Posted by
Andreas Bruckbauer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Ratiometric-FRET-on-Fluoview-tp7579865p7579880.html
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Switching filters will always be too slow in line by line aquisition, so i think you should only switch lasers. The protocol for CFP/YFP would be to aquire with 458 excitation the CFP emission at 475 - 525 nm and the YFP emission > 525 nm and the switch to 514.5 excitation and collect only the YFP emission at > 525 nm.
best wishes
Andreas
-----Original Message-----
From: Jean-Pierre CLAMME <
[hidden email]>
To: CONFOCALMICROSCOPY <
[hidden email]>
Sent: Tue, 5 Mar 2013 19:09
Subject: Re: Ratiometric FRET on Fluoview
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Hi Andreas,
The issue is not grouping channels in the sequential box and choosing line
and frame. The issue is changing filter sets quickly. To take the 3 images
DD, DA, AA the filter/light pathway has to be changed between images. DD
and DA or DD and AA can be taken in the same configuration, but AA and DA
can't. So the only way I can see is to use the Virtual channels and that
is too slow line by Line.
Best
JP
Confocal Microscopy List <
[hidden email]> wrote on
03/05/2013 07:01:06 AM:
> I have not used it for FRET, but we have a sequential tick box at
> the bottom of the window with the channel parameters. When ticked we
> can select "frame by frame" or "line by line" aquisition and how the
> channels will be grouped together.
> best wishes
> Andreas
>
> -----Original Message-----
> From: Jean-Pierre CLAMME <
[hidden email]>
> To: CONFOCALMICROSCOPY <
[hidden email]>
> Sent: Mon, 4 Mar 2013 22:44
> Subject: Ratiometric FRET on Fluoview
>
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> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
> Hello,
> I saw a paper about Ratiometric FRET (Roszik, Cytometer 2009) mentioning
> line by line acquisition of the 3 images (IDA, IDD, IAA). The authors
> mentioned the use of a LSM 510.
> I don't know the LSM510, but I have a fluoview 1000 and the only way I
can
> see how to do that is using the "virual Channels" . However that would
be
> image by image and not line by line.
> Does anyone has done ratiometric FRET on the fluoview and what method
did
> you use ?
> Thank you and Best regards,
> JP
>
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