Re: Intensity Calibration Microspheres

Posted by George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Intensity-Calibration-Microspheres-tp7590776p7590778.html

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I encourage more than 2 point calibration. Here is 5 intensities:

Ultra Rainbow Fluorescent Particle Slide, 5 Intensities ... FPS-5057-UR5
... I have one of these slides, and some data from FV3000RS and SP8 (not
turned into cohesive numbers like Claire's).

The spherotech fluorescent particle slides PDF

https://www.spherotech.com/2020%20Product%20Detail%20Pages/Spherotech%20Fluorescent%20Particle%20Slides.pdf

also has:

Rainbow Fluorescent Particle Slide, 6 Intensities, For log scale ...
FPS-3057-5LG

Rainbow Fluorescent Particle Slide, 5 Intensities in individual wells,
For linear scale ... FPS-3057-5LN

enjoy,

George

p.s. Ultra Rainbow has 8 fluorophores. I also now have:

Fluorescent PMMA Particle Slide, 9 different fluorescent beads; UV,
Light Yellow, Yellow, Nile Red, Pink, Purple, CyBlue, Sky Blue, & Jade Green

FPMAS-30M9

which have a couple of different intensities. All of these are 3.0 um
diameter ... the company is open to suggestions on other sizes,
products. The beads are also available in suspension ... one tube of
'the bright stuff' (and not so bright, too) COULD be purchased,
re-packaged (slides ... R.I. matched medium, small volume each)) and
distributed though - say - ABRF's LMRG.


On 4/17/2020 4:23 PM, Claire Brown, Dr. wrote:

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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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