Yes, we have in fact two blocking filters - 660nm or 710nm. So something like Alexa 633 or 647 should be detectable in theory. I wonder if any of you have experience exciting them or similar dyes with MP? Some papers seemed to have used A647, but the methods were not clearly described.
What about Alexa 405, Pacific Blue, or other blue dyes? We are trying to maximize the number of dyes that can use in the tissues; more specifically, something in combination with GFP/YFP/CFSE and a red/orange dye such as dsRed/RFP/CMPTX. Any suggestions will be appreciated.
Thanks,
Lily
-----Original Message-----
From: Guy Cox [mailto:[log in to unmask]]
Sent: Thursday, January 21, 2010 7:22 AM
To: [log in to unmask]
Subject: Re: probe for MP excitation
It depends on what filters are in your system. Since most Ti-S lasers
can be tuned shorter than 700nm you are unlikely to be able to get
anything longer than 650nm in the detectors. But of course if you are
willing to take on yourself the responsibility to fit custom filters you
could go lower - just remember if you tune your laser too short you will
fry your PMTs!
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Adrian Smith
Sent: Thursday, 21 January 2010 11:13 PM
To: [log in to unmask]
Subject: Re: probe for MP excitation
Good point!
I've been reading up on a few promising reagents recently only to
realise that the blocking filter rules them out.
But it does depends a little what you define as "far-red"... :)
Invitrogen have several products that they call "far-red" that have
plenty of emission <700nm (where I'm guessing most MP blocking filters
sit?)
I've read at least one paper that uses AlexaFluor 660 but that looks to
be optimal with OPO excitation at 1100nm (Andresen et al, Current
Opinion in Biotechnology 2009, 20:1-9).
I've spoken to a couple of people in recent weeks who've used AlexaFluor
647 with standard Ti-Sa excitation. We've only just started trying it
here and had mixed success (not entirely clear why yet). You do have to
look closely at the full transmission profiles of all the filters
(especially dichroics if you are using them for mutli-channel imaging)
in the pathway.
Regards,
Adrian Smith
Centenary Institute, Sydney, Australia
On 21/01/2010, at 10:51 PM, Guy Cox wrote:
> MP excited far-red dyes are not a starter since the blocking filters
> will block the signal as well. Blue should be easy - after all
everyone
> uses DAPI and Hoechst. I've also imaged blue FPs - not yet cloned,
> though.
>
> Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
> http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
>
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List
[mailto:[log in to unmask]]
> On Behalf Of Koo, Lily (NIH/NIAID) [E]
> Sent: Thursday, 21 January 2010 1:56 PM
> To: [log in to unmask]
> Subject: probe for MP excitation
>
> Dear all,
>
> I wonder if anyone had experience in trying dyes that are suitable for
> MP excitation and emit in the blue and/or far-red spectrum? Would any
> Alexa or LiveTracker dyes work well there?
>
> Thanks,
>
> Lily
>
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