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Argon ion laser - age/noise and effect on images..?

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Jacqueline Ross

Argon ion laser - age/noise and effect on images..?

Hi everyone,

I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.

The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.

What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.

Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.

Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.

Or if there are some tests that I can do to further define the issue?

The last measurements were as below for 8A with two different beam splitters;

488nm 0.51mW (NT 80/20) 2.31mW (MBS488)

Thanks in advance for any ideas.

Cheers,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI: +64 9 923 7438

Website: http://www.auckland.ac.nz/biru

Rosemary.White

Re: Argon ion laser - age/noise and effect on images..?

Hi Jacqui,

Could your laser have just gone out of alignment? This happens quite regularly on our old SP2.

cheers,

Rosemary

PI Int Dr

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 Jacqui Ross <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Friday, 18 November 2016 11:19 am
To: "[hidden email]" <[hidden email]>
Subject: Argon ion laser - age/noise and effect on images..?

Hi everyone,

I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.

The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.

Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.

Or if there are some tests that I can do to further define the issue?

The last measurements were as below for 8A with two different beam splitters;

488nm 0.51mW (NT 80/20) 2.31mW (MBS488)

Thanks in advance for any ideas.

Cheers,

Jacqui

Jacqueline Ross

Stephen Cody-2

Re: Argon ion laser - age/noise and effect on images..?

Hi Jacqui,

I would expect you'd see black horizontal lines if the laser was failing. If you are seeing a grainy image (I'm presuming a noisy image); increasing laser power though AOTF and decreasing PMT gain (HV) should make it go away.

Cheers Steve

Stephen H. Cody

On 18 Nov. 2016, at 8:26 am, [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 Jacqui,

Could your laser have just gone out of alignment? This happens quite regularly on our old SP2.

cheers,

Rosemary

PI Int Dr

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 Jacqui Ross <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Friday, 18 November 2016 11:19 am
To: "[hidden email]" <[hidden email]>
Subject: Argon ion laser - age/noise and effect on images..?

***** 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 everyone,

I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.

The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.

What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.

Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.

Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.

Or if there are some tests that I can do to further define the issue?

The last measurements were as below for 8A with two different beam splitters;

488nm 0.51mW (NT 80/20) 2.31mW (MBS488)

Thanks in advance for any ideas.

Cheers,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI: +64 9 923 7438

Website: http://www.auckland.ac.nz/biru

Zdenek Svindrych-2

Re: Argon ion laser - age/noise and effect on images..?

In reply to this post by Jacqueline Ross

never seen that myself (just a suspicion, sometimes, which I did not follow in any detail), but a quick check would be to use the PMT at very low gain with very high fluorescence intensity (e.g. rhodamine or fluorescein solution, open pinhole if needed). If the fluctuations don't decrease with lowering gain, something's wrong. You may also look at the frequency spectrum of the fluctuations (the fast scanning axis is your time axis), and it's dependence on zoom and scanning speed. Various sample thickness and pinhole sizes could elucidate sample vibration as the cause.

A fast photodiode above the objective lens, with an oscilloscope, should tell you the same thing...

Good luck, 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: 434-982-4869
Annual FRET Workshop: http://kcci.virginia.edu/workshop-2017

---------- Původní zpráva ----------
Od: Jacqui Ross <[hidden email]>
Komu: [hidden email]
Datum: 17. 11. 2016 19:20:34
Předmět: Argon ion laser - age/noise and effect on images..?

Hi everyone,

I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.

The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.

What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.

Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.

Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.

Or if there are some tests that I can do to further define the issue?

The last measurements were as below for 8A with two different beam splitters;

488nm 0.51mW (NT 80/20) 2.31mW (MBS488)

Thanks in advance for any ideas.

Cheers,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI: +64 9 923 7438

Website: http://www.auckland.ac.nz/biru

Craig Brideau

Re: Argon ion laser - age/noise and effect on images..?

