1 1 Configuring LabSpec 5 for Correct Spectral ID Searching The export procedure for the one click Spectral ID search is defined by the options set in the File gt Save As dialog window for a Grams spc file To ensure the one click Spectral ID search performs correctly please complete the following set up procedure in LabSpec 5 Open a spectrum in any format o Click on File gt Save As Select Grams spc from the Save as Type drop down box o Tick the box for Even spaced o Click Cancel If the Even spaced box is not ticked the Spectral ID search will not be performed correctly and spurious results may be displayed Page 114 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific 4 6 2 Baseline Correction Opens the Baseline dialog window Baseline correction allows a high background in a spectrum to be subtracted to yield a spectrum with a flat zero baseline The correction can be applied to a single spectrum or a multidimensional spectral array of spectra including time profiles Z depth profiles temperature profiles XY maps XZ and YZ slices and XYZ datacubes The example shown right illustrates a spectrum before and after baseline correction 1 000 2 000 3 00
11. on Zero to automatically subtract a constant Bete intensity value from the active spectrum or multidimensional spectral array so that the lowest intensity pixel is at zero When applied to a multidimensional spectral array each spectrum in the array is zeroed independently Get Click on Get to load the active spectrum in the Get main LabSpec 5 graphical user interface GUI into 39 the Corrector window Delete Click on Delete to clear the Corrector window Delete and remove the corrector spectrum displayed there Page 121 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA es Scientific Subtract Click on Subtract to subtract the corrector Subtract spectrum displayed in the Corrector window from the active spectrum or multidimensional spectral array Multiply Click on Multiply to multiply the active spectrum Multiply or multidimensional spectral array by the corrector spectrum displayed in the Corrector window Correct Click on Correct to correct the active spectrum or nae multidimensional spectral array for the contribution of the corrector spectrum displayed in the Corrector window The intensity of the corrector spectrum will be automatically adjusted before subtraction to best fit the spectrum If the Limits tick box is ticked the corrector i
12. the future Automotiva Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA annii scientific The example right shows the result of applying 10 000 Const 2 5 to a spectrum sane 6 000 4 000 Intensity cnt 2 000 0 10 000 8 000 6 000 4 000 Intensity cnt 2 000 The Const function can also be applied using the Multiply by Constant icon in the Graphical Manipulation toolbar see section 5 9 page 171 si Func 1 and Func 2 The Func 1 and Func 2 sections allow arithmetic functions to be created by the user and applied to the active spectrum The terminology used for these functions are as follows o Xand y refer to the intensity values of spectra which are open in LabSpec 5 x is the active data file and y is the other data file open in LabSpec 5 In the event that there is more than one spectrum which could be used for y a message box will ask for the y spectrum to be chosen from a list a b and c refer to the values of the first second and third axes of the active data file o Standard arithmetic functions are also possible including exp log sin asin cos acos tan atan abs sqrt etc As an example if Func 1 x y x refers to the intensity value at each pixel of the active spectrum and y refers to the intensity value at each pixel of another open spectrum Assume the two spectra have values
13. to Rectangle i close the dialog window afitth Height it aa 2d Min tine to cover zone 0 001 ae Set the DuoScan module as the active XY stage using the switch stage icon in the XYZ Coord section of the Control Panel see section 9 10 2 page 237 The laser spot position can now be controlled by inputting XYZ coordinates in the XYZ Coord section of the Control Panel see section 9 10 page 236 or by acquiring a Raman XY map multidimensional spectral array in the standard way In both cases the laser spot is now moved across the sample using the DuoScan mirrors The sample does not move Page 113 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA malian Scientific 4 6 Data Processing and Analysis Icons 4 6 1 Spectral ID Search 2 g Launch the one click Spectral ID database search for the active spectrum Clicking on this icon will initiate the following processes Export the active spectrum in Grams spc format Open Spectral ID Load the exported spectrum into Spectral ID Run a full soectrum matching search through the active databases Report the list of matching spectra with match quality scores O O O O Please see the HORIBA Scientific manual Using the Spectral ID Database Software with LabSpec 5 and full documentation provided with the Spectral ID database software for more
14. width at half maximum height FWHM and area Overlapping peaks can be fully deconvoluted through the peak fitting routine The Peak Searching and Fitting functions can be performed on both single spectra and multidimensional spectral arrays 4 6 7 1 Peaks Dialog Window The Peaks dialog window allows the peak searching labelling and fitting processes to be configured and applied and displays the peak parameters after fitting Options Search Function GaussLoren Approx W Use area F mr l Search Levell Size prit 3 Convert Peaks Clear e HEEL Fitting i l Variables Iteration 100 Options li Skip prt 0 Error 1 7371 7e 005 W Baseline Functions Help Close Page 134 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific Peak Options Select the appropriate function to be used for peak Options fitting from the Function drop down box Three Function GaussLoren default functions are provided We Use area o Gaussian o Lorentzian o Mixed Gaussian Lorentzian Other functions can be defined by clicking on Functions in the Peaks dialog window see 4 6 7 5 page 144 for full information If Use area is ticked the peak fitting routines will also calculate the area of the peak s When Use ar
