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

Posted by Zdenek Svindrych-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Argon-ion-laser-age-noise-and-effect-on-images-tp7586010p7586012.html

***** 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,

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: [phone]
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:  [phone]

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