Cameleon-Nanos : High Affinity GECIs

Takeharu Nagai’s lab has published in Nature Methods, Spontaneous network activity visualized by ultrasensitive Ca²⁺ indicators, yellow Cameleon-Nano, demonstrating a new set of calcium indicators based on yellow cameleon. Back when he was still Take-san, Take’s ability to churn out and manually screen hundreds of cameleon variants was impressive and inspiring. With high-throughput GECI pipelines now ramping up at Janelia, the idea of laboriously screening 200 variations on a theme (be it cameleons or GluSnFRs), seems a bit archaic. However, this paper is a good example of the progress that can still be made by understanding the needed sensor parameters and fiddling with the primary amino acid structure in a relatively low-throughput way. Take-sensei’s results are another example of the pramatic rule in protein design, “when in doubt, tinker with the linker.”

The cameleon-nano family achieves greater apparent calcium affinity than YC2.60-4.60, reaching levels of up to 15nM.  They did this by increasing the flexibility of the linker by extending the standard Tsien/Miyawaki/Baird Gly-Gly-Ser linker with additional glycines.  In this case, the longer the linker between the CaM and M13 segments, the greater the apparent affinity. Interestingly, improvement by increasing linker flexibility is precisely the opposite the advice Atsushi and Take gave me for achieving high ratio changes with FRET reporters.  Back at RIKEN in 2002, they suggested I use short, stiff linkers to restrict the rotational freedom of the fluorescent pairs.  Then one could find orientations where relative rotation of dipole moment gave much greater FRET changes than would be expected from changes in FP distance alone. Take and Atsushi’s big YC2.60/3.60 paper strongly supported this idea!  However, as our understanding of the ideal parameters of calcium sensor’s for in vivo imaging has grown, development directions have adjusted.

Cameleon-Nanos achieve higher signal/noise for sparse action potentials at the expense of linearity.  Like Fluo-4, the signal saturates at relatively low AP frequencies.  I think the absolute affinities measured for this family (15, 30, 50 and 140nM) should be considered very rough estimates. They extrapolated these values from stopped-flow binding experiments, because

Although we would like to measure the koff of YC2.60 and its high affinity variants such as YC-Nano15, we could not do it because it was very difficult to precisely control free Ca2+ concentration at around few tens of nM as far as we used EGTA (Kd for Ca2+ = 151 nM in 0.1 M ionic strength, pH 7.2 at 25 oC). For this purpose, much stronger Ca2+ chelator with a smaller Kd value was required. However there is no such Ca2+ chelator available now.

I’m not sure why they didn’t just use the higher affinity, Mg++ insensitive, chelator BAPTA to make the Kd measurements the right way, with a linear regression of log-log fluorescence/concentration values.  Due to instrument dead time, and the high affinity, I didn’t like stopped-flow based Kd measurements in the early GCaMP papers, and I don’t like them now.  Also, the apparent calcium Kd will be highly dependent on solution ionic strength and [Mg++] which is unreported. Despite these quibbles, which are important only inasmuch as they give insight into the mechanism of improvement and the direction of future development, the cameleon-nano family looks promising for mammalian brain imaging.  I still wonder if, assuming the reported Kd values are relevant in vivo, YC2.60 would be the best of the bunch, since cortical neurons have a resting Kd of ~50nM, which implies that a single AP transient of say 200nM free [Ca++] increase would push the calcium levels right up into the sweet-spot of YC2.60’s sensitivity.

This is all the more interesting given the recent results in YC3.60 imaging from Maz Hasan’s group.  Previously, he had shown that transgenic YC animals were pretty bad for imaging.  However, AAV-mediated gene delivery of YC3.60 has significantly improved the responses of the YC family. I’m not sure if they are really up to GCaMP3 levels under identical in vivo conditions, but they might have better long-term protein stability (or that might depend on which viral serotype is used.) What about cameleon-nanos, what about YC2.60?

CNiFERS of Acetylcholine and Attention

“If you find yourself needing to reread this paragraph, perhaps it’s not that well written. Or it may be that you are low on acetylcholine.” Acetylcholine (ACh) is a major modulator of brain activity in vivo and its release strongly influences attention. If we could visualize when and where ACh is released, we could more fully understand the large trial to trial variance found in many in vivo recordings of spike activity, and perhaps correlate that to attentional and behavioral states mediated by ACh transmission.