15. 0 Raman Shift cm 1 000 2 000 3 000 Raman Shift cm 4 6 2 1 Baseline Dialog Window The Baseline dialog window allows the baseline correction to be configured and performed Baseline Options Type Clear Degree Convert Bell mecan ee Te Attach Auta Style mm Copy Paste Help Close gu Page 115 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA es Scientific Options Set the parameters for the baseline Please see Options section 4 6 2 1 1 page 117 for information about T Pal the individual parameters and their affect on the z ee r baseline correction egee 5 Attach Yez Style Sub Click on Sub to subtract the current baseline from a the active spectrum Clear Click on Clear to clear the current baseline from Fs the active spectrum Convert Click on Convert to convert the active spectrum ama to the displayed baseline curve This function is useful to save a baseline curve in a standard spectrum file format Note that the active spectrum will be overwritten by the baseline curve Make sure that the file is saved with a different name to ensure the original spectrum data is not permanently overwritten and lost Auto Click on Auto to automatically fit a baseline to the ae active spectrum based on the parameters set in the
16. A ti Opens the Correction dialog window The correction functions allow spectra to be normalized and zeroed and for a substrate contaminant spectrum to be automatically subtracted 4 6 3 1 Correction Dialog Window The Correction dialog window allows the user to apply spectral normalization and correction to both single spectra and multidimensional spectral arrays Page 120 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific Correction i Get Delete Intensity cnt Subtract 2000 Mutioly Raman Shift erm 7 Corect Limits and Threshold From 240 62 To 2115 8 M Threshold 10 Normalisation Mode Sum Ares C Max Get Delete Subtract Mutiny Conect Normalize Click on Normalize to normalize each spectrum Normalise so that the total area sum or maximum signal of the spectrum is 100 If the Limits tick box is ticked the normalization will only be applied within the displayed limits In this case the spectrum will be normalized so that the area sum or maximum signal within the limits is 100 The limits can be adjusted by typing values in the Limits boxes or adjusting the cursor positions in the Corrector window The normalization mode can be selected from the Normalization section of the dialog window Zero Click
17. Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific Select Hor or Ver from the drop down box in the Profile Profile dialog window to create an intensity profile in the horizontal X axis direction or vertical Y axis direction respectively is E a E The profile is displayed in the Profile dialog window Standard data processing functions such as smoothing peak fitting or baselining and copy paste functions can be used with this profile Click Close to close the Profile dialog window Cose 4 6 9 Map Analysis Lab em t Opens the Map Analysis dialog window The Map Analysis dialog window displays the positions and settings for the Red Green and Blue cursors which are used to create intensity profiles and images from multidimensional spectral arrays including time profiles Z depth profiles temperature profiles XY maps XZ and YZ slices and XYZ datacubes The positions and settings can also be manually configured in this dialog window Map Analysis Opti phioris 7 Bazeline From To Red iv 1316 34 1343 52 Load Green E horz 1138 13 ene Blue iw 999 1 a4 1 0559 6 Green Blue Convert Help Spectrum y Corect Cancel Use If the Use box is ticked for a set of cursors Red Green or Blue then a profile or image will be generated displaying the average intensity between the
18. HORIBA asain Scientific o Mapping Pixel the scan area is set according to the pixel size specified in the Mapping Properties dialog window see section 4 5 5 page 97 The Width and Height boxes will be inactive and greyed out since the values are taken directly from the Mapping Properties dialog window Note that if SWIFT ultra fast Raman mapping is activated see section 4 5 5 page 97 the Width value will be set to 0O because SWIFT s continuous scanning in the X direction does not require DuoScan scanning in the X width direction o Video Cursor the scan area is set according to the dimensions and position of the Rectangular Mapping cursor see section 5 19 page 187 on the video window The Width and Height boxes will be inactive and greyed out since the values are taken directly from the video Rectangular Mapping cursor Click Start to activate the DuoScan scanning mirrors If the scan area has been set greater than the maximum allowable dimensions a warning message will be displayed This shows the maximum negative and positive displacement possible for the selected objective The area size set in the Height and Width boxes or defined according to the map pixel or defined by the rectangular video cursor must not be greater than the total displacement In the example shown right the maximum displacement is 10 um to 10 um meaning a total disp
