Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

cross talk/bleed in confocal


  • Please log in to reply
3 replies to this topic

#1 soymilk14

soymilk14

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 81 posts
1
Neutral

Posted 01 November 2009 - 09:54 AM

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.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,669 posts
396
Excellent

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

rhombus

    Rhombus/Uncle Rhombus

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 214 posts
20
Excellent

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

#4 soymilk14

soymilk14

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 81 posts
1
Neutral

Posted 02 November 2009 - 07:56 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


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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.