In reply to this post by Rosemary.White

As Rosemary says, it could be your alignment through the system generally. I would also check that the alignment and tuning of your AOTF is still ok. Otherwise grainy images are indicative of low signal, so the laser may just not be producing enough power. I recommend that equipment managers regularly measure the laser power at the sample plane of the microscope to determine long-term drift. If you have historical data available you can compare the current power out of the objective with power levels from when the system was operating better. This helps you diagnose if it is an excitation problem or an emission problem.

It is also possible to get Argon lasers refurbished for reasonable cost. This is typically half the price (or better) of a new laser and essentially brings it back to factory spec. I have had two Argon lasers in our lab sent out for refurbishment with excellent results.

Craig

On Thu, Nov 17, 2016 at 5:26 PM, <[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 Jacqui,

Could your laser have just gone out of alignment? This happens quite regularly on our old SP2.

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 Jacqui Ross <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Friday, 18 November 2016 11:19 am
To: "[hidden email]" <[hidden email]>
Subject: Argon ion laser - age/noise and effect on images..?

***** 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 everyone,

I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.

The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.

What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.

Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.

Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.

Or if there are some tests that I can do to further define the issue?

The last measurements were as below for 8A with two different beam splitters;

488nm 0.51mW (NT 80/20) 2.31mW (MBS488)

Thanks in advance for any ideas.

Cheers,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI: +64 9 923 7438

Website: http://www.auckland.ac.nz/biru

Jacqueline Ross

Re: Argon ion laser - age/noise and effect on images..?

In reply to this post by Jacqueline Ross

Hi everyone,

One further comment which I forgot to include which may be very relevant. We replaced the power supply for the argon ion laser last year after experiencing some fluctuations including black lines in the image. Although I thought that lasers and power supplies came as a pair, I was told that we didn’t need to replace the laser, only the power supply. If anyone else has knowledge about this, it would be very helpful.

Thanks to Patty who has advised me that she was also told that both needed to be replaced.

To Paul, Craig and Zdenek; thanks for your valuable suggestions for further testing. I will try these ideas next week.

Hi Rosemary, we do have our system serviced every year and the alignment is checked and should be OK as it was just serviced in September. However, I will go back to our engineer to check that. He also does the power level testing each time and I managed to get the historical data off him which is how I now know that it’s dropped to 1/3 of its previous power level.

Cheers,

Jacqui

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqui Ross
Sent: Friday, 18 November 2016 1:20 p.m.
To: [hidden email]
Subject: Argon ion laser - age/noise and effect on images..?

Hi everyone,

I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.

The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.

Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.

Or if there are some tests that I can do to further define the issue?

The last measurements were as below for 8A with two different beam splitters;

488nm 0.51mW (NT 80/20) 2.31mW (MBS488)

Thanks in advance for any ideas.

Cheers,

Jacqui

Jacqueline Ross

Jacqueline Ross

Re: Argon ion laser - age/noise and effect on images..?

In reply to this post by Stephen Cody-2

Thanks Steve,

I can reduce it by going slower and using more averaging. However, the PMT gain that I’m using is usually only 600V and to get a reasonable image, I’m having to slow right down and use averaging Line 8 on a system when I normally would get a good image with faster speed and Line 4 with similar labelling. It’s a bit puzzling but I’ve got a few tests to do now to see if I can get a better idea of what’s happening.

Cheers,

Jacqui

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Cody
Sent: Friday, 18 November 2016 2:47 p.m.
To: [hidden email]
Subject: Re: Argon ion laser - age/noise and effect on images..?

Hi Jacqui,

Cheers Steve

Stephen H. Cody

On 18 Nov. 2016, at 8:26 am, [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 Jacqui,

Could your laser have just gone out of alignment? This happens quite regularly on our old SP2.

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 Jacqui Ross <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Friday, 18 November 2016 11:19 am
To: "[hidden email]" <[hidden email]>
Subject: Argon ion laser - age/noise and effect on images..?

***** 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 everyone,

I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.

The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.

What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.

Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.

Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.

Or if there are some tests that I can do to further define the issue?

The last measurements were as below for 8A with two different beam splitters;

488nm 0.51mW (NT 80/20) 2.31mW (MBS488)

Thanks in advance for any ideas.

