interference fringes in SP5 scans at 10x

[Aryeh Weiss]

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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

[mmodel]

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Hello Aryeh,

You are doing fluorescence or reflection? If reflection then the fringes are almost unavoidable. For example, light reflecting from the front and from the back of the coverslip should interfere because of the long coherence length of the laser

Mike

---

From: Confocal Microscopy List <[hidden email]> on behalf of Aryeh Weiss <[hidden email]>
Sent: Sunday, November 20, 2016 7:25 AM
To: [hidden email]
Subject: interference fringes in SP5 scans at 10x

 

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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

[Aryeh Weiss]

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Thank you for your reply. This is fluorescence, not reflectance, imaging. The passbands are set to reject the excitation wavelengths.

--aryeh

On 20/11/2016 16:18, MODEL, MICHAEL wrote:

Hello Aryeh,

You are doing fluorescence or reflection? If reflection then the fringes are almost unavoidable. For example, light reflecting from the front and from the back of the coverslip should interfere because of the long coherence length of the laser

Mike

---

From: Confocal Microscopy List [hidden email] on behalf of Aryeh Weiss [hidden email]
Sent: Sunday, November 20, 2016 7:25 AM
To: [hidden email]
Subject: interference fringes in SP5 scans at 10x

 

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[hidden email]: listserv archives. confocalmicroscopy

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

[mmodel]

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Fluorescent light cannot produce interference. So this has to be reflection. It could be due to something with filters or from reflection from something inside. Have you try to remove the sample? Defocus the sample? Try single line scanning?

---

From: Aryeh Weiss <[hidden email]> on behalf of Aryeh Weiss <[hidden email]>
Sent: Sunday, November 20, 2016 9:49 AM
To: MODEL, MICHAEL; [hidden email]
Subject: Re: interference fringes in SP5 scans at 10x

 

Thank you for your reply. This is fluorescence, not reflectance, imaging. The passbands are set to reject the excitation wavelengths.

--aryeh

On 20/11/2016 16:18, MODEL, MICHAEL wrote:

Hello Aryeh,

You are doing fluorescence or reflection? If reflection then the fringes are almost unavoidable. For example, light reflecting from the front and from the back of the coverslip should interfere because of the long coherence length of the laser

Mike

---

From: Confocal Microscopy List [hidden email] on behalf of Aryeh Weiss [hidden email]
Sent: Sunday, November 20, 2016 7:25 AM
To: [hidden email]
Subject: interference fringes in SP5 scans at 10x

 

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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

[mcammer]

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Reflection.  We especially had this problem with the far-red lines with the SP2 and SP5 AOBS systems.  If you have one of the spectral systems, move the detection bounds further away from the laser lines.  Also, is there a polarizing filter that can be put in/out to help block laser reflection?  Don't recall if Leica had this.

 

               
_________________________________________
Michael Cammer, Optical Microscopy Specialist

http://ocs.med.nyu.edu/microscopy 

http://microscopynotes.com/

Cell: [phone]

---

From: Confocal Microscopy List [[hidden email]] on behalf of MODEL, MICHAEL [[hidden email]]
Sent: Sunday, November 20, 2016 10:08 AM
To: [hidden email]
Subject: Re: interference fringes in SP5 scans at 10x

***** 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. *****

Fluorescent light cannot produce interference. So this has to be reflection. It could be due to something with filters or from reflection from something inside. Have you try to remove the sample? Defocus the sample? Try single line scanning?

---

From: Aryeh Weiss <[hidden email]> on behalf of Aryeh Weiss <[hidden email]>
Sent: Sunday, November 20, 2016 9:49 AM
To: MODEL, MICHAEL; [hidden email]
Subject: Re: interference fringes in SP5 scans at 10x

 

Thank you for your reply. This is fluorescence, not reflectance, imaging. The passbands are set to reject the excitation wavelengths.

--aryeh

On 20/11/2016 16:18, MODEL, MICHAEL wrote:

Hello Aryeh,

You are doing fluorescence or reflection? If reflection then the fringes are almost unavoidable. For example, light reflecting from the front and from the back of the coverslip should interfere because of the long coherence length of the laser

Mike

---

From: Confocal Microscopy List [hidden email] on behalf of Aryeh Weiss [hidden email]
Sent: Sunday, November 20, 2016 7:25 AM
To: [hidden email]
Subject: interference fringes in SP5 scans at 10x

 

***** 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. *****

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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

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[manoj mathew]

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Dear Aryeh,

As mentioned already  fringes are almost always due to coherent excitation light from the laser/s entering the detectors. Fluorescence is non-choherent and cannot possibly give off fringes.

On SP5, essentially you have three barriers preventing the excitation light from entering the detectors:

1. AOBS

2. Notch filters 

3. Spectral Sliders 

This might be problem with the AOBS settings. The 405nm on Leica SP5 is not routed through the AOBS but through a separate primary dichroic mirror. This might explain why you don't see fringes with the 405nm.

