Re: interference fringes in SP5 scans at 10x

Posted by Rosemary.White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/interference-fringes-in-SP5-scans-at-10x-tp7586028p7586054.html

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Thanks everyone for this useful discussion. I was always convinced this was reflection, and now will check more closely when (not if…) we see this again.

cheers,

Rosemary

Dr Rosemary White

CSIRO Black Mountain

GPO Box 1700

Canberra, ACT 2601

Australia

T 61 2 6246 5475

E [hidden email]

From: Confocal Microscopy List <[hidden email]> on behalf of Sathya Srinivasan <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Wednesday, 23 November 2016 9:08 am
To: "[hidden email]" <[hidden email]>
Subject: Re: interference fringes in SP5 scans at 10x

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Hi Aryeh, 

There was a discussion about this issue during 2011:

Subject:		Troubleshooting: large fluorescent circle on an inverted microscope.
From:		Sandrine Pouvreau <[hidden email]>
Reply-To:		Confocal Microscopy List <[hidden email]>
Date:		Thu, 9 Jun 2011 09:22:10 -0500

Seems that discussion also concluded AOBS to be the problem:

https://lists.umn.edu/cgi-bin/wa?A2=ind1106&L=confocalmicroscopy&P=R3742&1=confocalmicroscopy&9=A&J=on&X=9C9E146E5DFC237030&d=No+Match%3BMatch%3BMatches&z=4

Good luck.

Sathya Srinivasan

ONPRC

On Sun, Nov 20, 2016 at 4:25 AM, Aryeh Weiss <[hidden email]> wrote:

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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