Voltage sensitive imaging powering up

8 07 2008

I’m starting to come around on voltage imaging. I haven’t been a fan of it for a number of reasons.

  • The response sizes suck.  Classic dyes and genetically encoded systems get a few percent fluorescence change at best. 
  • The response speeds suck. Measuring continuous current injections from -100mV to +150mV is not very interesting.  Action potentials are interesting.  But they are fast.
  • Toxicity. The dyes kill neurons, or strongly perturb their electrical properties.

OK, voltage-sensitive imaging isn’t totally useless, for example see Carl Petersen’s recent paper on Spatiotemporal Dynamics of Cortical Sensorimotor Integration in Behaving Mice (2007). But if the above problems could be solved, then voltage sensitive imaging would be a strong competitor to calcium imaging for the non-invasive, high-resolution monitoring of patterns of network activity. There has been considerable progress ameliorating these problems in the past few years, much of it by a consortium of labs (Isacoff, Knöpfel, Bezanilla, Miesenböck, and others) focused on these issues. (Umlaut’s apparently help in this field).

First, let’s look at a minor breakthrough for the fully genetically-encoded strategy.  In Engineering and Characterization of an Enhanced Fluorescent Protein Voltag Sensor (2007), The Knöpfel group tagged the recently discovered voltage sensitive phosphotase (Ci-VSP) with CFP and YFP FRET pairs in place of the phosphotase domain. This tagged protein expressed at the membrane much more efficiently than previous genetically encoded voltage sensors based on potassium channel subunits. By injecting physiological voltage changes and averaging 50-90 traces, they were able to pull out a few percent ratio change from a brief series of action potentials. Single spikes were resolvable.  Although this sensor (VSFP2.1) was pretty slow (tau > 10ms), this new substrate looked promising for future sensor development.

They have since sped the response up.  In Engineering of a Genetically Encodable Fluorescent Voltage Sensor Exploiting Fast Ci-VSP Voltage-Sensing Movements (2008 ), they determined that the gating motion of the voltage sensing component was very fast (~1ms), while the fluorescence change was slow (~100ms). So they did what any good FRET tinkerer would do, chop away at the linkers between FP components.  The sensor response improved, and they noticed that there was a disconnect between the speed of the CFP and YFP responses. Not only was CFP decreasing from enhanced FRET, it was being directly quenched by interactions with the lipid membrane. Chopping off the YFP from the the construct then dramatically increased the speed of the CFP quench. This improved sensor, VSFP3.1 has an activation time constant of 1.3ms, though it’s response magnitude is still quite small (a few % dF/F).

A hybrid approach to measuring electrical activity in genetically specified neurons (2005) has a much greater response magnitude. Pancho Bezanilla’s group exploited the rapid, voltage-dependent translocation of the small molecule quencher dipicrylamine (DPA) through the plasma membrane to change the fluorescence of membrane-teathered GFP in a voltage-dependent manner. Responses of the hybrid voltage sensor (hVOS) were relatively large (34% per 100mV) and fast (0.5ms). Single action potentials were detectable without averaging.  However, since DPA is a charged molecule, it significantly increased the capacitance of the membrane. The levels of DPA required to see large responses inhibited action potentials and were intolerable to neurons.

Last month in Rational Optimization and Imaging In Vivo of a Genetically Encoded Optical Voltage Reporter (2008 ), Sjulson and Miesenböck reported optimized parameters for the hVOS approach. They built a quantitative model of the quenching effects of DPA on membrane-teathered GFP.  The quenching is limited by the distance the DPA can approach the chromophore of GFP.  Only the closest DPA molecule to the chromophore significantly contributes to a GFP’s quenching. After lots of pretty heat maps and graphs, the model tells them to chop off the tail of EGFP to bring the C-terminal tethering sequence closer to chromophore. I should note that an 11 amino acid C-terminal truncation of ECFP has improved the response of a tremendous number of FRET reporters and has been standard practice for the last 8 years. By shortening the linker they manage to triple the response size. I’d suggest, if they haven’t already, to lop off another six amino acids (end the EGFP with …LEFVTAA) and see if works.  EGFP and ECFP usually tolerate it.

Using this optimized reporter, they are able to reduce DPA concentrations to levels that are usable in vivo, at least for a few minutes. They record fast optical responses to electrical activity in the Drosophila antennal lobe using 2uM DPA.  But after a few minutes, the DPA loaded neurons become strongly inhibited.


The bottom line? Voltage-sensitive imaging has seen big progress in the last few years, but still has a long way to go to gently record single APs in a dish or in vivo. Or does it?  I’m hearing whispers that a different group has developed a synthetic dye technique that is getting >10% dF/F to single APs with millisecond response times. Is it the real deal? Watch this space…



12 responses

8 07 2008

I’ve read a Roger Tsien portrait where he states that he came from chemistry to biology in the 70s because he wanted to develop a good voltage sensing dye. At the time he thaught it would be a few years of work… Thirty years later, we hopefully will see decisive advances in this field.

