interference fringes in SP5 scans at 10x

Posted by Aryeh Weiss on
URL: http://confocal-microscopy-list.275.s1.nabble.com/interference-fringes-in-SP5-scans-at-10x-tp7586028.html

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We are observing what appear to be interference fringes form reflected light in  our Leica SP5, with our 10x objective.

Example images can be seen using this link:
https://drive.google.com/drive/folders/0Bytu294eIohjVHQ1elVTSElscjg?usp=sharing

There is a README file that says which excitation lines were used.

Basically, when the 405nm excitation is used alone, we do not see the fringes. When the other lines (ie the "visible" lines, as opposed to what Leica calls UV, which is really V) are used, there are fringes even in the channel which is set to pass wavelengths shorter than the shortest excitation in use (in our case 488nm).

So, in brief, the "visible" laser lines all produce interference fringes in all channels. The fringe spacing changes with excitation wavelength (as expected), but not with scan speed. They are not apparent when working with 40x oil. However, I note that with the oil objective, there is much less reflection from the coverslip . We do not have other air objectives with which to test the system.

The fringes appear with various samples.

It is quite possible that this has always been there, but no one noticed because confocal at 10x is not done very often.

If anyone on the list can tell us more about these fringes, we would appreciate it.

Thanks in advance.
--aryeh