19. ORIBA HORIBA Scientific Select the baseline parameters within the Options section See section 4 6 2 1 1 page 117 for full details about the Baseline Correction Options The options can be modified at any point during the process of creating the baseline the displayed baseline will immediately update Click on Fit to fit a baseline curve to the spectrum Baseline points can be manually inserted adjusted and removed by using the Add baseline points icon see section 5 14 page 180 and Remove baseline points icon see section 5 15 page 181 in the Graphical Manipulation Toolbar o Click on the Add baseline points icon The cursor will change from the mouse cursor to the Add Baseline Points cursor Left click on the spectrum to add a baseline point to the displayed baseline curve lf there is no baseline curve on the spectrum the first left mouse click in this mode will create the baseline Hover the cursor over an existing baseline point When the cursor changes from the Add Baseline Points cursor to the Adjust Baseline Points cursor left click to drag the baseline point to a new position o Click on the Remove baseline points Icon Hover the cursor over an existing baseline point When the cursor changes from the mouse cursor to the Remove Baseline Points cursor left click to delete Baseline Options Type Degree Attach Style Fit yh
20. ach arrow The following peak labelling and fitting display components can be controlled Text Click on the Text Style drop down box to set the font font style font size and font color used to display the peak position on the spectrum If Show is ticked the peak position will be displayed on the spectrum Note that if Use data style is ticked the colour will be set to match the spectrum color If Use data style is unticked the colour will be set according to the selection made in the Style drop down box Arrow Click on the Arrow Style drop down box to set the colour width and line style for the arrow marker indicating the peak position If Show is ticked the arrow marker will be displayed on the spectrum Note that if Use data style is ticked the colour will be set to match the spectrum color If Use data style is unticked the colour will be set according to the selection made in the Style drop down box Shape Click on the Shape Style drop down box to set the colour width and line style for the individual peak shape s displayed on the spectrum If Show is ticked the peak shape s will be displayed on the spectrum Note that if Shape multicolor is ticked the colour will be automatically selected from a default palette in this case when multiple shapes are displayed on a single spectrum each shape will be a different color If Shape multicolor is unticked th
21. appearance Page 138 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific Formula Area Map GaussLoren 239521 GaussLoren 243576 p a Map i GaussLoren 23584 5 1123 54 Fan 43 1213 64 Tang 43 1331 63 6963 98 141 6898 9 1505 39 6246 16 1610 51 5597 4 GaussLoren 237322 5 GaussLoren 218590 1 GaussLoren 20005 8 Ld Each row displays the parameters for a single peak o p peak position in units as displayed on the spectrum s X axis typically Raman shift cm or nanometers nm o a peak amplitude in units as displayed on the spectrum s Y axis typically counts cnt or counts per second cnit s o W peak full width at half maximum height FWHM in units as displayed on the spectrum s X axis typically Raman shift cm or nanometers nm o g Gaussian contribution in a mixed Gaussian Lorentzian function The value of Gaussian contribution varies from O no Gaussian contribution fully Lorentzian through to 1 fully Gaussian The g column is only displayed when a mixed Gaussian Lorentzian function is selected for peak fitting in the main Peaks dialog window see section 4 6 7 1 page 134 o Formula the function used for the peak fitting as selected in the main Peaks dialog window see section 4 6 7 1 page 134 Gauss Gaussian
22. cified first using the Baseline dialog window see section 4 6 2 1 page 115 Search Click on Search to start the automatic peak n searching and identification routine Adjust the Size pnt and Level to control the searching and identification procedure see above for more information about these parameters Approx Click on Approx to run the peak approximation Approx routine which can be used to estimate the initial peak parameters prior to fitting Only the peak position and width parameters are adjusted Page 136 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific The peak approximation is a useful function to assist in complex peak fitting procedures Running the peak approximation routine prior to full fitting ensures that the starting parameters are realistic and close to their true values This reduces the possibility of the peak fitting routine locating an incorrect solution Fit Click on Fit to run the peak fitting routine which Fit can be used to calculate peak position amplitude full width at half maximum height FWHM Gaussian contribution and area The peak fitting routine can only be used if peaks have been located in the spectrum Peaks can be located automatically using the Search button or manually by using the Add peak icon in the