Cheers,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI: +64 9 923 7438

Website: http://www.auckland.ac.nz/biru

Jason Kirk

Re: Argon ion laser - age/noise and effect on images..?

Hi Jacqui,

First let me congratulate you on getting 8 years out of an Argon tube! Either it doesn’t get much use or you’re extremely lucky. Either way bravo!

You don’t say which model of confocal you are using but based on vintage and your clues regarding dichroics I am going to guess it is a Zeiss LSM 710.

Age will certainly have an effect on raw laser power, but you are still getting quite good power out of your laser - 2.31mW is quite a reasonable number from a 30mW laser. One thing that has not been suggested yet, and is specific to the LSM 7 series, is that in addition to the laser alignment into the scan head there are 2 steering mirrors downstream of the coupling port that control the alignment of the MBS to the pinhole. The 7 series has a fixed pinhole position so these mirrors are used to effectively align the laser to the pinhole. There is a calibration for this, however if your tech just replaced the power supply it may be that these alignments were not run again. You might try this - especially if in addition to signal loss it appears that your sample never really comes into focus. Even if you correct the focus drive if it still looks slightly out of focus this will likely be the fault of the pinhole alignment.

If the pinhole alignment is correct, then on older tubes don’t be afraid to just increase the AOTF transmission to compensate. If you are in fact just loosing power then there is no harm in correcting that with the AOTF adjustment. Remember that AOTF percentages are arbitrary and will be a function of raw input power.

Hope this helps!

-Jason

From: Confocal Microscopy List <[hidden email]> on behalf of Jacqui Ross <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Friday, 18 November 2016 11:19 am
To: "[hidden email]" <[hidden email]>
Subject: Argon ion laser - age/noise and effect on images..?

***** 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 everyone,

I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.

The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.

What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.

Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.

Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.

Or if there are some tests that I can do to further define the issue?

The last measurements were as below for 8A with two different beam splitters;

488nm 0.51mW (NT 80/20) 2.31mW (MBS488)

Thanks in advance for any ideas.

Cheers,

Jacqui
Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI: +64 9 923 7438

Website: http://www.auckland.ac.nz/biru

Glen MacDonald-2

Re: Argon ion laser - age/noise and effect on images..?

In reply to this post by Jacqueline Ross

*****
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 Jacqui,
How does this laser behave with the other lines? I recently had a multi-line argon scanning with blank lines and blocks of fields, but only with the 488 nm line. It was still delivering about 80% of new power. A laser power supply and alignment did not change the problem. The problem turned out to be the AOTF.

Regards,
Glen MacDonald
Digital Microscopy Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]

> On Nov 17, 2016, at 7:55 PM, Jacqui Ross <[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 everyone,
>
> One further comment which I forgot to include which may be very relevant. We replaced the power supply for the argon ion laser last year after experiencing some fluctuations including black lines in the image. Although I thought that lasers and power supplies came as a pair, I was told that we didn’t need to replace the laser, only the power supply. If anyone else has knowledge about this, it would be very helpful.
>
> Thanks to Patty who has advised me that she was also told that both needed to be replaced.
>
> To Paul, Craig and Zdenek; thanks for your valuable suggestions for further testing. I will try these ideas next week.
>
> Hi Rosemary, we do have our system serviced every year and the alignment is checked and should be OK as it was just serviced in September. However, I will go back to our engineer to check that. He also does the power level testing each time and I managed to get the historical data off him which is how I now know that it’s dropped to 1/3 of its previous power level.
>
> Cheers,
>
> Jacqui
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqui Ross
> Sent: Friday, 18 November 2016 1:20 p.m.
> To: [hidden email]
> Subject: Argon ion laser - age/noise and effect on images..?
>
> ***** 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 everyone,
>
> I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.
>
> The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.
>
> What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.
>
> Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.
>
> Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.
>
> Or if there are some tests that I can do to further define the issue?
>
> The last measurements were as below for 8A with two different beam splitters;
>
> 488nm 0.51mW (NT 80/20) 2.31mW (MBS488)
>
> Thanks in advance for any ideas.
>
> Cheers,
>
> Jacqui
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU)
> School of Medical Sciences
> Faculty of Medical & Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI: +64 9 923 7438
>
> Website: http://www.auckland.ac.nz/biru

Craig Brideau

Re: Argon ion laser - age/noise and effect on images..?

