Re: Intensity Calibration Microspheres

Posted by Patrick Van Oostveldt on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Intensity-Calibration-Microspheres-tp7590776p7590782.html

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Dear Claire,

Just a questions: do they have the same size?
Perhaps flow cytometry just measures the whole integrated intensity and not local fluorescence.
As fluorescence intensity is lower at higher temperatures it could be that during scanning they differently warm up.


Patrick Van Oostveldt
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> Op 17 apr. 2020 om 22:23 heeft Claire Brown, Dr. <[hidden email]> het volgende geschreven:
>
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> Hi All,
>
> We did some extensive intensity calibration experiments with different intensity green and red microspheres.
> The green microspheres were 3.7% and 35% relative intensities with an intensity ratio of 10.6. We measured the intensity ratio with lots of different microscopes and lots of different lenses and very consistently go a ratio of 8.
>
> I can't seem to figure this out at all. It means the bright beads have to be a bit dimmer than expected or the dim beads a bit brighter than expected.
> The calibration would have been done by ThermoFisher - I would guess they do this by flow cytometry.  Maybe it could just be that microscopy measures the intensities more accurately? Flow just gets on data point per sphere? Maybe the bright beads bleach relatively more than the dim ones when you are imaging on a CLSM?
>
> Any ideas are welcome!
>
> All the best,
>
> Claire
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