23. e colour will be set according to the selection made in the Style drop down box in this case when multiple shapes are displayed on a single spectrum each shape will be the same colour Sum Click on the Sum Style drop down box to set the colour width and line style for the sum spectrum i e the combination spectrum created by summing all the peak shapes displayed If Show is ticked the sum spectrum will be displayed on the spectrum Residual Click on the Residual Style drop down box to set the colour width and line style for the residual spectrum i e the difference between the sum spectrum and the raw data If Show is ticked the residual spectrum will be displayed on the spectrum Page 143 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA aan Scientific Format Click on the Format left hand Style drop down box to set the number of display characters for the nee peak position value sz Hep Arrow Cancel Shape Sum Residual Format lo V Use data style V Shape multicolor WV Attach arrow Click on the Format right hand Style drop down box to set the number of decimal places to be Heet ae displayed for the peak position value e setae Help Shape Sum Residual Format Auto gt io V Use data style V Shape multicolor MV Attach arrow Use data style When
24. ea is unticked only the default peak parameters will be calculated These are peak position amplitude i e maximum height full width at half maximum height FWHM and for mixed Gaussian Lorentzian functions the degree of Gaussian contribution Search Options Set the parameters used for automatic peak search searching and identification The search routine Level O00000 locates local intensity maxima and assigns these as peaks Size pnt 5 A local maximum must be greater than a certain E percentage of the maximum intensity in the whole spectrum The Level parameter defines this percentage of maximum spectral intensity Typically as the Level is increased only the most intense peaks will be identified If low intensity peaks need to be identified Level should be reduced A local maximum is assumed to exist within a finite number of adjacent data points The Size pnt parameter defines this number Typically as the Size pnt is increased only peaks which are widely separated will be identified If close lying peaks need to be identified Size pnt should be reduced The Level and Size pnt values can be set by typing a value in the appropriate box or using the appropriate scroll bar The peak labelling displayed on the active spectrum will update continuously so that the result can be monitored Page 135 Explore the future Automot
25. elf The profile is created in either a horizontal X axis or vertical Y axis direction in the image depending on the selection made in the drop down box is E a E o Hor horizontal X axis o Ver vertical Y axis Corr Click on Corr to modify the image based on Cor manipulation made to the profile For example if the profile displayed in the Profile dialog window is smoothed clicking on Corr will apply the same smoothing function to the entire image in the horizontal or vertical dimensions according to the selection of Hor or Ver in the drop down box 4 6 8 1 Displaying an Intensity Profile from an Image To display an intensity profile from an image such as optical image or two dimensional Raman mapped image open the image file In the case of a two dimensional Raman mapped image click on the image window either Map or Score Select the component from which the profile is to be created using the tags in the right hand Data bar see section 6 3 page 198 Ensure the cursor mode in the image window is set to Cross right click and select Cursor and then choose Cross from the Style drop down box Open the Profile dialog window by clicking on the Fa Profile icon in the Icon bar Position the cursor at the point of the image from where the profile is to be created Page 146 Explore the future Automotive Test Systems Process amp
26. ental Medical Semiconductor Scientific HORIBA HORIBA ws Scientific Double click on the parameter name box to allow Variables the name to be edited Type in the desired category name Cancel Click on any other parameter name box to register ESZE the new name oO Zzalo o o s o a ia e ila e le elal The name given to the new parameter must exactly match the name used in definition of functions in the Peak Functions dalog window see section 4 6 7 5 page 144 The init min and max values must be set to appropriate values 4 6 7 3 3 Deleting Parameters from the Variables Dialog Window Parameters can be deleted by right clicking on the vannis parameter name boxes which is to be deleted and selecting Remove row w 0 001 Cancel 0 m mary iT miny tu mary iA n The parameter will be deleted from the Variables Variables 2 Cancel dialog window AATA Ar Aa Page 142 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific 4 6 7 4 Peak Options Dialog Window The Peak Options dialog window allows control of the display options for the peak labelling and fitting Options Style Text 16 Times Arrow Shape Sum Residual Format Auto 10 z V Use data style l Shape multicolor WV Att
27. essary to update the actual data in the array by clicking on Correct If this is not done when the cursor on the profile image is moved the modifications will be lost the next time the spectrum is displayed in the Point window it will return to its original form Page 148 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA
28. ible in the Corrector window click on the Center Cursors icon in the Icon bar Limits and Threshold Set the limits for Normalize and Correct functions ticking the Limits tick box and typing the From minimum and To maximum limit values The limits can also be set by adjusting the position of the cursors in the Corrector window As the cursors are moved the From and To values shown in the Limits and Threshold section are continuously updated Set the threshold value by typing in the desired level in percent in the Threshold box Normalization Select the normalization mode for the Normalize function o Sum normalize so that the sum of the spectrum intensity is 100 o Area normalize so that the area of the spectrum is 100 o Max normalize to the maximum intensity point in the spectrum Limits and Threshold Aulomotive Test Systems Process amp Environmental Medical Semiconductor Scientific LabSpec 5 user manual Correction FeO e Delete Intensity ont Subtract pz _ se pee sieve 5 2000 Multiply Raman Shift cm 4 Correct Threshold From 1240 62 To 2115 8 k Threshold 10 are Help Normalisation Mode Sum C Area C Max Close Hell e t Limits and Threshold From 240 62 To 2115 8 v Threshold 10 Limits and Threshold From 240 62 To 21158 Threshold 10 NSormalisation Mode f
