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Two photon microscope

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Nathan Perkins edited this page May 26, 2016 · 9 revisions

Preface and warning

Lasers are dangerous. Class 4 lasers are especially dangerous. Be sure to complete the laser safety training prior to using the two photon microscope, and adhere to all safety protocols.

A few components of the two photon microscope are fragile, and care must be used to avoid damaging the components. Specifically, the photomultiplier tubes, when active, are extremely light sensitive. Be sure to only activate them once the lights are off and the enclosure is closed.

Components

Below are relevant components that will be referred to throughout the instructions:

Chiller

Chiller

Laser control

Laser control

Front of rack

Front of rack

Back of rack

Back of rack

Powering on

  1. Ensure the large Spectra-Physics box under the air table is on. If it is off, turn on and give it several hours to power up and stabilize.
  2. Check the chiller to ensure it has sufficient water. It needs to be filled about once a month. The water level should be between the two horizontal lines (highlighted in the image above).
  3. Open the nitrogen tank. The nitrogen flow should be halfway (5) on the Spectra-Physics Model 3910 box.
  4. Power on the laser (by holding the white "laser power" button until "laser emission" flashes). Be sure to place the laser in use sign on the door.
  5. Slowly increase the laser power, by about 1W every 5-10 minutes until it reaches 5W.
  6. The National Instruments PXI box should already be on. If not, turn it on.
  7. Power on the the Sutter resonant controller, the Sutter manipulator controller, the Thorlabs shutter control, the Conoptics beam controller, the Thorlabs piezo controller, the Hamamatsu power supply (the yellow button must be off before powering on), and the two-photon mirror controller.
  8. Power on the computer. Be sure the NI box is on before the computer and remains on the whole time the computer is on. It will not be recognized if powered on after the computer.
  9. Open the main laser shutter (put on your eye protection before this step). From the laser controller, click the button that corresponds with setup and use the up / down buttons to toggle the shutter open.
  10. Once the computer has booted, mode lock the laser (see instructions in next section).
  11. To begin imaging, either click the "ScanImage" shortcut on the desktop or open MATLAB and type scanimage to launch the two photon software.

Desktop

Mode lock laser

Throughout the imaging session, the laser must be mode-locked at the desired wavelength. Once mode locked, it should remain so until the main laser shutter is closed. If the main laser shutter is reopened, it will have to be mode locked again.

To mode lock the laser, while wearing safety goggles:

  1. Turn on the secondary monitor and open the "SPLICCO" software that shows analysis of the laser wavelengths.
  2. Go to the Spectra-Physics Tsunami box with the two knobs labeled "prism" and "slit" (note: do not adjust the blue or green knobs, the Tsunami box contains carefully aligned lasers that require a specialist to setup).
  3. The goal is to get a gaussian distribution in the plot shown by "SPLICCO", centered around the desired wavelength. If the laser was recently used, it might be possible to turn both the prism and slit knobs simultaneously and easily get this. Otherwise, it takes some finesse and practice adjusting the knobs to get the laser to mode-lock. Generally, lower wavelengths (<800nm) are easier.

SPLICCO

Usage

  1. If the "Shutter Enabled" light on the Thorlabs shutter control is illuminated, disable the secondary shutter by clicking the button labeled "enable" on the Thorlabs shutter control until the "Shutter Enabled" light turns off.
  2. Switch the two-photon mirror control to the "WF" mode, and turn on the fluorescence light source.
  3. Select an appropriate filter cube and use the eyepieces to find and focus on the sample.
  4. Turn off the fluorescence light source and switch the two-photon mirror control to the "2P" mode.
  5. Close the curtain or enclosure.
  6. Enable the secondary shutter by clicking the button labeled "enable" on the Thorlabs shutter control. This allows ScanImage to control the shutter.
  7. Turn off the lights in the room and close the enclosure.
  8. Turn down the voltage on the PMT (~0.1V). Press the "yellow" button the Hamamatsu PMT power supply to enable the PMT.
  9. Press the "Focus" button in ScanImage to start the resonant scanner and to begin displaying the signal from the two-photon.
  10. Slowly increase the voltage on the PMT until you can see signal.
  11. You can now use ScanImage to acquire images and volumes, control the Fast-Z, zoom, etc. Their documentation describes more of the functionality available: ScanImage documentation.

Some tips for ScanImage:

  • Turn off BiDi mode.
  • Adjust the white value for Channel 1 to be larger (~1000).
  • If you see a flashing image, then the beam strength needs to be decreased and the PMT voltage needs to be increased.
  • Currently, the ScanImage beam power (in the "POEWR CONTROL" window) is not fully setup, but will be in the future.

ScanImage

Powering off

  1. Exit ScanImage and Matlab using the "File" menu from the "MAIN CONTROLS" window of ScanImage.
  2. Close the main laser shutter.
  3. Power off the computer. Be sure it fully shuts down before continuing.
  4. Power off the Sutter resonant controller, the Sutter manipulator controller, the Thorlabs shutter control, the Conoptics beam controller, the Thor labs piezo controller and the Hamamatsu power supply (ensure the yellow button is off before powering off). The the National Instruments PXI box can remain on.
  5. Slowly turn down the laser power to 0.2 watts. Once the laser power reaches this power level, press the power button to turn off the laser. Remember to remove the laser warning sign from the door.
  6. Close the nitrogen tank using the tank value and the small black value coming out of the regulator.
  7. If you used a water objective, drying the water objective by moving lens paper over the objective slowly. Do not push the paper directly against the objective, but instead hold it taut across the tip of the objective. Do not reuse the same portion of the paper multiple times.

Imaging options

Currently, we have a 20x water-immersion objective to use for imaging.

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