Back in grad school, when I was desperately trying to figure out what biological question to answer with my GluSnFR glutamate sensor, I ended up in a meeting with Kleinfeld, his grad student Lee Schroder and Palmer Taylor. We plotted a strategy to make a FRET sensor for acetylcholine.  Palmer had recently solved crystal structures of an acetylcholine binding protein bound to agonists and antagonists.  Snails secrete this binding protein into their ACh synapses to modulate their potency.  The structures showed a conformational change upon agonist binding.  The hope was that by fusing CFP and YFP to the most translocated bits of the protein, they would be able to see an ACh dependent FRET change.  I was skeptical that it would work, as the translocation was much less than with calmodulin-M13 or periplasmic binding proteins used in Cameleon and GluSnFR, but thought was at least worth a shot.  FRET efficiency is highly dependent on dipole orientation, not just dipole distance, and you never know how a small conformational change might rearrange the FP dipoles…

Of course, the simple idea didn’t work.  Instead of giving up on the first dozen attempts, they kept plugging away at alternative strategies for measuring ACh release, and eventually succeeded.  In this Nature Neuroscience report, An in vivo biosensor for neurotransmitter release and in situ receptor activity, Nguyen et al demonstrate a mammalian cell based system for optically measuring ACh levels in an intact brain.  They coexpressed M1 muscarinic receptors with the genetically-encoded calcium indicator TN-XXL in HEK293 cells.  ACh binding to the M1 receptor induced IP₃-mediated calcium influx.  This calcium rise was then picked up by the TN-XXL and reported as a change in CFP/YFP fluorescence.  The crazy part is that they took this cell culture assay and implanted the cells into the brains of living rats!

The CNiFER in vivo experimental paradigm

In culture, the response was highly sensitive and monotonic (for phasic response section, EC₅₀ of 11 nM, a Hill coefficient of 1.9 and a maximum of ΔR/R = 1.1). In vivo, using two-photon imaging through a cortical window, they were able to see clear ACh responses in frontal cortex from electrical stimulation of the nucleus basalis magnocellularis, typically 200-μs current pulses of 200 μA @ 100Hz for 20-500ms.

This was essentially a in vivo proof of principal experiment, showing that one could image ACh release in spatially and temporally precise regions of the brain.  However, the imaging was done under urethane anesthesia, which is a much different brain state than an awake, behaving animal.  Are CNiFERs sensitive, powerful and stable enough to determine behavioral states via imaging in an awake animal?  Would expressing GCaMP3 (an indicator with greater fluorescence dynamic range) improve the performance of the CNiFER system? We used a very similar assay with ACh applied to HEK cells during the initial screens for better GCaMPs. Or, is the performance more limited by the properties of the M1 receptor and the adapting nature of IP3-mediated calcium dynamics?  CNiFERS provide an interesting platform for looking at ACh and potentially other G-protein mediated signaling, but it remains to be seen if labs that aren’t as technically proficient with two-photon rig will find it more useful than cyclic voltammetry for measuring acetylcholine levels.

Nature Neuroscience, 13 (1), 127-132 DOI: 10.1038/nn.2469
Nguyen, Q., Schroeder, L., Mank, M., Muller, A., Taylor, P., Griesbeck, O., & Kleinfeld, D. (2009). An in vivo biosensor for neurotransmitter release and in situ receptor activity

Background : Perceval, the ATP:ADP sensor

Recently, Brain Windows mentioned the report A genetically encoded fluorescent reporter of ATP:ADP ratio. We invited Dr. Jim Berg, the lead author of the study to provide additional background to our readers. Below, Jim provides a fascinating look at rationale behind sensor development.  I really like that they came at this problem with a biological question in mind, something I would recommend before anyone start the development of a genetically encoded indicator.