29. ing can remove or alter features in a spectrum Median smoothing should be used with care Despike Click on Despike to remove random spikes also Despike known as cosmic rays from a spectrum A spike is calculated as a pixel which has an intensity greater than the average spectrum intensity Height The Despike function removes the spike and replaces it with a weighted average of the surrounding pixels Note that in some cases the Despike function can remove or alter features in a spectrum particularly when the spectrum is comprised of very sharp peaks Despike should be used with care Please also see section 3 5 4 7 page 37 for more information about other spike filter options in LabSpec 5 Page 126 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific 4 6 5 Fourier Transform fim bt Opens the Fourier Transform dialog window The Fourier Transform function allows smoothing of a spectrum based on direct Fourier data transformation applying filter and apodization functions The spectrum is converted into its real and imaginary Fourier functions which essentially represents the spectrum as a combination of wave patterns of varying frequency Smoothing can be applied by removing high frequency contribution corresponding to noise and leaving medium and low frequency contribution corres
30. ion 4 6 7 3 page 140 for more information Options Click on Options to open the Peak Options Dire dialog window to view and set display options for the peak labelling and fitting See section 4 6 7 4 page 143 for more information Functions Click on Functions to open the Peak Functions Functions dialog window to view and create user defined peak shape functions See page 4 6 7 5 page 144 for more information 4 6 7 2 Peak Parameters Dialog Window The Peak Parameters dialog window displays peak position amplitude full width at half maximum height FWHM Gaussian contribution and area for all peaks labelled on a spectrum The parameters can be manually adjusted and fixed so that the parameter is not varied during the fitting routine The Peak Parameters dialog window for individual spectra e g Spectrum window or Point window of a multidimensional spectral array has the following appearance a i S ix Fis Formula Area 1509 82 0 047 2904 10 2257 1 GaussLoreni 0 30009 1619 5 0 037 9798 9 25232 1 P GaussLoreni 0 210299 1565 0 243844 17 8693 0 55855 P GaussLoreni 3 6618 1613 09 0 390541 14 838 0 0919826 GaussLoreni 5 72321 1622 61 0 375234 12 9164 0 52442 GaussLoreri 4 15512 1653 15 0 253714 18 9257 O6f0445 GaussLoreri 5 32977 The Peak Parameters dialog window for multidimensional spectral arrays e g the Splm window of a multidimensional spectral array has the following
31. ive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA malian Scientific Fitting Options Set the parameters used for the peak fitting Fitting routine This routine uses a Levenberg Marquarat teratom mo o ooo non linear peak fit algorithm and iteratively adjusts all peak parameters to minimise the standard error Skip pnt O E 1 73717 005 The maximum number of iterations can be set by ii typing an appropriate number in the Iteration box W Baseline Typically the larger the iteration number the more accurate the final fit result will be but the longer the process will take The algorithm can be set to miss out data points within the spectrum in order to speed up the process Skip pnt is used to define how many points are missed o Skip pnt 0 all data points are used for the fitting o Skip pnt 1 every second data point is used for the fitting routine o Skip pnt 2 every third data point is used for the fitting routine Typically as Skip pnt is increased the fit results will be less accurate but the process will be faster The Error box displays the Standard Error between the fit result and the raw data The smaller the Standard Error the more accurate the fit result If the Baseline box is ticked the peak fitting routine will additionally fit the specified baseline The baseline must be spe
32. lacement of 20 um DuoScan Zone is out of limits 10 10 pm The Raman measurement spectrum or multidimensional spectral array can now be set up and acquired in the normal way Remember that the spectrum recorded will be an average spectrum from the scanned area Click Stop to stop the DuoScan scanning mirrors Page 112 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific 4 5 10 1 2 Point Mode When DuoScan is used in Point Mode it is possible to move the laser spot to any position on eeeeeeece the sample surface within the maximum possible A cue bum scan displacement In this mode DuoScan can eeeeoecece be used as a substitute to the standard motorized Ae ypte gt stage for point by point XY mapping multidimensional spectral array acquisition this function is particularly useful when it is not possible to move the sample underneath the laser beam or ultra fine step size is required down to DuoScan s minimum step size of 50 nm To use DuoScan in Point Mode the scanning T T mirrors must be static To confirm this open the DuoScan XY scanning dialog window by clicking on the DuoScan icon If the DuoScan mirrors are scanning the STOP XY Scanning ke button will be active Click on STOP to stop the m Zone DuoScan scanning and then click CLOSE