Very good point Glen! If the other lines from the same argon are not showing the same power drop then it might be your AOTF tuning at the rogue wavelength. On the other hand, the tertiary lines (like 457nm) tend to go first if it is the tube itself that is failing. Given the age of your laser it is likely you have lost gas pressure over time, or your electrodes are just wearing out. Both of these issues can be fixed by having the laser reconditioned.

Craig

On Fri, Nov 18, 2016 at 9:27 AM, Glen MacDonald <[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 Jacqui,
How does this laser behave with the other lines? I recently had a multi-line argon scanning with blank lines and blocks of fields, but only with the 488 nm line. It was still delivering about 80% of new power. A laser power supply and alignment did not change the problem. The problem turned out to be the AOTF.

Regards,
Glen MacDonald
Digital Microscopy Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]

> On Nov 17, 2016, at 7:55 PM, Jacqui Ross <[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 everyone,
>
> One further comment which I forgot to include which may be very relevant. We replaced the power supply for the argon ion laser last year after experiencing some fluctuations including black lines in the image. Although I thought that lasers and power supplies came as a pair, I was told that we didn’t need to replace the laser, only the power supply. If anyone else has knowledge about this, it would be very helpful.
>
> Thanks to Patty who has advised me that she was also told that both needed to be replaced.
>
> To Paul, Craig and Zdenek; thanks for your valuable suggestions for further testing. I will try these ideas next week.
>
> Hi Rosemary, we do have our system serviced every year and the alignment is checked and should be OK as it was just serviced in September. However, I will go back to our engineer to check that. He also does the power level testing each time and I managed to get the historical data off him which is how I now know that it’s dropped to 1/3 of its previous power level.
>
> Cheers,
>
> Jacqui
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqui Ross
> Sent: Friday, 18 November 2016 1:20 p.m.
> To: [hidden email]
> Subject: Argon ion laser - age/noise and effect on images..?
>
> ***** 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 everyone,
>
> I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.
>
> The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.
>
> What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.
>
> Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.
>
> Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.
>
> Or if there are some tests that I can do to further define the issue?
>
> The last measurements were as below for 8A with two different beam splitters;
>
> 488nm 0.51mW (NT 80/20) 2.31mW (MBS488)
>
> Thanks in advance for any ideas.
>
> Cheers,
>
> Jacqui
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU)
> School of Medical Sciences
> Faculty of Medical & Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI: +64 9 923 7438
>
> Website: http://www.auckland.ac.nz/biru

Arvydas Matiukas

Re: Argon ion laser - age/noise and effect on images..?

In reply to this post by Jacqueline Ross

Hi Jacqui,

I recently replaced a failed argon laser head after 6 years and about 5000 hrs of operation.

Before the failure I noticed higher tube current, lower output power and higher temporal

power variation (slow, on the minute time scale). Image quality was somewhat degraded

but not directly attributable to the laser noise (high frequency noise) and requiring significantly

slower scan and/or more averaging.

Based on my experience granularity of confocal images is related to the problems on the

detection path: noisy detector, degraded emission filter, beamsplitters, pinholes, and other components

depending on the design of your confocal. Especially when your laser output at focal

plane is in correct mW range. Additionally to other listers advice I suggest to run a simple scan

with (strong) backreflection of laser beam (from mirror slide or just coverslip edge)

and low detector voltage settings (manually configure

optics allowing the backreflected laser beam to reach the detector). That would diagnose

if low noise level images can be acquired.

Another potential problem outside the detection path may be AOTF failure as mentioned by Glen.

Our confocal experienced AOTF problems due to failing fan and AOTF overheating. I would

suggest to check that the ambient temperatures around/inside AOTF and laser head are

in the correct range and do not fluctuate.

