WITec Alpha 300R Quick Operation Summary October 2018

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1 WITec Alpha 300R Quick Operation Summary October 2018 This document is frequently updated if you feel information should be added, please indicate that to the facility manager (currently Philip Carubia, B57 Bard Hall, office and cell: ). 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 WITec 300R 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 SB30. Safety: This instrument uses Class IIIb lasers, so please be alert to the safety issues you learned in the EH&S laser safety course. Use of laser safety eyewear is not required but common sense is. Please think twice and act once. 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 glass disposal container on the floor near the base of the system. Also dispose of wipes, tape, etc. when you are done with them. About the WITec Raman system The WITec Raman is an advanced confocal Raman microscope that combines fast imaging, high spectral sensitivity and spatial resolution with excellent analysis software into a system designed around imaging. The system has high confocality and boasts a minimum depth slice of ~350 nm. This enables the user to take 3D Raman images with sub-micron resolution. The system consists of three main parts: lasers, microscope, and spectrometers. Lasers: The system currently has two lasers 532 nm and 785 nm. Both lasers are located on the benchtop next to the computer tower. Laser light going both from the laser to the microscope and from the microscope to the spectrometer is fiber coupled. This provides several advantages: First, the confocality comes from the aperture created by focusing on the skinny fiber. Second, the fibers provide high throughput over long distances. Third, the fiber protects users from laser light. The fiber coupler for the laser is located on the front side of the laser next to the computer tower. The fiber coupler is delicate and sensitive. Please DO NOT BUMP OR TRY TO ADJUST THE FIBER COUPLERS! The laser is pictured below. The user will use three things on the laser. 1) Attenuator: The attenuator is the small micrometer knob on the front of the laser located between the laser body and the fiber coupler. Users will use the attenuator in conjunction with the laser power meter to adjust the laser fluence incident on the sample. Turning the attenuator clockwise decreases the laser power and counterclockwise increases the laser power. 2) Shutter: The shutter is the silver knurled knob located just behind the attenuator on the laser body. The shutter is open when the slot in top of the knob is aligned with the fiber axis and closed when the slot is perpendicular to the fiber axis. The shutter should be either open or closed; there is no intermediate setting. 3) Power: Both lasers can be turned on and off using the key on the back side of the laser body. Turning the key 90 clockwise will turn the laser on and 90 counter clockwise will turn it off. For very stable power the laser should be given ~5 minutes of warmup time. Page 1 of 7

2 Image1: laser body with attenuator, shutter and power key positions. Microscope: Labelled parts and pieces of the microscope are shown in the image below with descriptions from the bottom up. X-Y stage: The sample stage is automated in X and Y directions and is controlled either by the software or the X-box controller. The Z control actually raises the entire microscope head. Pressing up on the controller makes the gap between the objective and the sample get bigger. To protect the objective, use caution when moving the stage or microscope head. Objective turret: the objective turret can house up to 6 objectives. There are five standard objectives though the majority of spectra will be acquired using either the 50X LWD or the 100X. Users will use the 100X objective ONLY for very flat samples. Do not use this objective for powders, liquids, or samples that have long range curvature or surface roughness that is larger than 20 µm Users may use the 50X LWD for all other samples. Bright field/raman selector: BF (Bright field) position on left is for white light imaging and the right position is for Raman Imaging. F-stop and Aperture: Use the F-stop to help focus on a difficult sample. Use the aperture to increase visible light contrast or to make the software slider more sensitive. Visible light analyzer: Analyzer for viewing samples in a crossed polarized configuration. Pull knob toward front of scope to put the analyzer in beam path. Remove the analyzer for Raman imaging. Page 2 of 7

