New stable genetically-encoded Ca sensor

A FRET-Based Calcium Biosensor with Fast Signal Kinetics and High Fluorescence Change — Mank et al. 90 (5): 1790 — Biophysical Journal

Relevant details (from the discussion):

Above we reported the generation of a FRET-based calcium biosensor employing TnC as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. Its off-rate is significantly faster than those of previous double chromophore sensors and even outmatches the fastest single fluorophore sensors to date.

Although it is faster than what was previously available, it would be nice if the off-rate was even faster:

Its off-rate was extremely fast, optimally fitted with a double exponential with a dominating {tau} of 142 ms (A1 = 0.63) and a minor {tau} of 867 ms (A2 = 0.06) (Fig. 2 D). Mutation of the N-cap residue 131 of helix G within TnC from isoleucine to threonine (35Go) yielded an indicator of higher calcium affinity with a Kd of 1.7 µM (Fig. 2 B) and shifted the Hill slope to 1.1, although at reduced maximal fluorescence change of 270%. TN-XL expressed well in primary hippocampal neurons at 37°C. Fluorescence was evenly distributed, filling all neuronal processes, with no signs of aggregation. The nucleus was devoid of fluorescence. Repeated stimulations with high potassium followed by repeated washouts demonstrated stable baselines over long recording sessions and reproducible signals after stimulation. Moreover the signals induced by high potassium were more than doubled compared to TN-L15.

Hippocampus response to KCl application

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9 thoughts on “New stable genetically-encoded Ca sensor

  1. 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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  2. 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.

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  3. 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.

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  4. 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.

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  5. 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.

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  6. 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.

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