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Hello Gary:
We use a Hioki 3664 Optical power meter, instead of using one special calibration slide we use the PMT, with a fixed (1%) laser power, with every light inside the dark room turned off, 10x SuperApochromat objective selected, same HV, gain and offset every year and 10 repeatitions for each laser, we accept a maximum in the obtained standar deviation obtained for this sampling method.
In this way we check two things, sensibility and stability of the PMT detector, and a cross reference for the power arriving from the laser to the detector, then we compare the results of the PMT check with the measurement of the optical power meter and if the power meter shows that there is not signifcant change of the laser power (also 10 measurements are recorded and statistically analized) we have to go back to objective cleaninless, glasses in the way, dic analyzer, polarizer in the way, closed or partially closed diafragms, and last of all the PMT (never have been the trouble those).
For analizing the spectral analizer of the confocal, we do something simple, place a good mirror, clean, on top of the 10x SuperApochromat objective, use the 20/80 dichroic mirror (20% of the excitation goes trough and 80% of the emission goes back to PMT detector) and turn all the laser lines on, put a 100% mirror sending all the light back to CH1 and do a full range (400nm to 800nm) spectral scan, 256x256pix image, with a 5nm band width and a 1nm step, in this way you should obtain high peaks on your laser lines points and no emissión out of them, so if the 543nm peak is in the 544nm or 542nm place, you know you have to realign and recalibrate your spectral detection.
I hope this helps you and if you know something that can improve or make this easier please let me know
Regards
Gabriel OH
> Date: Wed, 10 Aug 2011 15:57:41 -0400
> From:
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> Subject: Color Registration and Laser Power Measurement
> To:
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>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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>
> Greetings,
>
> I am preparing to check the color registration, beam alignment and
> laser power of a Zeiss LSM 710 NLO Upright Multiphoton Microscope.
>
> I think that the FocalCheck fluorescence microscope test slide #1,
> Molecular Probes F36909, is a suitable calibration slide, and that the
> Coherent FieldMate meter and PS19 sensor combination is a good option
> for laser power measurement.
>
> Could you tell me what calibration slide and laser power meter you use
> for such an application?
>
> Thank you very much!
>
> Gary G. Li, PhD
> Microscopy Core Facility Manager
> NIH/NIDA at Baltimore, MD 21224
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