Re: Two-photon microscope questions

Posted by Peter Rupprecht on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-photon-microscope-questions-tp7583010p7583019.html

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Dear Heping,

To your 2nd question:
>> 2) What is a common procedure to image the back aperture of the objective to the input window of the PMT?
>> My first thought is to create a collimated source with size > than
>> back aperture and shine directly into the back aperture with objective off (to see spot size
>> at PMT input). Would this be correct?

This sounds difficult to me. The detector size is usually so big that it would be difficult to find the optimal alignment by this method.

For a quick alignment, I would, if the excitation light path is perfectly aligned, put a homogeneous plastic fluoslide under the objective and align the detection pathway such that the picture you get looks as good as possible. Do this with the lowest zoom setting (highest scanning angle), otherwise it will always look perfect. With "good", I mean a FOV with the highest brightness in its center. Simply play around with mirror angles while imaging and check the picture live. Usually, due to aperture clipping effects while scanning, the FOV is not totally homogeneous. This also depends on the highest zoom factor allowed by your scanners or your software. -- If someone has a better idea, I'd be happy to learn them!

As already mentioned, I would also vote for putting the dichroic as close to the objective as possible.


As a sidenote, I have always been wondering why some 2P-microscopes I've seen use such long detection pathways (>15 cm) for 2P microscopy, with all the tubes and inner parts being completely black. Wouldn't it be much better to coat the full detection pathway surfaces with an aluminium or silver coating in order to trap every scattered photon until it impinges on the detector? Has anyone tried this out and compared it to non-coated surfaces for highly scattering conditions?
Best,
Peter