RENISHAW INVIA RAMAN SPECTROMETER
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1 STANDARD OPERATING PROCEDURE: RENISHAW INVIA RAMAN SPECTROMETER Purpose of this Instrument: The Renishaw invia Raman Spectrometer is an instrument used to analyze the Raman scattered light from samples to infer the chemistry and structure of the material of interest. Location: 381 Chemistry Research Laboratory Primary Staff Contact: Weiqiang Ding (304) cell Secondary Staff Contact: Harley Hart (412) cell The Shared Research Facilities are operated for the benefit of all researchers. If you encounter any problems with this piece of equipment, please contact the staff member listed above immediately. There is never a penalty for asking questions. If the equipment is not behaving exactly the way it should, contact a staff member. WARNING: This system uses a Class IIIb laser and emits visible and invisible radiation. Laser safety goggles MUST be worn when operating the laser as an eye hazard exists. START-UP NOTE: The RAMAN spectrometer, microscope, and laser should be powered down when not in use. If the power has been left on to any of these items, please inform a MCF staff member. 1. Turn on the computer if it is not already on and log in to the computer. 2. Log in your session on the FOM. Write down your name and sample/laser information on the log book located on the sample preparation bench beside the computer cart. 3. Turn ON the laser safety warning light (switch by the door next to the room light switch) to warn any individuals who need to enter the lab during your session. 4. Turn ON the orange microscope power switch on the left-side of the optical microscope base (Figure 1a). 5. Turn ON the power toggle switch on the front-side of the PRIOR control box located to the left of the microscope (Figure 1b). 6. Toggle the power switch on the right-hand side of the RAMAN spectrometer set-up to ON (Figure 2a). Allow 20 minutes for the CCD detector to cool down to its operating temperature. 1
2 (a) (b) Figure 1. (a) Optical microscope power; and (b) motor control box power. (a) (b) Figure 2. (a) Raman spectrometer power switch (marked); (b) Motor reference options window. 7. Start the WIRE 3.4 software on the computer desktop. 8. The software will give options as to which motors to reference (Figure 2b). Select Reference All Motors and then click OK. The tool will initialize all motors of the spectrometer. If the motors fail to reference, contact a MCF staff member for help. LASER SETUP 1. Check and remove any item placed in the path of the laser beam on the optical table (Figure 3). Such item will cause dangerous reflections of the laser beam that poses a serious eye hazard. 2
3 Figure 3. Laser setup on optical table. 2. If you need to use 532 nm laser, first find and put on a pair of laser safety glasses for 532 nm laser (orange colored lenses). Laser safety glasses are stored on the sample preparation bench beside the computer cart. 3. Switch the 532 nm laser power supply toggle switch to the ON position (located on the optical table, Figure 4a). The power light indicator on the front of the power supply will light up. 4. Wait 30 Seconds to allow the power supply to stabilize. 5. Turn on the laser power supply key to the ON position. The laser light indicator will light up (Figure 4b). Allow 3 minutes for the laser to stabilize. (a) (b) Figure nm laser power supply: (a) power switch in the back; (b) laser in the ON position. 6. At the bottom of the Wire 3.4 program, select the 1800 l/mm grating and 532 nm laser edge (Figure 5). Figure 5. Grating and laser edge selection at bottom of the Wire software program. 3
4 SWITCHING LASER WARNING: Do NOT use 785 nm laser unless you are trained by MCF staff to do so. 1. To use 785 nm NIR laser, find and put on a pair of laser safety glasses for 785 nm laser (green colored lenses). Laser safety glasses are stored on the sample preparation bench beside the computer cart. 7. Turn on the laser power key at the back of the red laser (Figure 6a). 8. Wait 30 seconds to allow the power supply to stabilize. 9. Turn on the red laser by press the Laser ON button (Figure 6b). (a) (b) Figure 6. (a) 785 nm laser power on (red LED); (b) 785 nm laser on (green LED). 10. At the bottom of the Wire program, select the 1200 l/mm grating and 785 nm laser edge (Figure 7). A Change Spectrometer Lens Set window pops up for replacing the A(1)B(1)C(1) lenses with New Lens Set. Figure 7. Grating and laser edge selection options. 11. Find the Raman front door lock key (Figure 7a) in the black Raman toolbox on optical table. Use the key to open up the Raman instrument front door (Figure 7a). 12. Locate the accessory box labeled A(1)B(1)C(1) and accessory box labeled A(3) (Figure 7b) on the optical table beside the spectroscope. 4
