Comments on: New stable genetically-encoded Ca sensor http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/ at the intersection of neuroscience and AI. Sun, 27 Jul 2008 09:22:35 +0000 http://wordpress.org/?v=2.5.1 By: disney pixar cars toys http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-530118 disney pixar cars toys Thu, 25 Oct 2007 18:19:04 +0000 http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-530118 <strong>disney pixar cars toys </strong> disney pixar cars toys webstuds 07 disney pixar cars toys

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]]> By: Andrew Hires http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-46159 Andrew Hires Sun, 04 Mar 2007 07:16:05 +0000 http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-46159 Yes you can do FRET imaging with 2-photon, using these CFP-YFP based sensors such as TnC and Cameleons. The excitation spectrum of fluorescent proteins are much greater in 2p mode, so it is impossible to fully isolate the donar excitation. So you will have some cross-excitation of the acceptor which will degrade your maximum ratio changes. However, it has been done with genetically encoded sensors as early as 1999. See this paper for reference. Fan, G.Y., Fujisaki, H., Miyawaki, A., Tsay, R.-K., Tsien, R.Y., and Ellisman, M.H. 1999. Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with yellow cameleons. Biophys. J. 76: 2412-2420. Yes you can do FRET imaging with 2-photon, using these CFP-YFP based sensors such as TnC and Cameleons. The excitation spectrum of fluorescent proteins are much greater in 2p mode, so it is impossible to fully isolate the donar excitation. So you will have some cross-excitation of the acceptor which will degrade your maximum ratio changes. However, it has been done with genetically encoded sensors as early as 1999. See this paper for reference.

Fan, G.Y., Fujisaki, H., Miyawaki, A., Tsay, R.-K., Tsien, R.Y., and Ellisman, M.H. 1999. Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with yellow cameleons. Biophys. J. 76: 2412-2420.

]]> By: Guest http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3469 Guest Fri, 26 May 2006 03:37:25 +0000 http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3469 Aha. Well, I suppose the details would depend highly on the kind of fluorophore, and the kind of system... Aha. Well, I suppose the details would depend highly on the kind of fluorophore, and the kind of system…

]]> By: Dan Dright http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3468 Dan Dright Fri, 26 May 2006 02:32:52 +0000 http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3468 via http://www.fretimaging.org : Fluorescence Resonance Energy Transfer (FRET) imaging is a powerful microscopy technique that overcomes some of the usual limitations of light microscopy to allow researchers to visualize and quantify protein associations under physiological conditions in individual cells. Also known as Förster Energy Transfer (FET), FRET utilizes radiationless energy transfer between proteins tagged with mutant forms of jellyfish green fluorescent proteins (GFP). FRET imaging can be done using wide field, confocal, and <b>two-photon microscopy systems.</b> via http://www.fretimaging.org :

Fluorescence Resonance Energy Transfer (FRET) imaging is a powerful microscopy technique that overcomes some of the usual limitations of light microscopy to allow researchers to visualize and quantify protein associations under physiological conditions in individual cells. Also known as Förster Energy Transfer (FET), FRET utilizes radiationless energy transfer between proteins tagged with mutant forms of jellyfish green fluorescent proteins (GFP). FRET imaging can be done using wide field, confocal, and two-photon microscopy systems.

]]> By: Dan Dright http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3355 Dan Dright Wed, 24 May 2006 02:37:42 +0000 http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3355 I gotcha. I didn't mean to be thick. It comes naturally. :) It seems to me that it might limit the resolution of the technique. Let me ask. The fret guy was in a pissy mood today. Didn't get his FRET. I gotcha. I didn’t mean to be thick. It comes naturally. :)

It seems to me that it might limit the resolution of the technique.

Let me ask. The fret guy was in a pissy mood today. Didn’t get his FRET.

]]> By: Guest http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3323 Guest Tue, 23 May 2006 20:28:58 +0000 http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3323 Hi Dan, What I meant was: suppose the donor is excited by wavelength lambda_short, and the acceptor is excited by wavelength lambda_long. However, the breadth of the spectrum will be very large for both the donor and acceptor, and so when illuminating the donor in two-photon mode, the infrared light may directly excite the acceptor. Hi Dan,

What I meant was: suppose the donor is excited by wavelength lambda_short, and the acceptor is excited by wavelength lambda_long. However, the breadth of the spectrum will be very large for both the donor and acceptor, and so when illuminating the donor in two-photon mode, the infrared light may directly excite the acceptor.

]]> By: Dan Dright http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3192 Dan Dright Mon, 22 May 2006 02:29:12 +0000 http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3192 Wait. I reread your question. Yes.But that's the whole mechanism of FRET, no? More interesting would be an array to determine directionality of conformational changes. Wait. I reread your question. Yes.But that’s the whole mechanism of FRET, no?

More interesting would be an array to determine directionality of conformational changes.

]]> By: Dan Dright http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3191 Dan Dright Mon, 22 May 2006 01:58:19 +0000 http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3191 We do FRET in our lab. I will find out for you. We do FRET in our lab. I will find out for you.

]]> By: Guest http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3151 Guest Sun, 21 May 2006 21:12:48 +0000 http://neurodudes.com/2006/05/21/new-stable-genetically-encoded-ca-sensor/#comment-3151 Does anyone know if FRET can be used with two-photon? I have been thinking about this kind of strategy for a long time, but of course the key problem is the breadth of two-photon excitation spectra; it's unlikely for some dye pairs, that the right wavelength could be picked to do two-hv excitation. I'm curious to know if there is a published work describing that this can be done, or proving that it's a waste of time... Does anyone know if FRET can be used with two-photon? I have been thinking about this kind of strategy for a long time, but of course the key problem is the breadth of two-photon excitation spectra; it’s unlikely for some dye pairs, that the right wavelength could be picked to do two-hv excitation. I’m curious to know if there is a published work describing that this can be done, or proving that it’s a waste of time…

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