Posted by
PAVAK SHAH on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Intensity-Calibration-Microspheres-tp7590776p7590783.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
Hi Claire,
There are a number of possible ways to measure bead fluorescence, it would
be helpful if you described your pipeline in detail. Are you segmenting
beads by intensity and summing up the total fluorescence per bead? Or
taking the average? Are you performing background subtraction? As Patrick
inquired, are the beads the same size?
The likeliest explanation, given that you're consistently underestimating
the ratio between them is that there is a fixed background in the image.
For example, if the dim beads represent 100 counts absolute and the right
beads 1060 for a ratio exactly of 10.6, a background of 50 counts would
give you an apparent ratio of 8 instead. To correct for this you can either
subtract the background or do a 3-point calibration with 0-intensity ROI's
of the same size as your beads randomly distributed in the image background.
Best,
Pavak
On Fri, Apr 17, 2020, 1:24 PM Claire Brown, Dr. <
[hidden email]>
wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
http://www.imgur.com and include the link in your posting.
> *****
>
> 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
> Join BioImaging North America (BINA):
https://www.bioimagingna.org/join> Join Canada BioImaging (CBI):
https://www.canadabioimaging.org/contact>