5 (a) (b) Figure 7. (a) Open Raman enclosure lock with key; (b) Accessory boxes for A1/B1/C1 lens (for 532 nm laser) and A3/B3/C3 lens (for 785 nm laser). 13. WARNING: Do NOT perform the following steps unless you are trained by MCF staff to do so. Remove the cover of the grating chamber. Find and remove the A(1) B(1) C(1) lens (Figure 8). Use extreme care while removing C(1) lens to avoiding hitting the prism. Store them in the box labeled A(1)B(1)C(1). 14. Mount A(3) B(3) C(3) lenses at their specific positions (Figure 8). 15. Put the grating chamber cover back. Close the instrument door and lock the door with the key. A C B Figure 8. Raman instrument chamber with A(1), B(1) and C(1) lenses installed for 532 nm laser. 5
6 SYSTEM CHECK 1. Turn the objectives to the back to open up the sample stage area (Figure 9). 2. Lower the stage of the microscope using the Z-position adjustment knobs on the side of the microscope. 3. Place the silicon reference standard (stored in the black box on the computer cart) on the stage. Use the stage clip to secure the glass slide. Figure 9. Lowered sample stage with objectives in the rear. 4. Move the 5x objective into position so that it is above the silicon reference sample. NOTE: Whenever the sample objective is changed on the turret, the objective setting needs to be changed in the Sample Review window. This will adjust the scale on the video window (see Figure 6b). 5. On the filter wheels of the microscope, adjust the filter wheels to the following positions (Figure 10a): Upper filter wheel = 2 Lower filter wheel = 1 This allows for viewing of the sample using the white light source of the microscope and video camera (Figure 10b). (a) (b) Figure 10. (a) Filter wheels in positions 2 and 1; (b) Focused silicon reference sample in video window. 6
7 6. Focus on the surface of the silicon using height adjustment knob on the side of the microscope. It may be necessary to adjust the F-stop wheel on the bottom left of the microscope to reduce the light intensity. 7. Once the sample is in focus, rotate the objective turret to the 20x position and focus on the sample. Then rotate the turret to the 50x objective and focus the sample. WARNING: Adjust the stage height SLOWLY so as NOT to crash the objective onto the sample, which will damage the objective. 8. In the software program, select Measurement > NEW Measurement > 532nm-1800 set-up. This set-up is the saved measurement set-up for the silicon reference sample. 9. On the filter wheels of the microscope, adjust the filter wheels to the following positions to activate Raman collection channel (Figure 11a): Upper filter wheel = ARROW Lower filter wheel = 4 The laser spot will appear in the crosshairs (Figure 11b). Using the stage height adjustment knob, focus the laser spot. (a) (b) Figure 11. (a) Filter wheels positions for measurement; (b) Centered laser spot in the Video window. NOTE: If the laser spot is outside of the crosshairs, DO NOT ADJUST THE CROSSHAIR POSITION. Contact a MFCF staff for help in adjusting the laser position. 9. Select Tools > Calibration > Quick Calibration and the tool will set the offset to the silicon reference peak to 521 l/mm (Figure 12). 7
8 Figure 12. Silicon reference sample spectral scan. 10. After the calibration, lower the stage height using the adjustment knobs on the side of the microscope and rotate the objective turret so that an empty objective position is above the sample. 11. Release sample clip and remove the silicon reference sample and place it back into the storage box. The tool is calibrated for the processing of samples. SPECTRAL ACQUISITION There are several types of measurements available for users are as follows: Spectral Acquisition standard method for spectral acquisition Image Acquisition collection of filter spectra. Mapping Acquisition collection of spectral acquisitions over an area of the sample. If the mapping technique is desired to be used, contact a Shared Facilities staff member. NOTE: The following SOP is for a Spectral Acquisition. For instruction on the Mapping and Image Acquisition techniques, please contact a MFCF staff member. 1. Place sample onto a glass microscope slide located on the sample prep table. 