3 Camera selector: Directs light to the camera when in the out position. True surface: the True Surface function allows the software to track with the surface of uneven samples. Pull the knob out to allow the true surface laser to be incident on the sample surface (True surface also requires activation and adjustment in the software). True surface will only function with a magnification > 20X. True surface may have trouble focusing on rough samples or samples with large steps. Laser power meter: Pull the lever out to direct light to the meter. The meter reader is located on the benchtop to the right of the microscope. The power for the meter reader is located on the left side of the meter. Rayleigh filter: Turn wheel to appropriate laser. Calibration source: This function is only used for calibrating spectra with an external calibration source. If you have questions about this capability please contact the facility manager. Spectrometer selection: Pull knob for appropriate laser wavelength. Be sure that the knob for the unused spectrometer is pushed in. Spectrometers: The two spectrometers are located on the blue table to the left of the microscope. Cameras for both spectrometers operate at sub-ambient conditions. When starting the software there is a cooling step that takes about 5 minutes to complete before the system is ready to acquire spectra. Spectrometers and options are as follows: UHTS600: Visible light spectrometer optimized for best sensitivity between 450nm and 750nm. 600mm focal length. Three grating options: 300l/mm, 1200l/mm and 1800l/mm. UHTS400: NIR spectrometer optimized for best sensitivity between 630nm and 1100nm. 400mm focal length. Two grating options: 300l/mm and 1200l/mm. No specific spectrometer selection is required. Setting the microscope appropriately for the laser that you will be using will automatically select the appropriate spectrometer. NOTE: The spectrometers are sensitive! Please do not use the spectrometers as a table for your samples! The spectrometers should always be on. If they are off they should only be turned on by the facility manager. Page 3 of 7

4 Software: The software that controls the system is control5. Open it from the icon on the desktop. There are five main windows. Control, WITec video control, Stitching image, Project manager, and Messages. WITec Video Control: is the main interface for stage and microscope control. Come here for objective selection, true surface interface, light intensity, ETC. Stitching image: stitched and single images will display in separate windows that can be used to navigate the sample when in Raman mode. Project manager: All data will display in project manager including Raman maps and single white light images. Control: Is the main interface for image and spectral acquisition and is described in further detail below. Page 4 of 7

5 Control functions: Control is the main interface for a number of functions in the Scan table: Scan table provides adjustment for the Z-axis coordinate system. Only make changes to scan table with the permission of the facility manager as the can lead to greater probability of crashing objectives. Sample positioning: Sample positioning to controls the X-Y stage. Use it to: Move to positions Reset the coordinate system to zero (set zero) Move to specific positions in the stitching image window (listen position). Spectrograph 1: Use Spectrograph to control spectrometer functions. Spectrograph will control the spectrometer specified under configurations. Choose grating type under grating. Center wavelength will center the specified wavelength on the detector also centering it in the acquired spectrum. Spectral unit can be changed to the users preferred unit Spectral center will center the specified spectral unti on the detector also centering it in the acquired spectrum. Oscilliscope: The oscilloscope function will allow a user to view a real-time Raman spectrum of the sample. This can be very helpful for maximizing signal as well as looking for regions of interest. Image stitching: Use image stitching to join multiple images into one larger image. Specify stitched geometry by specifying area and center or through listen position. A larger image taken with a lower magnification objective is required to use the listen position option. Large area scan: The large area scan function is for Raman mapping. Set scan method. Continuous is typical and fastest. Set Geometry either by specifying area and center or through listen position. Specify spectral resolution by setting points/line and lines/image Page 5 of 7

