Re: Argon ion laser - age/noise and effect on images..?
Posted by
Jason Kirk on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Argon-ion-laser-age-noise-and-effect-on-images-tp7586010p7586018.html
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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
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