9 07 2008

Modern dyes are fast and sensitive, read up on Fromherz`s Annine Dyes. They are easily fast enough to capture action potentials, and the sensitivity is up to 59%/100mv , not just a few percent change. Kuhn. Biophys.J. 2004
Unfortunately, the dyes are new and the article published in chemistry journals, far from where potential neurobiologist might stumble upon them.

9 07 2008

just found the link to RY Tsien interview :

9 07 2008

Chistophe, I think its an intrinsically difficult problem. You have to put charges in the membrane, and that will always increase the capacitance. So you’ve just got to make the fluorescence change maximally efficient.

Dave, Thanks for the input on the ANNINE dyes. I wasn’t aware of them. Looks like Kuhn, Denk and Bruno have already tried it in vivo. Evoked responses in the barrel cortex give <1% fluorescence change. In vivo two-photon voltage-sensitive dye imaging reveals top-down control of cortical layers 1 and 2 during wakefulnes

16 07 2008
Sensing salty currents with Mermaids « Brain Windows

[…] engineered by the Miyawaki lab, mUKG and mKOk, and graft them onto the Ci-VSP scaffold used in VSFP2.1 (also developed at RIKEN).  The green and orange fluorescent proteins undergo significant FRET […]

18 07 2008

Andrew: Yep, the in-vivo performance was very low, but I suspect that this problem will be solved with Annine-6 Plus, a new version of the dye as well as further practical experimention. Keep in mind that in the Kuhn paper J. Phys. Chem. B 2003, 107, 7903-7913, the Annine dyes were compared directly with the currently used ANEPBS dyes, and they demonstrated 3 times the voltage sensitivity. In this paper, http://www.biophysj.org/cgi/content/full/90/10/3608, they were able to take voltage readings with a 60ns pulse.
With Annine 6, the sulphate headgroup probably is not charged enough to prevent flip-flop, where the chromaphore reverses its orientation in the lipid bilayer. As this progresses, you end up with an even number of up and down chromaphores, and the change in fluorescence cancels out. The new Annine-6 Plus has a dicationic headgroup that improves solubility and makes flipflop less favourable.
Im not so sure that the current design of voltage sensitive proteins will ever work well. It seems to me that trying to wiggle a huge beta-barreled gfp with a voltage gated channel designed to move just enough to let ions through is streching it a bit. Personally, I would use a single fixed fluorescent fret acceptors, and on the movable section of the voltage gated channel add a tetracystein moiety and go with Tsien’s bivalent arsenicals as fret donors. their small size should get them closer to the fluorescent protein, and not hinder the movement of the channel so much…

26 06 2009
Guixue Bu

I come across this blog when I googled ANNINE-6plus. It is very interesting.

I actually have the first experience with both ANNINE-6 and ANNINE-6plus. I used ANNINE-6 in 2006 and then ANNINE-6plus in 2007. They are just simply the best voltage dyes. Both dyes are commercially available now. Check out my conference proceeding:

I also have preliminary data of the action potentials recorded from isolated dorsal ganglia neurons and neurons within intact dorsal ganglia.

You may find that there are a few critical issues associated with microscopic imaging of action potentials of neuron and myocyte. I figured them out in my dissertation research. Please let me know if you are interested in further discussion on this topic. My email address is gbu@iupui.edu.

3 03 2013

Thanks for the info!

20 05 2010

FYI, the proceedings mentioned above are now at: http://www.cinc.org/archives/2007/pdf/0357.pdf

We’d like to try the Annine-6plus but it doesn’t seem so easy to buy…

29 11 2010
Guixue Bu

We published the microscopic optical imaging of cardiomyocyte action potentials with ANNINE-6 & ANNINE-6plus in a paper entitled “Uniform action potential repolarization within the sarcolemma of in situ ventricular cardiomyocytes” (Biophysical Journal, 96(6):2532-46). It also has a filtering algorithm which allows you to significantly improve the SNR of your signal.

An study published early this year in The Journal of Neuroscience found that ANNINE-6plus has less effect on GABA(A) receptor function, suggesting less neurophysilogical side effect of ANNINE-6plus.

ANNINE-6plus has been available for some time. You can only buy it from Germany.

3 05 2012

http://www.sensitivefarbstoffe.de/ That is the link to buy Annine-6-plus (possibly the least informative page on the internet).

3 03 2013

You’re right, I’m in Germany and this is still hard to get hold of. I think I will just synthesise it myself. Thanks for the info here! Also the PGH dyes are also very great. with incremental improvements in sensitivity. This has great potential with room for improvements.

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