Alignment protocol for Leica SP8 (Confocal, 2-photon & CARS)

[Martin Hunter]

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Hi,

I am managing a confocal lab facility with a Leica SP8 system, with capabilities for 2-photon and CARS microscopy.

Does anyone have suggestions on how to verify alignment of the lasers, especially for 2-photon and CARS 3D imaging?

Back when we had a Leica SP2, we got trained in the protocol to ensure the excitation lasers were properly aligned. However, Leica is no longer offering that training to us, saying we should just ask them to come and check it for us.

Thanks,

Martin Hunter
Tufts Advanced Microscopic Imaging Center (TAMIC)
Medford, MA

[Craig Brideau]

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I have been finding lately that all the major microscope companies have been reluctant to let anyone tinker with their equipment in any way. I'm not sure if this is a business or liability issue, but the support for this kind of activity has vanished in the last few years.

The easiest way to align two beams is to shoot both through two pinholes/apertures or closed irises some distance apart. Of course you need to exercise proper beam safety etc. so if you don't know what you are doing don't do this as you might hurt yourself or the equipment.</disclaimer>

If you can find two apertures in your system you can use those to do the alignment. A power meter at the output or a camera with attenuator can let you safely 'see' the product of the beams traveling through the apertures.

Craig

On Thu, Dec 1, 2016 at 9:35 AM, Martin Hunter <[hidden email]> wrote:

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Hi,

I am managing a confocal lab facility with a Leica SP8 system, with capabilities for 2-photon and CARS microscopy.

Does anyone have suggestions on how to verify alignment of the lasers, especially for 2-photon and CARS 3D imaging?

Back when we had a Leica SP2, we got trained in the protocol to ensure the excitation lasers were properly aligned. However, Leica is no longer offering that training to us, saying we should just ask them to come and check it for us.

Thanks,

Martin Hunter
Tufts Advanced Microscopic Imaging Center (TAMIC)
Medford, MA

[weber]

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You can use your 2P in reflection mode ! So use this mode and put a mirror. Set your focus with a 10X objective.
Right now you can use your 2P laser (as it possible around 700nm or less) use less 1% of the power. Low gain and increase it to show the saturation. Really easy to do and you can verify your 2P laser alignment. If your beam is not at the center of the field add a lens (1m length) in the pathway with two precise screw. You can find them in Thorlabs company.