33. lows individual spectral windows in an Extended Range spectrum acquisition to be glued together to yield a single spectrum This process can also be applied automatically during an Extended Range acquisition see section 3 5 6 page 49 Equally the Combine function can be used to create an average spectrum from all open spectra Set the options as desired Iv Adiust intensity levels p Combine l z Go Mw Remove combined datas o Adjust intensity levels if this box is ticked the baselines of the individual spectral windows will be adjusted prior to gluing Page 133 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA malian Scientific to yield a seamless final spectrum See section 3 5 6 3 page 53 for full information about this mode o Remove combined datas if this box is ticked the individual spectra or spectral windows will be deleted after combination leaving only the single combined spectrum on screen Combine Click on the adjacent Go button to apply the M Adjust intensity levels Combine process i Remove combined datas 4 6 7 Peak Searching and Fitting oy ut Opens the Peaks dialog window The Peak Searching and Fitting module allows peaks in a spectrum to be automatically labelled by their position and full peak fitting can be carried out to fully characterise peak parameters such as position amplitude full
34. nction can be used to copy peak parameters from one spectrum and paste them to another spectrum using Paste Page 139 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA SoVentittse Paste Click on Paste to paste parameters in the Peak Paste Parameters dialog window This function can be used to paste peak parameters copied using Copy from one spectrum to another Apply Click on Apply to update the peak s displayed Aaa on the spectrum according to parameters in the Peak Parameters dialog window This button must be used when parameters are manually adjusted in the Peak Parameters dialog window The peaks displayed on the spectrum will not reflect the new parameters until Apply is clicked 4 6 7 3 Peak Variables Dialog Window The Variables dialog window displays the initial parameters used in the peak fitting procedure and the minimum and maximum values they can take during the fitting procedure In most cases the default values are suitable for general peak fitting routines but in specific cases the initial parameters and their minimum and maximum values can be manually adjusted as required Warables The Variables dialog shows the initial init values and minimum Min maximum Max values for each parameter o p peak position in units as displayed on the spectrum s X axis t
35. nd replaces those pixels with the polynomial curve The window where this operation is applied is moved across the entire spectrum The Degree and Size must be set to an appropriate level Typically the smaller the Degree and the larger Size the more significant the smoothing Note that in some cases smoothing can remove or alter Page 125 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductar Scientific HORIBA HORIBA malian Scientific features in a spectrum Smoothing should be used with care Der1 Click on Der1 to convert the active spectrum to Del its first derivative function At each pixel position the derivative is calculated using a defined range Size of pixels either side of it Der2 Click on Der2 to convert the active spectrum to De2 its second derivative function At each pixel position the second derivative is calculated using a defined range Size of pixels either side of it Median Click on Median to apply a non linear median Median smoothing function to the active spectrum Median smoothing replaces a spectrum pixel intensity value by the median of intensity values within a defined range Size either side of it This replacement process is repeated for all pixels in the spectrum Typically the larger the Size the more significant the median smoothing Note that in some cases median smooth
36. nded for spectra with signal to noise 2 20 o Lite recommended for very noisy spectra with signal to noise lt 20 In addition both algorithms can be used with an integrated Despike function to remove random spikes also known as cosmic rays See also section 3 5 4 7 page 37 for more information about other spike filter options in LabSpec 5 Note that the Denoise function can be automatically applied to all acquired data through the Acquisition gt Options dialog window see section 3 5 4 15 page 46 If a spectrum has had the Denoise function automatically applied through Acquisition gt Options it cannot have the function applied again through the Filtration dialog window Using the Denoise Function Select the desired Denoise algorithm from the Filtration Denoise drop down box Denoise lt tandard Standard Des ike Er Lite Lite Despike Smooth D sd Der size 3 v Der2 Height 30 Median Despike Help Close Click Apply to apply the Denoise algorithm to the Apply active spectrum 4 6 4 1 2 Matrix Operations The functions described below require the Degree Size and Height values to be set in the drop down boxes as described in the text Smooth Click on Smooth to apply a Savitsky Golay Smooth smoothing function to the active spectrum Savitsky Golay smoothing fits a polynomial function of a specified Degree through a range Size of adjacent pixels a
