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Basic setup of the system

  • Turn on all devices
    • Extension cord – Brillouin laser and spectrometer (left power strip)
    • Extension cord – Nikon microscope (right power strip)
    • Brillouin PC – on the right
  • Make sure that the USB cables from the Nikon microscope are connected to the Brillouin PC
    • There is a USB switchboard on top of the Brillouin PC.
    • Light 2 – Brillouin, Light 1 – Spinning disk
  • Start the LEON software
    • If the VIPA spectrometer shows “Waiting for device” close the software, restart the spectrometer, and start the software again.
    • If the Laser emission button is greyed out, make sure that:
      • The TOPAS software is disconnected from the laser.
      • Restart the laser module: turn it off and on using the switch directly on the laser module (bottom box on the platform) and disconnect the USB cable.
      • Restart the PC and then start LEON again.
  • Wait at least 60 minutes until the system is warmed up.
  • Enable emission in the System tab
    • If the option is greyed out, it is probably because the TOPAS software is already on.
    • Turn off LEON and TOPAS, then turn LEON on again.
  • Start the TOPAS software.
  • Connect to the laser: Menu – Connect
  • Load the configuration: Menu – Configuration Manager – click on 10 mW (the lowest laser intensity) – click on Load – close.
  • Display Laser Control and Signal Display: right-click on DLpro (S/N 23575) – Laser Control – Repeat – Signal Display
  • Lock the laser frequency: right-click on Fine In 1 (V) on the left Y-axis – Autoscale – Tools – Lock Points – then choose the lowest point on the red curve – Lock Enable
  • Close the TOPAS software.
  • Back in the LEON software: Lock Etalon
    • The laser and spectrometer settings from the previous measurement should load, but it is crucial to leave the system warming up for at least 60 minutes.
    • You may see intensity fluctuations in the Brillouin signal. This means that the system is not fully stabilized yet. Be patient.
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Calibration of the Brillouin signal

  • If the Brillouin signal was correctly set in the previous measurement, it is recommended that you do not adjust the VIPAs or laser settings. If you do not see any Brillouin signal, contact the facility assistance.
  • Place a Petri dish with water on the microscope and focus near the surface
    • Click on Scan in the System tab and then go to the Acquisition tab.
    • In the Beam Path Control window (top left), click on Devices and turn on the Laser Shutter.
    • In the Spectrogram window (top right), click Preview On.
    • Find the spot with the highest reflection according to the spectrogram. This is the surface.
  • Go back to the System tab and move the focus a few µm up so that the signal is not too high.
  • In the main window, click the top-left icon and change the view from Brightfield to Spectrogram.
  • Set the contrast to 100–150, set the exposure to 100 ms, and click Preview On (bottom right).
  • Adjust the VIPA 1 and VIPA 2 tilt sliders so that the main quadrant is centered.
  • Adjust the VIPA 1 and VIPA 2 shift sliders so that all corner points (Rayleigh peaks) have similar brightness.
  • Adjust the Laser Fiber Couplers X, Y, and Z so that the overall signal is optimized.
    • Start with X and slowly move in one or the other direction to find the maximum. If you cannot find it, return to the original position.
    • Repeat the process for Y. If you find a better position in Y, go back to X and repeat the adjustment.
    • Finish with Z.
  • Adjust the Optibridge Fiber Couplers X, Y, and Z so that the Brillouin peaks are clearly visible.
    • Usually, there is no need to change these sliders significantly.
  • Turn off the preview, change the preset to Scan, and go to the Acquisition tab.
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Adjusting the laser spot

  • If you change the objective, or if you see that the Brillouin image is shifted relative to the brightfield image, adjust the position of the laser spot accordingly:
  • Choose the Brightfield preset in the Acquisition tab; set FilterTurret1 to empty.
  • Focus on the surface.
  • Set up the laser spot: Laser Shutter – Open – Camera Filter – Out
    • Exposure time – 0.1 ms
  • There will be two large dots and one small dot between them. In Laser Settings (Beam Path Control), click Reset and place the cross on the smaller laser spot.
  • Put the Camera Filter back and continue the measurement.
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Changing the laser power

  • In System tab, unlock the etalon.
  • Open TOPAS software and conect to the laser: Menu – Connect
  • Load the configuration: Menu – Configuration Manager – click on desired power – click on Load – close.
  • Display Laser Control and Signal Display: right-click on DLpro (S/N 23575) – Laser Control – Repeat – Signal Display
  • Lock the laser frequency: right-click on Fine In 1 (V) on the left Y-axis – Autoscale – Tools – Lock Points – then choose the lowest point on the red curve – Lock Enable
    • If you don’t see a red curve, or there is visible noise, you can try increase or decrease Set Curent settings in Laser control window in steps of 0.1 mA (put the cursor behind the second digit and use the arrows)
    • Whether you must adjust it up or down you need to try out, but you will see the effect immediately
  • After locking the laser frquency, go to LEON software and Lock Etalon
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Setting up the experiment and measurement

  • Choose the appropriate objective (in Beam Path Control Presets) and apply the corresponding immersion medium (oil, silicone oil, or water).
  • Turn on the Brightfield preview (Brightfield panel on the right side) and turn on the light on the microscope or in Beam Path Control (DiaSwitch and DiaLamp).
  • Focus on your sample and find your region of interest.
  • Using your mouse and the Scan Control panel (bottom left), specify your measurement region, resolution, and Z-stack if needed.
  • Open the Laser Shutter and turn on the Spectrogram to check the Brillouin signal.
    • If you do not see anything in the spectrogram, make sure that the Laser Shutter is open.
    • If you do not see Brillouin peaks in the spectrogram, increase the exposure time up to 300 ms. The higher the exposure, the longer the measurement time.
    • Change the final exposure time in the ScanControl panel.
    • If the Brillouin peaks are still low, you can increase the laser power (default is 10 mW, maximum is 30 mW), but be aware that higher laser power can lead to phototoxicity.
  • Click Ctrl+N, or click on Create in the Experiment tab.
  • Find your data folder or create one in D://UserData/ and name your data file.
  • You may now save your Brightfield image (Brightfield panel) and start the Brillouin measurement (Scan Control panel).
  • After the measurement is finished, you can continue saving Brillouin measurements in the current file or start a new one by pressing Ctrl+N, or by clicking on Create in the Experiment tab.
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