 

A pixel-by-pixel ratio of the 490 nm excitation image by the 430 nm excitation image from two cultured HEK293 cells expressing Perceval during control conditions (left) and after 40 min of metabolic inhibition with 5 mM 2-deoxyglucose (right)

 

Here’s a little insight into why we decided to develop a fluorescent sensor for cellular energy, and how Perceval evolved. One of the primary research interests of the Yellen lab is the interaction between diet and epilepsy. The ketogenic diet, a high fat, low carbohydrate regimen, is remarkably effective at reducing seizure number. We are investigating how the transition in brain metabolism from glucose to a mixture of glucose and ketone bodies (the metabolically active byproduct of fat metabolism) could lead to a change in neuronal excitability. Previously, we described how acute application of ketone bodies reduces the excitability of substantia nigra neurons, an effect that relies on the opening of ATP-sensitive potassium (K_ATP) channels. Our hypothesis is that the inhibition of glycolysis by ketone body metabolism leads to a reduction in sub-membrane ATP, resulting in an opening of K_ATP channels and a decrease in neuronal excitability. This relies on the controversial idea that sub-membrane ATP is provided by glycolysis (possibly by glycolytic enzymes tethered to the membrane), and that the diffusion of ATP is restricted between the submembrane space and bulk cytoplasm, and concept known as “compartmentation”. To fully test this hypothesis, we required an optical sensor for ATP levels.

When planning these experiments, our first thought was to use Luciferase to detect different subcellular ATP levels. For a number of reasons, primarily Luciferase’s weak signal, we decided that a fluorescent sensor for ATP would be much more useful for our application. Our initial approach was a FRET-based design, with CFP and YFP tethered to a bacterial periplasmic binding protein that dimerized upon ATP application. Although these sensors gave some encouraging results, we never got the change in signal that would be required for cellular assays. We then adopted the ‘circularly permuted fluorescent protein (cpFP) approach that had previously produced sensors for calcium (pericam) and hydrogen peroxide (HyPer). We inserted the yellow fluorescent protein cpmVenus into the loop of the bacterial ATP binding protein, GlnK1 (involved in the regulation of ammonia transport) and found that application of small amounts of ATP to the purified sensor led to a substantial change in the excitation spectrum of the sensor. The affinity of the sensor for ATP was extremely high, orders of magnitude more sensitive than would be appropriate for cellular assays. We also found that our sensor responded to ADP application, only with a much smaller fluorescence change. It was then that we determined that these two perceived negatives (too high affinity and ADP binding) would lead to a sensor that reports the ratio of ATP to ADP. In a bit of good fortune, our design for an ATP sensor had in fact given us a sensor for the more valuable ATP:ADP ratio. After tinkering with our sensor by semirandom mutagenesis of the GlnK1 portion of the protein, we expressed the improved sensor, which we named Perceval (for permuted reporter of cellular energy value) into cultured cells and monitored a change in fluorescence with metabolic inhibition.

Right now, we are excited to use Perceval to investigate neuronal/glial metabolism in mammals. We may target subcellular ATP by either tethering Perceval to a membrane protein, or by using TIRF microscopy. In addition, we are continuing to design improved versions of Perceval, as well as sensors for other metabolic intermediates. We also hope that these sensors will be useful in applications beyond neuronal metabolism, from studies of cancer cells to bacterial metabolism.

Symposium : A Revolution in Fluorescence Imaging

This coming Tuesday and Wednesday (Feb 17th & 18th) at UCSD, there will be a symposium honoring Roger Tsien, featuring presentations from 32 former and current members of the Tsien Lab. The topics are quite diverse, concentrated in genetically-encoded indicators, but also featuring fluorescent cell penetrating peptides for cancer therapy, photophore ligases for imaging synaptic development, and even a radical new design for the internal combustion engine.

The quality of speakers and subjects looks to be outstanding.  Here is a complete schedule.  You may notice that at 11:15 AM on Tuesday in Price Center East Ballroom, I will be presenting recent progress we have made in the development of genetically-encoded calcium indicators and their application to in vivo imaging.  Don’t miss that one!  🙂  Roger’s talk, which will assuredly be equal parts absorbing, humorous, and illuminating, is at 4pm Wednesday in the Price Center Theater.

If you live in Southern California and are interesting in imaging technology, there isn’t a better place to be than this symposium.  If you can’t make it, Brain Windows will have a full write-up following the event.

Here is the un-official schedule.

Tuesday February 17th – Price Center East Ballroom

9:00 -9:05 Varda  Levram -Ellisman Opening

9:05-9:15 Palmer Taylor

Designing the next generation of genetically encoded sensors

9:15-9:30 Roger Heim

FRET for compound screening at Aurora/Vertex

9:30-9:45 Amy Palmer

Designing and using genetically encoded sensors: Lessons I learned from Roger

9:45-10:00 Robert Campbell

Beyond brightness: colony screens for fluorescent protein photo stability and biosensor FRET changes

10:00-10:15 Colette Dooley

GFP sensors for reactive oxygen species: Tying up loose ends and looking forward.