Regards,

Arvydas

Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
SUNY Upstate Medical University
Neuroscience & Physiology Dept

Room 4607 IHP

505 Irving Ave

Syracuse, NY 13210

Email: [hidden email]

>>> Jacqui Ross <[hidden email]> 11/17/2016 10:55 PM >>>

Hi everyone,

One further comment which I forgot to include which may be very relevant. We replaced the power supply for the argon ion laser last year after experiencing some fluctuations including black lines in the image. Although I thought that lasers and power supplies came as a pair, I was told that we didn't need to replace the laser, only the power supply. If anyone else has knowledge about this, it would be very helpful.

Thanks to Patty who has advised me that she was also told that both needed to be replaced.

To Paul, Craig and Zdenek; thanks for your valuable suggestions for further testing. I will try these ideas next week.

Cheers,

Jacqui

Hi everyone,

I'm hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.

The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.

What I'm noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I've checked the other detectors and it doesn't seem to be related to the PMTs.

Much slower speeds and more averaging do improve the images but I'm puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn't normally need to go slower/average as much.

Can anyone tell me whether the age of the laser can cause issues like this? I'm wondering if we should just replace it. We only normally replace lasers when they expire.

Or if there are some tests that I can do to further define the issue?

The last measurements were as below for 8A with two different beam splitters;

488nm 0.51mW (NT 80/20) 2.31mW (MBS488)

Thanks in advance for any ideas.

Cheers,

Jacqui

Jacqueline Ross

Jacqueline Ross

Re: Argon ion laser - age/noise and effect on images..?

In reply to this post by Craig Brideau

Thanks Glen, Jason and Craig for your suggestions. I’m not sure if the alignments were run after we got our new power supply but our engineer is quite fastidious so I’d be surprised if he didn’t check this. I’ll test out the other lines and see what the performance is there.

Cheers,

Jacqui

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Saturday, 19 November 2016 7:03 a.m.
To: [hidden email]
Subject: Re: Argon ion laser - age/noise and effect on images..?

Craig

On Fri, Nov 18, 2016 at 9:27 AM, Glen MacDonald <[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 Jacqui,
How does this laser behave with the other lines? I recently had a multi-line argon scanning with blank lines and blocks of fields, but only with the 488 nm line. It was still delivering about 80% of new power. A laser power supply and alignment did not change the problem. The problem turned out to be the AOTF.

Regards,
Glen MacDonald
Digital Microscopy Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]

> On Nov 17, 2016, at 7:55 PM, Jacqui Ross <[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 everyone,
>
> One further comment which I forgot to include which may be very relevant. We replaced the power supply for the argon ion laser last year after experiencing some fluctuations including black lines in the image. Although I thought that lasers and power supplies came as a pair, I was told that we didn’t need to replace the laser, only the power supply. If anyone else has knowledge about this, it would be very helpful.
>
> Thanks to Patty who has advised me that she was also told that both needed to be replaced.
>
> To Paul, Craig and Zdenek; thanks for your valuable suggestions for further testing. I will try these ideas next week.
>
> Hi Rosemary, we do have our system serviced every year and the alignment is checked and should be OK as it was just serviced in September. However, I will go back to our engineer to check that. He also does the power level testing each time and I managed to get the historical data off him which is how I now know that it’s dropped to 1/3 of its previous power level.
>
> Cheers,
>
> Jacqui
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqui Ross
> Sent: Friday, 18 November 2016 1:20 p.m.
> To: [hidden email]
> Subject: Argon ion laser - age/noise and effect on images..?
>
> ***** 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 everyone,
>
> I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.
>
> The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.
>
> What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.
>
> Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.
>
> Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.
>
> Or if there are some tests that I can do to further define the issue?
>
> The last measurements were as below for 8A with two different beam splitters;
>
> 488nm 0.51mW (NT 80/20) 2.31mW (MBS488)
>
> Thanks in advance for any ideas.
>
> Cheers,
>
> Jacqui
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU)
> School of Medical Sciences
> Faculty of Medical & Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI: +64 9 923 7438
>
> Website: http://www.auckland.ac.nz/biru

Craig Brideau

Re: Argon ion laser - age/noise and effect on images..?

