Intensity Calibration Microspheres
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Claire Brown |
Intensity Calibration Microspheres
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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 |
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Scott Henderson-2 |
Re: Intensity Calibration Microspheres
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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 Claire, Just curious - were the measurements done in water? Regards, Scott ----------------------- Scott Henderson, Ph.D. Professor, Dept. Molecular Medicine Director, TSRI Microscopy Core Room MB-22 Scripps Research 10550 North Torrey Pines Rd. La Jolla, CA 92037 858-784-8163 On Apr 17, 2020, at 1:23 PM, Claire Brown, Dr. <[hidden email]<mailto:[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 |
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George McNamara |
Re: Intensity Calibration Microspheres
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In reply to this post by Claire Brown
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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. ***** 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: > ***** > 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 |
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Patrick Van Oostveldt |
Re: Intensity Calibration Microspheres
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In reply to this post by Claire Brown
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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. ***** 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 Sint-Denijslaan 199 9000 Gent Gsm +32 487656381 Verstuurd vanaf mijn iPhone > Op 17 apr. 2020 om 22:23 heeft Claire Brown, Dr. <[hidden email]> het volgende geschreven: > > ***** > 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 |
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PAVAK SHAH |
Re: Intensity Calibration Microspheres
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In reply to this post by Claire Brown
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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 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 > |
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Claire Brown |
Re: Intensity Calibration Microspheres
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In reply to this post by Claire Brown
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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. ***** No they were done in CyGel from Biostatus Ltd. I guess there could be some quenching from the medium. |
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Claire Brown |
Re: Intensity Calibration Microspheres
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In reply to this post by Claire Brown
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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. ***** Thanks George. We are looking in 3D so would need individual samples. Can you get all the different brightness beads in suspension? |
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Claire Brown |
Re: Intensity Calibration Microspheres
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In reply to this post by Claire Brown
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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. ***** The imaging was done on over 40 different microscopes with different magnifications, immersion medium and NA and instrument seeings. This is why we used the intensity ratio. the dim and bright beads were in the same sample though. |
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Claire Brown |
Re: Intensity Calibration Microspheres
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In reply to this post by Claire Brown
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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. ***** They are the same size, but perhaps the brighter ones could heat up more? Not sure how much heating you might get with a green laser though. Could also be flow get the max intensity at the middle of the sphere but in microscopy we measure the whole thing. however, I would think that any difference like that would disappear with the ratio. |
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Claire Brown |
Re: Intensity Calibration Microspheres
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In reply to this post by Claire Brown
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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. ***** Hello Pavak, We did correct for background so that is not it. As I said to Patrick they are also the same size. I guess we could also have the wrong lot information. We got them from a 3rd party not directly from the company. Thanks, Claire |
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George McNamara |
yes, most of Spherotech's products are beads in suspension already for flow cytometry ... Re: Intensity Calibration Microspheres
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In reply to this post by Claire Brown
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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. ***** yes, most of Spherotech's products are beads in suspension already for flow cytometry ... if not in their catalog, easy enough for them to make suspensions and bottle (ok, tube) them. On 4/20/2020 6:58 PM, Claire Brown 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. > ***** > > Thanks George. We are looking in 3D so would need individual samples. Can you get all the different brightness beads in suspension? |
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