Check the following AOBS settings:

1. Check if it is in reflection or fluorescence mode. You need to select the fluorescence mode

2. Check if appropriate notch filter (to cut of excitation light) is inserted. 

Also check your emission spectral slider settings:

If they are not properly set (sufficiently away from the excitation wavelengths) it could allow excitation light into the detectors. 

However even if the spectral sliders are set properly there could be issues if the spectral calibration of the system is off. Spectral calibration is done through the service software and needs to be done periodically along with pinhole alignment etc. by the service engineer. 

Best Regards,

Manoj

On Sun, Nov 20, 2016 at 8:38 PM, MODEL, MICHAEL <[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. *****

Fluorescent light cannot produce interference. So this has to be reflection. It could be due to something with filters or from reflection from something inside. Have you try to remove the sample? Defocus the sample? Try single line scanning?

---

From: Aryeh Weiss <[hidden email]> on behalf of Aryeh Weiss <[hidden email]>
Sent: Sunday, November 20, 2016 9:49 AM
To: MODEL, MICHAEL; [hidden email]
Subject: Re: interference fringes in SP5 scans at 10x

 

Thank you for your reply. This is fluorescence, not reflectance, imaging. The passbands are set to reject the excitation wavelengths.

--aryeh

On 20/11/2016 16:18, MODEL, MICHAEL wrote:

Hello Aryeh,

You are doing fluorescence or reflection? If reflection then the fringes are almost unavoidable. For example, light reflecting from the front and from the back of the coverslip should interfere because of the long coherence length of the laser

Mike

---

From: Confocal Microscopy List [hidden email] on behalf of Aryeh Weiss [hidden email]
Sent: Sunday, November 20, 2016 7:25 AM
To: [hidden email]
Subject: interference fringes in SP5 scans at 10x

 

***** 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. *****

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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

[Aryeh Weiss]

***** 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. *****

Thanks to the many members of the list that have replied to my post.

As many have pointed out, fluorescence is not coherent, and  the fringes must be from reflections.

One suggestion has been to check the AOBS alignment, and we will try to do that. A misaligned AOBS might be passing some
of the reflected excitation light.

We made sure that the spectral bands were not close to the excitation lines, so it is unlikely to to direct leakage of the excitation.

I was not aware that the SP5 has notch filters to block the excitation light. I thought that the AOBS and knife edges were sufficient.

So we have something to check (the AOBS), and we will report on whether this was the problem.

--aryeh

On 20/11/2016 20:08, Manoj Mathew 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. *****

Dear Aryeh,

As mentioned already  fringes are almost always due to coherent excitation light from the laser/s entering the detectors. Fluorescence is non-choherent and cannot possibly give off fringes.

On SP5, essentially you have three barriers preventing the excitation light from entering the detectors:

1. AOBS

2. Notch filters 

3. Spectral Sliders 

This might be problem with the AOBS settings. The 405nm on Leica SP5 is not routed through the AOBS but through a separate primary dichroic mirror. This might explain why you don't see fringes with the 405nm.

Check the following AOBS settings:

1. Check if it is in reflection or fluorescence mode. You need to select the fluorescence mode

2. Check if appropriate notch filter (to cut of excitation light) is inserted. 

Also check your emission spectral slider settings:

If they are not properly set (sufficiently away from the excitation wavelengths) it could allow excitation light into the detectors. 

However even if the spectral sliders are set properly there could be issues if the spectral calibration of the system is off. Spectral calibration is done through the service software and needs to be done periodically along with pinhole alignment etc. by the service engineer. 

Best Regards,

Manoj

On Sun, Nov 20, 2016 at 8:38 PM, MODEL, MICHAEL <[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. *****

Fluorescent light cannot produce interference. So this has to be reflection. It could be due to something with filters or from reflection from something inside. Have you try to remove the sample? Defocus the sample? Try single line scanning?

---

From: Aryeh Weiss <[hidden email]> on behalf of Aryeh Weiss <[hidden email]>
Sent: Sunday, November 20, 2016 9:49 AM
To: MODEL, MICHAEL; [hidden email]
Subject: Re: interference fringes in SP5 scans at 10x

 

Thank you for your reply. This is fluorescence, not reflectance, imaging. The passbands are set to reject the excitation wavelengths.