37. ntensity adjustment will be calculated only within the displayed limits The limits can be adjusted by typing values in the Limits boxes or adjusting the cursor positions in the Corrector window This function is useful if a substrate diluent or contaminant spectrum needs to be removed from a sample spectrum Threshold Click on Threshold to threshold the active Threshold multidimensional spectral array The threshold function will be applied to any spectra within the array which has a maximum intensity relative to the most intensity spectrum in the array less than the displayed threshold value set in the Limits and Threshold section The threshold function will convert the spectrum to have zero intensity throughout its spectral range Page 122 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA Explore the future HORIBA Scientific Corrector Window The current corrector spectrum will be displayed in the Corrector window Click on Get to load the active spectrum in the main LabSpec 5 graphical user interface GUI into the Corrector window Click on Delete to clear the Corrector window and remove the corrector spectrum displayed there The spectrum in the Corrector window can be manipulated using the standard Graphical Manipulation Toolbar icons see section 5 page 164 If the cursors are not vis
38. nual HORIBA Scientific Shapes Formula To define a custom peak shape formula type the shape name in a Name text box and input the formula in the Formula text box The name will be displayed in the main Functions drop down box in the main Peaks dialog window Any parameter can be used but it must also be defined as a parameter in the Peak Variables dialog window see section 4 6 7 3 page 140 Note that certain parameters are predefined o p peak position in units as displayed on the spectrum s X axis typically Raman shift cm or nanometers nm o a peak amplitude in units as displayed on the spectrum s Y axis typically counts cnt or counts per second cnt s o W peak full width at half maximum height FWHM in units as displayed on the spectrum s X axis typically Raman shift cm or nanometers nm 4 6 8 Profile E g Opens the Profile dialog window The Profile dialog window displays an intensity profile across an image such as optical image or two dimensional Raman mapped image Profile Profile z E E Page I 145 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA es Scientific Profile The Profile window displays the image intensity Profile profile at the cursor position The scales for the X and Y axes are taken directly from the image its
39. ponding to Raman peaks The Fourier Transform smoothing function can be performed on both single spectra and multidimensional spectral arrays 4 6 5 1 Fourier Transform Dialog Window The Fourier Transform dialog window displays the Fourier functions of the active spectrum and provides Apodization and Filter options for the transformation Fourier Fourier Data Ok aa Help c Cancel Options Lirnit 37 7358 Go Apod No Filter No Fourier Data The Fourier Data window displays the real and Fourier Data i A za imaginary Fourier functions of the active spectrum sae 100 The red cursor can be used to set the high frequency limit frequencies above the limit position will be removed from the spectrum Intensity Typically the lower the cursor position the more A smoothing is applied At position 0 the spectrum is i 50 100 fully smoothed to a flat line At position 100 the spectrum is fully unsmoothed Page 127 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA Tews Scientific Limit The Limit allows manual control of the limit cursor Limi 37 7358 Go displayed in the Fourier Data window Type in the required cursor position value ranging between 0 and 100 and click on Go Apod Select the type of apodization to be used in the spo No Fourier transformation from the Apod
40. the degree of smoothing to be monitored The limit can be set in two ways o Click and drag the red cursor in the Fourier Data window o Type in the required limit into the Limit box and click Go When the desired smoothing is achieved click OK to permanently apply the smoothing to the active spectrum and close the Fourier Transform dialog window Alternatively click Cancel to close the dialog window without applying any smoothing to the spectrum The spectrum will be left unchanged LabSpec 5 user manual Fourier Fourier Data x10 Options Limit 37 7358 Go Apod No v Filter No 7 r Options Fourier Fourier Data x10 Options Limit 137 7358 Go Apod No Filter No Fourier Fourier Data x10 N Options Limit 37 7356 Go Apod No 7 Filter No v Page 129 Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA annia Scientific 4 6 6 Math tr RES 23 Opens the Math dialog window Arithmetic functions can be applied to the intensity values of an active spectrum Additionally the Extended Range spectral acquisition Combine data function can be applied post acquisition through the Math dialog window The Math functions can be performed on both single spectra and multidimensional spectral arrays 4 6 6 1 Math Dialog Window The Math dialog window contains text input boxes so that arithme
41. tic functions can be created and then applied to the active data Consat Const Func Func 2 W Adjust intensity levels Combine _ l e Remove combined datas He Const The Const function adds a constant intensity value to all pixels in the active spectrum Type in the desired constant and click on the kons E adjacent Go button to apply the function to the active spectrum A positive constant will be added to the spectrum intensity values a negative constant will be subtracted from the spectrum intensity values Page 130 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific The example right shows the result of applying Const 1200 to a spectrum 10 000 Intensity cnt 5 000 10 000 5 000 Intensity cnt The Const function can also be applied using the Add Constant icon in the Graphical Manipulation toolbar see section 5 8 page 170 Const The Const function multiplies all pixel intensity values in the active spectrum by a constant value Type in the desired constant and click on the adjacent Go button to apply the function to the active spectrum A constant greater than zero will increase the spectrum intensity whilst a constant less than zero will decrease the spectrum intensity Const Page I 131 Explore
