hello everyone,
i am new in learning confocal, i just want to ask the method on how to check and make sure that there is no or minimal cross talk or bleedthrough?
thank you so much.
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3 replies to this topic
#2
bob1
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Posted 01 November 2009 - 03:42 PM
There shouldn't be, the LASER light is very specific wavelengths so there should be no excitation outside of the LASER you are using. Note that some of the flurophores have similar excitation ranges, it is best to avoid using similar ones on the same sample.
#3
rhombus
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Rhombus/Uncle Rhombus
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Posted 02 November 2009 - 03:00 AM
Dear soymilk14,
You need to do Sequential Scanning. This means that the scans for each of the fluorophores (Dapi, FITC and TRITC) are done one by one and the software then collects them together.
Bob1 is right in that the emission spectrums for both Blue and Green, and Green and red overlap. This is a way of reducing the overlapping light. This is particularly important if you are looking at Co-localisation.
Hope this is useful.
Rhombus
You need to do Sequential Scanning. This means that the scans for each of the fluorophores (Dapi, FITC and TRITC) are done one by one and the software then collects them together.
Bob1 is right in that the emission spectrums for both Blue and Green, and Green and red overlap. This is a way of reducing the overlapping light. This is particularly important if you are looking at Co-localisation.
Hope this is useful.
Rhombus
#4
soymilk14
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Posted 02 November 2009 - 07:56 AM
rhombus, on Nov 2 2009, 03:00 AM, said:
Dear soymilk14,
You need to do Sequential Scanning. This means that the scans for each of the fluorophores (Dapi, FITC and TRITC) are done one by one and the software then collects them together.
Bob1 is right in that the emission spectrums for both Blue and Green, and Green and red overlap. This is a way of reducing the overlapping light. This is particularly important if you are looking at Co-localisation.
Hope this is useful.
Rhombus
You need to do Sequential Scanning. This means that the scans for each of the fluorophores (Dapi, FITC and TRITC) are done one by one and the software then collects them together.
Bob1 is right in that the emission spectrums for both Blue and Green, and Green and red overlap. This is a way of reducing the overlapping light. This is particularly important if you are looking at Co-localisation.
Hope this is useful.
Rhombus
Thanks Rhombus and Bob1. I know i need to learn more, i am still starting... thanks for sharing this info with me. Can you recommend any good reading material for confocal? I would highly appreciate it. thanks!
regards,
soymilk14
