Re: 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-tp7586028p7586040.html

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Thank you for your detailed reply.

Looks like a consensus has formed to check the AOBS and AOTF tuning, so we will do that.
It occurs to me that one way to check if it is leakage through the AOBS and AOTF is to set the passbands  so that there are no laser lines that overlap the passband. For us, that could be 520-550, since we have a multiline argon-ion, 561, 594 and 633 lasers. In that case, I would expect to see the fringes disappear or be very much weaker. However, we also see the fringes in the far red channel, where there are no laser lines. Maybe a mistuned AOBS also affects the direction (angle) of the reflected light?

Best regards,
--aryeh

On 20/11/2016 21:48, [hidden email] wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Aryeh,

we've seen something similar when one of our AOBS drivers failed. The microscope still worked with bright samples, but the reflections were quite strong.

You can try to switch between fluorescence and reflection mode, obviously, the reflections should be much stronger in the 'reflection' mode. But it was the other way round in our case....

Or the AOBS may need some tuning (just like AOTFs do). I don't know whether there are any notch filters, we have the 'X' configuration (with white light laser), so no notch filters here.

The microscope also comes with a diagnostic software that lets you diagnose and tune AOBS (among other things), but there is not much documentation available. Your local Leica rep should be able to help you.

Good luck!

Best, zdenek

--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, VA
http://www.kcci.virginia.edu/
tel: [phone]
Annual FRET Workshop: http://kcci.virginia.edu/workshop-2017

---------- Původní zpráva ----------
Od: Aryeh Weiss [hidden email]
Komu: [hidden email]
Datum: 20. 11. 2016 7:26:36
Předmět: interference fringes in SP5 scans at 10x

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

-- 
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]