6 Quick steps for taking a measurement: The following describes the alignment of the alpha300 system in order to obtain a confocal Raman image, perform a line scan, a time series or capture a single Raman spectrum. The first of the following steps describe the procedure for focusing the microscope on the sample using white light illumination followed by the focusing of laser radiation. 1. Power up the computer and start WITec Control. Select the appropriate Raman mode for the laser that you will be using from the Configurations-menu. The CCD camera will be cooled to its minimum stable temperature automatically 2. Place the shutter into the laser beam path 3. Turn the laser power on by turning the key on the back 90 clockwise. 4. Mount the sample on the scanning stage. 5. Rotate the microscope turret until an appropriate objective is in the working position. Make sure you use the microscope objective in the proper way (e.g. use a cover slip if a cover slip corrected objective is used). 6. Pull out the video rod to direct light to the color video camera. 7. Move the Raman/bright field (R/BF) slider to the bright field position. 8. Adjust the illumination to the required level using the illumination menu item in the Control Window or with the controller up or down arrow. 9. Observe the image of the color video camera on the computer monitor using the Video Control Window in the WITec Control software. 10. Focus on the surface of your sample with the Microscope Z stage. This can be done in two different ways: a. Using the Z microscope control in the Graphic Control Window. Clicking on the arrows will move the stage in the indicated direction. b. Using the remote control with the Z Microscope selected as the controlled device. Using the left joystick, the Z-focusing-stage can be moved up or down. c. Notes on focusing the microscope: i. If possible, move the objective initially away from the sample to avoid a collision between the objective and the sample. ii. It can sometimes be very difficult to focus on flat and clean surfaces. The field stop diaphragm can help to overcome these difficulties as outlined in the following: To focus, close the field stop diaphragm. Approach the sample until the edge of the field stop appears focused. At this point, the sample is also in focus. This is because the field stop is positioned at the back focal plane of the objective. 11. Move the R/BF slider to the Raman position. 12. Turn off the white light illumination 13. Select an appropriate grating through the Spectrograph menu. Start with a low dispersion grating (300l/mm) that covers a large frequency range and move it to an appropriate wavenumber position (e.g. using a 532nm laser and the 300 l/mm grating, using 2050 rel. 1/cm as the spectral center is a good start). 14. Before opening the laser shutter, set the laser power to a value appropriate for the sample. If using the Silicon test sample, it should always be checked with maximum laser power. Open the laser shutter. 15. Start the oscilloscope in WITec Control using the Start Oscilloscope button in the oscilloscope menu of the Control Window. The integration time can be adjusted in the oscilloscope menu of the Control Window and should initially be set to s. You should now see a Raman spectrum of Silicon with the characteristic 1st order Si line at _520 cm-1, updated every 0.05s to 1s depending on the selected exposure time. Page 6 of 7

7 16. Adjust the focus of the microscope using the microscope Z stage (as described in point 9) to maximize the signal. 17. Once the optimum signal intensity is achieved, stop the oscilloscope with any Stop button or icon. 18. The parameters for an image scan, line scan, a single spectrum or a time series should then be defined. If an image scan is performed, the measurement parameters in the image scan menu of the Control Window should be adjusted (see WITec Control manual Section for a description of the individual parameters). If the (X), (Y) and (Z,Microscope) positions indicated in the scan table menu of the Control Window differ from the position entered as Center (X), Center (Y) and Center (Z) in the image scan menu, the scan table will move to a different position before starting the scan. This might then lead to data acquisition at the wrong position/area. Use the Center at Current Pos. button in the image scan menu of the ControlWindow to avoid this displacement. HINT Typical values for a confocal Raman imaging measurement are: a. Points per line: b. Lines per image: c. Width: 10-50μm d. Height: 10-50μm e. Integration Time: ms 19. You may now begin your measurement by opening the laser shutter and clicking on the corresponding Start button. 20. If an Image Scan is performed, you will see a FILTERMANAGER and two GRAPH windows: One of these will display the spectrum of your sample updated depending on the selected integration time. 21. A variety of images can be calculated from the spectra and displayed during the scan, without affecting the spectra acquisition. This is done by creating a new filter in the FILTER MANAGER window and selecting the appropriate spectral range in a displayed spectrum. Please refer to the WITec Project user manual (the functionality in WITec Control is identical) for more information about the variety of data evaluation tools available in this powerful software. Through the definition of filters in the FILTER MANGER, image data objects will be created which can be displayed in image windows. These image windows will display the calculated value of the Raman signal of your sample at each pixel. Clicking on any position in this image will display the spectrum obtained at this specific position in the second GRAPH window. This action does not affect the data acquisition. Finishing your session: When you have completed your measurements please be sure to complete the following steps to shut the system down: Close all laser shutters and turn off all lasers by turning the key counterclockwise. Move the microscope head up and place the 5X objective in the beam path. Turn off the visible light in the WITec video control. Close Control5 by going to file>exit. This is important as it will stop the camera cooling and if not completed the cameras will stay at -60 C. CLEAN UP! The lab should be left as clean as you found it. Disable Coral. Page 7 of 7