42. two cursors The cursor positions are set by clicking the Red Green or Blue cursor icons in the left hand Graphical Manipulation bar see section 5 2 page 165 and dragging the cursors to the desired positions on either the Splm or Point windows or by typing in desired values into the From and To boxes in the Map Analysis dialog window Note that if Use is unticked for all three cursors and Green Blue is also unticked the Map window will not be displayed To redisplay the Map window make sure that at least one of the Use boxes are ticked or that the Green Blue box is ticked Page 147 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA HORIBA ws Scientific Baseline If the Baseline box is ticked for a set of cursors Red Green or Blue then the cursor region is first baselined before calculation of the average intensity between the cursors This mode is useful to ensure that the image created truly reflects peak intensity and not general background intensity perhaps from fluorescence or photoluminescence Fromand To Displays the beginning From and end To spectral positions for a set of cursors Red Green or Blue These can be manually adjusted by typing in desired values and clicking OK Green Blue If the Green Bl
43. ue box is ticked an additional profile image is displayed showing the ratio of average intensities of the Green and Blue cursors i e Intensitycreen Intensityg_y_e This display is useful to visualize a change in peak ratios within a multidimensional spectral array To remove the Green Blue intensity profile image untick the box Spectrum If the Spectrum box is ticked the Point window is displayed showing the spectrum at the current cursor position Note that if the Spectrum box is unticked the Point window will not be displayed To redisplay the Point window tick the Spectrum box In the event that the Point window is accidentally deleted within the main LabSpec 5 graphical user interface GUI use the following procedure to re display the window open the Map Analysis dialog window untick the Spectrum box click OK re open the Map Analysis dialog window tick the Spectrum box click OK the Point window is now displayed in the main LabSpec 5 graphical user interface GUI O O O O O O O Correct Click on Correct to update the multidimensional or spectral array with the Point spectrum after it has been processed modified in some way for example smoothing or baselining The Point window only displays data held within the multidimensional spectral array so if the spectrum in the Point window is modified it is nec
44. ussian Lorentzian function In the example shown above the initial value of g is 0 5 and the Gaussian contribution can be varied from 0 no Gaussian contribution fully Lorentzian through to 1 fully Gaussian during the fitting procedure If Copy is ticked for a parameter the min and max values will be displayed individually for each peak in the Peak Parameters dialog window This allows min and max parameters to be set individually for each peak Note that the Copy box must be ticked before the peaks are automatically located using Search or manually located using the Add peak icon in the Graphical Manipulation toolbar see section 5 10 page 172 4 6 7 3 1 Setting the Init Min and Max Values in the Variables Dialog Window Left click in the desired parameter init min or max box and insert the desired value Add information for each parameter as required 4 6 7 3 2 Adding Parameters to the Variables Dialog Window Additional categories can be added by right Variables clicking on one of the parameter name boxes and selecting Insert row Cancel C i r A new row will be inserted above the selected BEms position and will display the same name and init min and max values as the original parameter ap oo oo eos kz ala e a E a al Page 141 Explore the future Automotive Test Systems Process amp Environm
45. ypically Raman shift cm or nanometers nm In the example shown above the initial value of p is the X axis position x at which the peak is initially located or positioned and the position can be varied from p 5 to p 5 during the fitting procedure o W peak full width at half maximum height in units as displayed on the spectrum s X axis typically Raman shift cm or nanometers nm In the example shown above the initial value of w is 3 and width can be varied from 0 001 to 200 during the fitting procedure o a peak amplitude in units as displayed on the spectrum s Y axis typically counts cnt or counts per second cnt s In the example shown above the initial value of a is the Y axis position y at which the peak is initially located or positioned and the amplitude can be varied from 0 to the maximum Y axis value maxy during the fitting procedure Page 140 Explore the future Automotive Test Systems Process amp Environmental Medical Semiconductor Scientific HORIBA LabSpec 5 user manual HORIBA Scientific o b c d parameters within the Gaussian Lorentzian equations In the example shown above the initial values of b c and d are 0 and they can be varied from either the inverse of the maximum Y axis value maxy or the minimum Y axis value miny to the maximum Y axis value maxy during the fitting procedure o g Gaussian contribution in a mixed Ga

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