Re: Intensity Calibration Microspheres **vendor reply**

[Kilgore, Jason A.]

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** vendor reply **

Hi, Claire,

If you contact me offline, and share the catalog and lot number, I can look up the lot specifications and quality testing methods and get back to you, since I'm with Thermo Fisher / Molecular Probes Tech Support.

Jason
[hidden email]

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Life Sciences Solutions
 

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Claire Brown, Dr.
Sent: Friday, April 17, 2020 1:23 PM
To: [hidden email]
Subject: Intensity Calibration Microspheres

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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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