It would be very odd if one line from the argon is problematic while the others are fine. In the unlikely case you see this, you either have a bad combiner dichroic or an AOM issue. If all the lines are low power then it is either tube failure or alignment of the laser itself into the laser launch/combiner unit.

Best of luck!

Craig

On Sun, Nov 20, 2016 at 9:20 PM, Jacqui Ross <[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. *****

Thanks Glen, Jason and Craig for your suggestions. I’m not sure if the alignments were run after we got our new power supply but our engineer is quite fastidious so I’d be surprised if he didn’t check this. I’ll test out the other lines and see what the performance is there.

Cheers,

Jacqui

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Saturday, 19 November 2016 7:03 a.m.
To: [hidden email]
Subject: Re: Argon ion laser - age/noise and effect on images..?

***** 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. *****

Very good point Glen! If the other lines from the same argon are not showing the same power drop then it might be your AOTF tuning at the rogue wavelength. On the other hand, the tertiary lines (like 457nm) tend to go first if it is the tube itself that is failing. Given the age of your laser it is likely you have lost gas pressure over time, or your electrodes are just wearing out. Both of these issues can be fixed by having the laser reconditioned.

Craig

On Fri, Nov 18, 2016 at 9:27 AM, Glen MacDonald <[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 Jacqui,
How does this laser behave with the other lines? I recently had a multi-line argon scanning with blank lines and blocks of fields, but only with the 488 nm line. It was still delivering about 80% of new power. A laser power supply and alignment did not change the problem. The problem turned out to be the AOTF.

Regards,
Glen MacDonald
Digital Microscopy Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]

> On Nov 17, 2016, at 7:55 PM, Jacqui Ross <[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 everyone,
>
> One further comment which I forgot to include which may be very relevant. We replaced the power supply for the argon ion laser last year after experiencing some fluctuations including black lines in the image. Although I thought that lasers and power supplies came as a pair, I was told that we didn’t need to replace the laser, only the power supply. If anyone else has knowledge about this, it would be very helpful.
>
> Thanks to Patty who has advised me that she was also told that both needed to be replaced.
>
> To Paul, Craig and Zdenek; thanks for your valuable suggestions for further testing. I will try these ideas next week.
>
> Hi Rosemary, we do have our system serviced every year and the alignment is checked and should be OK as it was just serviced in September. However, I will go back to our engineer to check that. He also does the power level testing each time and I managed to get the historical data off him which is how I now know that it’s dropped to 1/3 of its previous power level.
>
> Cheers,
>
> Jacqui
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqui Ross
> Sent: Friday, 18 November 2016 1:20 p.m.
> To: [hidden email]
> Subject: Argon ion laser - age/noise and effect on images..?
>
> ***** 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 everyone,
>
> I’m hoping that someone can help me to confirm whether an effect I am seeing with one of our confocal microscopes is likely to be due to the ageing of an argon ion multiline laser.
>
> The laser is about 8 years old and the power level has dropped to about 1/3 of the original output but we are still able to excite the fluorophores very well at AOTF controlled percentages <5%.
>
> What I’m noticing is that when I use the argon ion laser, e.g. 488nm line, the images look much more granular than when using other lasers when similar voltages are being used at the PMT. I’ve checked the other detectors and it doesn’t seem to be related to the PMTs.
>
> Much slower speeds and more averaging do improve the images but I’m puzzled by this as the issue is there even when the Master gain/voltage is at a level which I wouldn’t normally need to go slower/average as much.
>
> Can anyone tell me whether the age of the laser can cause issues like this? I’m wondering if we should just replace it. We only normally replace lasers when they expire.
>
> Or if there are some tests that I can do to further define the issue?
>
> The last measurements were as below for 8A with two different beam splitters;
>
> 488nm 0.51mW (NT 80/20) 2.31mW (MBS488)
>
> Thanks in advance for any ideas.
>
> Cheers,
>
> Jacqui
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU)
> School of Medical Sciences
> Faculty of Medical & Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI: +64 9 923 7438
>
> Website: http://www.auckland.ac.nz/biru