Another trick is to frequency-double your Ti:Saph for single photon
confocal work. You need a KTP crystal or the like, costing about
$1000. It will halve the wavelength of your laser, whatever your
laser is tuned to. You have to rotate the crystal to match the
frequency, but it is pretty easy to do with a basic rotation stage.
Craig
On Thu, Nov 12, 2009 at 11:22 AM, Ann Haberman <[log in to unmask]> wrote:
> Hi Jamie,
> Is mKO the same as Kate? I afraid that I don't have any experience with mKO,
> but do know that you will not be able to image mCherry with your laser.
> mCherry requires wavelengths longer than 1,000 nm.
> I do have a dsRed variant to suggest though! The dsRed express variant sold
> by Clontech is very bright and easy to excite with both a two-photon laser
> and the 568 laser. It is compatible with excitation of GFP and CFP at a
> wavelength of 865 nm with the 2p laser. I would definitely suggest that
> they buy this fluorophore instead!
> There is a strain of mice that expresses this ubiquitously sold by Jax made
> by the Nagy lab. I use it all the time and love it. The fluorescent protein
> itself was engineered by the Glick lab (chicago I think)
> best,
> Ann
>
> I have a researcher trying to plan future experiments with fluorescent
> proteins and we are trying to determine what the best options would be based
> on our equipment set. They are considering mCherry vs mKO and are leaning
> toward mKO (I think there is an mKO2 now) because the literature shows it to
> be brighter and more stable. They would like to do in vivo work with 2
> photon (we have a Prairie Ultima 2 with a Coherent Chameleon XR laser: 720 -
> 980nm). Ideally, they would also like to do single photon confocal, but we
> are limited to a 568 laser at the moment (hoping to upgrade soon), which
> seems to be just a bit too high for mKO.
>
> Has anyone worked with these proteins (especially in vivo) who could give me
> an idea of the excitation peaks with standard confocal and 2-P?
> Characterization of mCherry is pretty good, but there is very little to go
> on with the mKO. Any help pointing to some literature would be most
> appreciated as well.
>
> James E. Hayden, RBP, FBCA
>
> Manager
>
> Microscopy Core Facility
>
> The Wistar Institute
>
> 3601 Spruce St.
>
> Philadelphia, PA 19104
>
> (215)898-3887
>
> [log in to unmask]
>
> --
>
> Ann Haberman, PhD
> Department of Laboratory Medicine
> Yale University School of Medicine
> 1 Gilbert St.
> TAC S541
> New Haven, CT 06510
>
> 203-785-7349
> 203-785-5415 (fax)
> [log in to unmask]
>
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