2. Turn the objective turret so that scope objectives are in the rear. This is to prevent accidental crashing of the objectives into the sample. 3. Lower the stage of the microscope using the stage height adjustment knobs on the side of the microscope. Pull back the sample clip on the stage, and place the glass slide on the stage. Release the stage clip to secure the glass slide. 4. Move the 5x objective into position so that it is above the sample. NOTE: Whenever the sample objective is changed on the turret, then the objective setting needs to be changed in the Sample Review window. This will adjust the scale on the video window. 5. On the filter wheels of the microscope, adjust the filter wheels to the following positions: 8
9 Upper filter wheel = 2 Lower filter wheel = 1 This allows for viewing of the sample using the white light source of the microscope and video camera. 6. Using the stage height adjustment knob on the side of the microscope, focus on the surface of the sample. It may be necessary to adjust the F-stop on the bottom left of the microscope in order to focus on the surface of clear or highly reflective samples. 7. Once the sample is in focus, rotate the objective turret to the 20x position and focus the sample using the stage height adjustment knob. Then rotate the turret to the 50x objective and focus the sample using the stage height adjustment knob. CAUTION: Adjust the stage height slowly so as to NOT crash the objective into the sample as damage to the objective can occur. 8. Select Measurement > NEW > Spectral Acquisition and set the scan to the desired parameters in the Spectral Acquisition Setup window: NOTE: The APPLY button must be pressed to save any changes made to the acquisition parameters. 1). RANGE TAB (a) (b) Figure 13. Range Tab windows for (a) Static scan and (b) Extended scan. 9
10 Grating Scan: i. Static Scan: The user sets the center point of the Spectrum Range and the system will set the scan range 500 cm -1 on the center point. This scan can have an exposure time of less than one second per accumulation. ii. Extended Scan: The user sets the upper and lower limit of the Spectrum Range and the spectrometer will scan continuously over the range. This scan is limited to exposure times of 10 seconds or greater. Confocality: sets the sample volume of the sample collected i. Standard: Uses a larger volume to increase signal strength. ii. High: Reduces the volume and signal strength to increase depth resolution. Configuration: i. Laser name: 532nm notch ii. Grating Type: 1800 l/mm (vis) 2) ACQUISITION TAB Figure 14. Acquisition Tab window. Title and Description: Name the acquisition and give a description of the scan. This information will be saved with the acquisition. Exposure Time: This is the amount of exposure time at the detector where longer exposure times will improve the signal-to-noise ratio. Accumulations: This is the number of scan repetitions added together to improve signalto-noise ratios and for the removal of cosmic rays. If the cosmic ray removal box is selected, then two additional scans will be processed in addition to the number of accumulations selected 10
11 Objective: This is the objective selected in the Sample Review window and must be changed every time the user changes the objective manually on the microscope. Laser Power: This is the percentage of laser power being used for the scan where higher powers translate into better signal-noise ratios. Start with lower laser powers on initial scans if the sample is susceptible to being damaged by the laser. Cosmic Ray Removal: Select this box to have the spectrometer take two additional scans to remove random cosmic peaks Close Shutter on Completion: Select this box to close the laser at the end of the acquisition and limit laser exposure on the sample. 3) FILE TAB Autosave: Select this box so that the acquisition will be saved to the folder with filename designated by the user. Select the AUTO INCREMENT box to save every scan acquisition with a sequential numbered filename. 4) TIMING TAB Figure 15. File Tab window. Sample Bleaching: This is used to decrease fluorescence from the sample by exposing the sample to the sample to the laser prior to taking a measurement. Select this box and set the time if this issue is occurring on the sample. Time Series Measurements: This allows for the user to set a specified time in between scans. 11
