http://confocal-microscopy-list.275.s1.nabble.com/Ratiometric-FRET-on-Fluoview-tp7579865p7579897.html
Agreed about FRET with a dichroic-based beam splitter on the emission side such as a DualView or QuadView. No commercial interest, but our group uses a DV2 widefield/TIRF system for quantitative FRET just about all day every day. As the executives say, drive for show and putt for dough. In general I'd always go widefield if quantitative is more important than spatial.
JP, unless I remember wrong the 510 has its detectors in a series with switchable dichroic filters between each detector (for example, a 515-ish nm longpass dichroic between detectors 1 and 2 if you want to see cerulean/venus/FRET). Thus it collects each emission channel simultaneously and can go as fast as it can scan a line and alternate lasers, which pass through a two-notch excitation filter. Creulean-FRET bleed-through is still a bear of course, but it always is, and there are long established ways to correct that.
Dept. of Pharmacology & Chemical Biology
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> If speed is the issue and you can illuminate with broad spectrum light, you
> might want to consider the QV2 from Photometrics:
>
http://www.photometrics.com/products/multichannel/>
> It allows a single camera to simultaneously image up to 4 channels by
> splitting the chip into quadrants and splitting the light into four paths
> that each pass through a different emission filter before hitting the CCD.
>
> Just clarify, I have never personally used one of these in a research
> project, but I have worked with one in developing software support for the
> resulting images. I do not work for Photometrics.
>
> Chris Tully
> Microscopy and Image Analysis Expert
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> On Tue, Mar 5, 2013 at 2:09 PM, Jean-Pierre CLAMME <
>
[hidden email]> wrote:
>
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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:
>>
>>> Andreas Bruckbauer <
[hidden email]>
>>> Sent by: Confocal Microscopy List <
[hidden email]>
>>>
>>> 03/05/2013 07:05 AM
>>>
>>> Please respond to
>>> Confocal Microscopy List <
[hidden email]>
>>>
>>> To
>>>
>>>
[hidden email]
>>>
>>> cc
>>>
>>> Subject
>>>
>>> Re: Ratiometric FRET on Fluoview
>>>
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>>
>>> 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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>>> 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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