CFP/YFP
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Everyone, Please help. What could be a good experimental setup to do live cell imaging using CFP and YFP on two separate channels such excitation wavelength and emission filters? Park |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Ok, do you want to do confocal or two photon? Can you afford an off-the-shelf system, or are you building your own from scratch, or do you have an existing system you could modify? Craig On 9/21/07, Lee, Park Joo <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Everyone, > > Please help. What could be a good experimental setup to do live cell imaging > using CFP and YFP on two separate channels such excitation wavelength and > emission filters? > > Park > |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Another important question is the temporal and spatial resolution you require. Do you need to look at organelles in one yeast moving "real time" or fields of COS, Hela or fibroblasts over many hours? > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Ok, do you want to do confocal or two photon? > Can you afford an off-the-shelf system, or are you building your own > from scratch, or do you have an existing system you could modify? > > Craig > > > On 9/21/07, Lee, Park Joo <[hidden email]> wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi Everyone, >> >> Please help. What could be a good experimental setup to do live cell >> imaging >> using CFP and YFP on two separate channels such excitation wavelength >> and >> emission filters? >> >> Park >> > _________________________________________ Michael Cammer http://www.aecom.yu.edu/aif/ |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Park, based on your post, I assume you want to use a confocal, since you are talking about channels and excitation wavelength?! Which type of system are you using? In general, I can not recommend to simultaneously acquire CFP and YFP, since there is quite some overlapping between the emission of those both dyes (see http://www.mcb.arizona.edu/ipc/fret/index.html). Sequential detection would be the way to go. You can do this line-by-line with a confocal, or with dual dichroic plus fast emission filter wheel on a camera-based system, if you would like to follow rapid events. Common excitation wavelengths for CFP are 405/440/458, for YFP 514nm. The optimal filter setup is based on your system (i.e. dichroic performance). Fluorchrome databases like the one I mentioned above are very useful for finding the best combination. cheers, Michael > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Another important question is the temporal and spatial resolution you > require. Do you need to look at organelles in one yeast moving "real > time" or fields of COS, Hela or fibroblasts over many hours? > > > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Ok, do you want to do confocal or two photon? >> Can you afford an off-the-shelf system, or are you building your own >> from scratch, or do you have an existing system you could modify? >> >> Craig >> >> >> On 9/21/07, Lee, Park Joo <[hidden email]> wrote: >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> Hi Everyone, >>> >>> Please help. What could be a good experimental setup to do live cell >>> imaging >>> using CFP and YFP on two separate channels such excitation wavelength >>> and >>> emission filters? >>> >>> Park >>> >> > > > _________________________________________ > Michael Cammer http://www.aecom.yu.edu/aif/ |
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In reply to this post by Lee, Park Joo
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal CFP and YFP together with the 2-Photon are probably not the best choice, because you cannot excite the dyes one after each other. And on the emission side you will always get bleed-through from CFP to YFP channel. cheers, Michael [hidden email] wrote: > Thanks Mike. I was thinking about doing it either on confocal or on twp photon. The sequential detection you mentioned sounds very interesting. Maybe I should try it out. > > Park > > -------------- Original message ---------------------- > From: Michael Weber <[hidden email]> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi Park, >> >> based on your post, I assume you want to use a confocal, since you are >> talking about channels and excitation wavelength?! Which type of system >> are you using? >> >> In general, I can not recommend to simultaneously acquire CFP and YFP, >> since there is quite some overlapping between the emission of those both >> dyes (see http://www.mcb.arizona.edu/ipc/fret/index.html). Sequential >> detection would be the way to go. You can do this line-by-line with a >> confocal, or with dual dichroic plus fast emission filter wheel on a >> camera-based system, if you would like to follow rapid events. Common >> excitation wavelengths for CFP are 405/440/458, for YFP 514nm. The optimal >> filter setup is based on your system (i.e. dichroic performance). >> Fluorchrome databases like the one I mentioned above are very useful for >> finding the best combination. >> >> cheers, >> Michael >> >> >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> Another important question is the temporal and spatial resolution you >>> require. Do you need to look at organelles in one yeast moving "real >>> time" or fields of COS, Hela or fibroblasts over many hours? >>> >>> >>> >>>> Search the CONFOCAL archive at >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>>> >>>> Ok, do you want to do confocal or two photon? >>>> Can you afford an off-the-shelf system, or are you building your own >>>> from scratch, or do you have an existing system you could modify? >>>> >>>> Craig >>>> >>>> >>>> On 9/21/07, Lee, Park Joo <[hidden email]> wrote: >>>>> Search the CONFOCAL archive at >>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>>>> >>>>> Hi Everyone, >>>>> >>>>> Please help. What could be a good experimental setup to do live cell >>>>> imaging >>>>> using CFP and YFP on two separate channels such excitation wavelength >>>>> and >>>>> emission filters? >>>>> >>>>> Park >>>>> >>> >>> _________________________________________ >>> Michael Cammer http://www.aecom.yu.edu/aif/ |
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