12 5) TEMPERATURE Figure 16. Timing Tab window. This is currently NOT an option to users as a temperature stage is required. 6) FOCUS TRACK TAB This is used to maintain the laser focus position for time and mapping acquisitions. See Module #7 of user manual (located on the computer desktop) and/or contact a MFCF staff member for additional information. 7) ADVANCED TAB Scan Type: When an Extended Scan has been selected in the RANGE TAB, then the user has the option of using a Step Scan or a Synchroscan (or Continuous Scan). The recommended option is the Synchroscan, but if saturation of the detector is occurring, then the Step option should be selected so exposure time under 10s can be used. Camera Gain: Select the HIGH sensitivity option. Pinhole: The system has a motorized pin-hole assembly and this function is used to for improving the profile of the laser beam. The option IN is recommended. Laser Focus: This option controls the beam expander. A setting of 100% is a completely defocused beam, while 0% is a tightly focused beam. If the sample being used is subject to damage by the laser, then a highly defocused beam is recommended to reduce the power density at the sample. Image Capture: This feature can be used to have white light images of the sample taken before or after an acquisition. 12
13 Figure 17. Advanced Tab window. 9. On the filter wheels of the microscope, adjust the filter wheels to the following positions: Upper filter wheel = ARROW Lower filter wheel = 4 The laser spot will appear in the crosshairs (Figure 18). Using the stage height adjustment knob, focus the laser spot. Figure 18. Centered laser spot in Video window. NOTE: If the laser spot is outside of the crosshairs, DO NOT ADJUST THE CROSSHAIR POSITION. The laser position needs adjusted and a Shared Facilities staff member needs to be contacted. 10. Turn off the room light (light switch located by the door). 11. Choose the RUN icon on the toolbar to run the measurement. The acquisition can be stopped at any time by pressing the ABORT icon. 12. If another acquisition is required and spectral acquisition settings need to be changed, select Measurement > Set-up Measurement option. The scan parameters from the previous acquisition will still be stored in the Spectral Acquisition Set-up window. If the scan parameters need to be saved for later use, select Measurement > Save Measurement. 13
14 13. To remove the sample from the microscope stage, lower the stage height using the adjustment knobs on the side of the microscope and rotate the objectives turret so that an empty objective position is above the sample. 14. Release the sample clip and remove the microscope slide from the stage. 15. See Module #3 of user manual for data manipulation techniques under the Analyze tab of the Wire 3.2 software. 16. If another sample is to be processed, then proceed back to step #1 of this section. If all necessary acquisitions, have been acquired, then proceed to the SYSTEM SHUT DOWN section of the SOP. NOTE: Users should make sure that they copy their data off the computer in a timely manner. The Shared Research Facilities is a multi-user facility and therefore cannot guarantee that the saved file will not be modified or deleted. DATA OPTIMIZATION 1. To improve signal to noise ratio a. Increase exposure time b. Increase number of accumulations 2. To eliminate strong background due to fluorescence a. Decrease laser power b. Quench fluorescence by exposing sample to incident laser light for a period of time c. Change excitation wavelength 3. To avoid saturated signal a. Reduce laser power b. Reduce exposure time 4. To avoid laser ablation on the sample a. Use a lower magnification objective lens b. Defocus the laser spot c. Reduce laser power 14
15 DATA ANALYSIS NOTE: Below lists two basic data processing procedures. Please refer to M3- Data manipulation module (on computer desktop) for additional information or contact a MFCF staff member. 1. Baseline subtraction 1) Open the spectrum in the Viewer. 2) Select Processing > Subtract Baseline. A new Viewer opens with the spectrum in the top half and the results of any baseline subtraction in the lower (Figure 15). 3) The default baseline is fitted between the two end points of the spectrum. Use left mouse click to add more points to the baseline. 4) Use right mouse click to get the context menu. Select Properties > Cubic Spline Interpolation from the menu. 5) Select Accept from the context menu. 6) You will be asked to keep the correction. NOTE: If you select Yes, you will overwrite the original file. You may need to keep a backup or rename the file. 2. Smoothing 1) Select Processing > Smooth. Figure 19. Baseline subtraction example. 2) A new window will open with the sample spectrum at the top and the result spectrum below (Figure 16). 3) To modify the degree of smoothing, select Properties from the context menu (right mouse click) to see the Smooth Properties window. 4) You can save the resulting smoothed spectrum by select Processing > Smooth > Accept > Current dataset. 15