--aryeh

On 20/11/2016 16:18, MODEL, MICHAEL wrote:

Hello Aryeh,

You are doing fluorescence or reflection? If reflection then the fringes are almost unavoidable. For example, light reflecting from the front and from the back of the coverslip should interfere because of the long coherence length of the laser

Mike

---

From: Confocal Microscopy List [hidden email] on behalf of Aryeh Weiss [hidden email]
Sent: Sunday, November 20, 2016 7:25 AM
To: [hidden email]
Subject: interference fringes in SP5 scans at 10x

 

***** 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. *****

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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

[Zdenek Svindrych-2]

***** 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]

[Aryeh Weiss]

***** 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. *****

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]

[manoj mathew]

***** 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. *****

Clarification to my previous post:

We have a Leica SP5 with STED attachment and it comes with notch filters. I now believe they are probably specific to the STED configuration and not a standard option.

If the notch filters are installed they are accessible not through the AOBS settings (sorry my bad in the previous post) but through an icon called Analyzer Filter below the AOBS icon on the configuration window.

AOBS and spectral sliders alone should be able to block out all excitation light without the need for the notch filters. They are probably more useful when the STED laser is ON. 

Best,
Manoj

On Mon, Nov 21, 2016 at 8:52 AM, Aryeh Weiss <[hidden email]> wrote:

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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]

[Sathya Srinivasan]

***** 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, 

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:

***** 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. *****

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

[Rosemary.White]

***** 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. *****

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

***** 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, 

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:

***** 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. *****

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

[Steffen Dietzel]

***** 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. *****

Aryeh,

in the SP8, additional notch filters are used only to block out 2-photon lasers or STED depletion lasers, but not for the normal excitation. I would imagine it is the same for the SP5.

Concerning the interference phenomenon, this may be a stupid question, but could interference or another involved process somehow change the wavelength of the excitation light from a single wavelength peak to a low intensity broader profile? When I see this phenomenon, I have the impression it is strongest close to the excitation wavelength, on both sides of it. By excitation with a pulsed laser we can gate it out if we dismiss the first 0.3 ns after the pulse, so it is clearly not fluorescence, as was already said by others.

Steffen

Am 20.11.2016 um 19:34 schrieb Aryeh Weiss:

***** 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. *****

Thanks to the many members of the list that have replied to my post.

As many have pointed out, fluorescence is not coherent, and  the fringes must be from reflections.

One suggestion has been to check the AOBS alignment, and we will try to do that. A misaligned AOBS might be passing some
of the reflected excitation light.

We made sure that the spectral bands were not close to the excitation lines, so it is unlikely to to direct leakage of the excitation.

I was not aware that the SP5 has notch filters to block the excitation light. I thought that the AOBS and knife edges were sufficient.

So we have something to check (the AOBS), and we will report on whether this was the problem.

--aryeh

On 20/11/2016 20:08, Manoj Mathew 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. *****

Dear Aryeh,

As mentioned already  fringes are almost always due to coherent excitation light from the laser/s entering the detectors. Fluorescence is non-choherent and cannot possibly give off fringes.

On SP5, essentially you have three barriers preventing the excitation light from entering the detectors:

1. AOBS

2. Notch filters 

3. Spectral Sliders 

This might be problem with the AOBS settings. The 405nm on Leica SP5 is not routed through the AOBS but through a separate primary dichroic mirror. This might explain why you don't see fringes with the 405nm.

Check the following AOBS settings:

1. Check if it is in reflection or fluorescence mode. You need to select the fluorescence mode

2. Check if appropriate notch filter (to cut of excitation light) is inserted. 

Also check your emission spectral slider settings:

If they are not properly set (sufficiently away from the excitation wavelengths) it could allow excitation light into the detectors. 

However even if the spectral sliders are set properly there could be issues if the spectral calibration of the system is off. Spectral calibration is done through the service software and needs to be done periodically along with pinhole alignment etc. by the service engineer. 

Best Regards,

Manoj

On Sun, Nov 20, 2016 at 8:38 PM, MODEL, MICHAEL <[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. *****

Fluorescent light cannot produce interference. So this has to be reflection. It could be due to something with filters or from reflection from something inside. Have you try to remove the sample? Defocus the sample? Try single line scanning?

---

From: Aryeh Weiss <[hidden email]> on behalf of Aryeh Weiss <[hidden email]>
Sent: Sunday, November 20, 2016 9:49 AM
To: MODEL, MICHAEL; [hidden email]
Subject: Re: interference fringes in SP5 scans at 10x

 

Thank you for your reply. This is fluorescence, not reflectance, imaging. The passbands are set to reject the excitation wavelengths.

--aryeh

On 20/11/2016 16:18, MODEL, MICHAEL wrote:

Hello Aryeh,

You are doing fluorescence or reflection? If reflection then the fringes are almost unavoidable. For example, light reflecting from the front and from the back of the coverslip should interfere because of the long coherence length of the laser

Mike

---

From: Confocal Microscopy List [hidden email] on behalf of Aryeh Weiss [hidden email]
Sent: Sunday, November 20, 2016 7:25 AM
To: [hidden email]
Subject: interference fringes in SP5 scans at 10x

 

***** 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. *****

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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