16 Figure 20. Curve smoothing example 3. Zap Use Zap function to remove stray bands such as cosmic rays from the spectrum. 1) Open the spectrum of interest. 2) Select Processing > Zap from the menu. A new Viewer will open with the sample spectrum at the top and the result spectrum below (Figure 17). 3) The upper spectrum has a zap region between two vertical black lines. Grab each line in turn and adjust the position of the zap region to just enclose the band to be removed. You may need to use the zoom function to isolate the band. Figure 21. Zap function example. 16
17 4. Curve-fitting 1) Select Analysis > Curve Fit to open the curve fit window. 2) Zoom into a region that contains the band and some baseline data at either side. 3) Use the mouse to position the approximate centre of the band. 4) Click to add the band and repeat for the centers of other bands if there are multiple bands. 5) You can adjust the center or width markers for each band using the mouse and cursor to get a better rough fit. 6) Use mouse right click to get the menu and select Start Fit to fit the peak (Figure 18). 7) You can save the curve fit file from the context menu. Figure 22. Curve fitting example. 5. Peak pick 1) Select Analysis > Peak pick to open the curve fit window (Figure 19). 2) You may adjust the thresholds by selecting Analysis > Autoset thresholds. Figure 23. Peak pick example. 17
18 SYSTEM SHUT DOWN 1. Turn OFF the laser. 2. Turn the laser power supply key to the OFF position. The power light indicator on the front of the power supply will turn off. 3. If you used 785 nm laser, set the laser and grating back to 532 nm/1800l/mm in the WIRE 3.4 program. Follow the accessory switch procedure to replace the A(3)B(3)C(3) lens with A(1)B(1)C(1) lens. 4. Toggle the power switch on the right side of the RAMAN spectrometer set-up to OFF. 5. Turn OFF the power switch on the front-side of the PRIOR control box. 6. Turn OFF the power switch on the left-side of the white light source for the microscope. 7. CLOSE the WIRE 3.4 software on the computer desktop. 8. Check that all required parameters are recorded in the log book. Sign out on the log book. 9. Clean up work area by doing the following: a. Safety glasses are returned to the proper case on the sample preparation bench next to the computer cart. b. The silicon reference sample is placed back in the storage case located on top of the computer cart. 10. Properly dispose of any used materials i.e. wipes, glass slides, etc. 11. Sign out of the FOM. Report any problems in the log off window. 12. Turn off the Laser Warning light (switch by the door) white exiting the lab. 18
19 EMERGENCY PROCEDURES If no one is available and the machine is not acting as expected, the user should do the following: Press ABORT on the WIRE 3.2 software. Turn the key to the laser power supply to the OFF position. Turn off the x-rays by pressing the red OFF button on the power controller. Do not leave the machine running in an abnormal state. If the machine cannot be placed in the default state, immediately contact: Primary Staff Contact: Weiqiang Ding (304) Secondary Staff Contact: Harley Hart (412) Then, if possible, the user should stay by the tool while trying to contact a Shared Facilities staff member. If it becomes necessary to leave the tool then the user should leave a large, legible note at the RAMAN stating the tool is DOWN. The user should then complete a Tool Issue Report form and submit this form to ESB G75D. The user should also add comment using the FOM software indicating the tool status. If a dangerous situation is evident (smoke, fire, sparks, etc), ONLY if it is safe to do so, the user should turn off power to the system by switching the toggle switch on the right side of the tool and the laser power supply key and toggle switch to the OFF position. The user should then notify all other persons within the Hodges Hall 325 lab to evacuate and leave the lab immediately. The user should then contact proper emergency personnel from a safe place. 19
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