10:15-10:30 Peter Wang

Fluorescent Proteins and FRET biosensors for visualizing cell motility and mechanotransduction

Fluorescent proteins in neuroscience

11:00-11:15 Brian Bacskai

Aberrant calcium homeostasis in the Alzheimer mouse brain

11:15-11:30 Andrew Hires

Watching a mouse think: Novel fluorescent genetically-encoded calcium indicators applied to in vivo brain imaging

11:30-11:45 Alice Ting

Imaging synapse development with engineered photophore ligases

11:45-12:00 Rex Kerr

3D calcium imaging in C. elegans

Clinical applications

12:00-12:15 Todd Aguilera

Activatable Cell Penetrating Peptides for use in clinical contrast agent and therapeutic development

12:15-12:30 Quyen Nguyen

Surgery with Molecular Fluorescence Imaging Guidance

Fluorescent probes (Chemistry)

1:30-1:45 Tito Gonzalez

Voltage-Sensitive FRET Probes & Applications

1:45-2:00 Paul Negulescu

From watching ions to moving them

2:00-2:15 Timothy Dore

Roger-Inspired Photochemistry: Releasing Biological Effectors with 2PE

2:00-2:15 Joe Kao

Electron Paramagnetic Resonance Imaging in Living Animals

2:15-2:30 Brent Martin

Chemical probes for studying protein acylation

2:30-2:45 Jianghong Rao

Non-GFP based probes for imaging of the hydrolytic enzyme activity

Cellular research with and without Fluorescent probes

3:15-3:30 Carsten Schultz

Cell membrane repair visualized by GFP fusion proteins

3:30-3:45 David Green

Transcriptomes and Systems Biology: application to early mammalian embryogenesis

3:45-4:00 Clotilde Randriamampita

Paradoxical aspects of T cell activation revealed with fluorescent proteins

4:15-4:30 Wen-Hong Li

Studying dynamic cell-cell communication in vivo by Trojan-LAMP

4:30-4:45 Martin Poenie

Aim and Shoot: Two roles for dynein in T cell effector function

4:45-5:00 Gregor Zlokarnik

From bla to blah, blah in 20 years

5:00-5:15                        James Sharp

President, Zeiss MicroImaging Gmbh

February 18 2009 – Leichtag 107

Cellular research with and without fluorescent proteins

9:00-9:15 David Zacharias

Fluorescent Proteins, Palmitoylation and Cancer: two out of three ain’t bad

9:15-9:30 Jin Zhang

Visualization of Cell Signaling Dynamics: A Tale of MAPK

9:30-9:45 Paul Sammak

Nuclear organization and movement in pluripotent stem cells measured by Histone GFP H2B

Branching out

9:45-10:00 Yong Yao

NIH Toolbox Program

10:00-10:15 Oded Tour

The Tour Engine – A novel Internal Combustion Engine with the potential to boost efficiency and cut emissions

Into the future

10:45-11:00 Xiaokun Shu

Visibly and infrared fluorescent proteins: photophysics and engineering

11:00-11:15 Michael Lin

Engineering fluorescent proteins for visualizing newly synthesized proteins and improving FRET-based biosensors

11:15-11:30 Jeremy Babendure

Training our next generation of Fluorescent Protein Enthusiasts

Main Event – Price Center Theater

4:00-5:00 Roger Tsien

Chancellor invitational lecture 2008 Nobel Prize in Chemistry

A brief history of calcium imaging

A few months ago I threw together a short presentation on the history of calcium imaging for a journal club here at Janelia. It is incomplete. It lacks notes. It is technical. It focuses much attention on early genetically-encoded indicators. However, calcium imaging is so intertwined with the work of Roger Tsien, my Ph.D. thesis advisor, and since he just won the Nobel Prize, I thought it might be of interest to some of the audience of Brain Windows. It does provide a little bit of background for some of the more recent developments chronicled on this site.

Enjoy.

The great GECI shootout

Dierk Reiff’s lab has done another head-to-head in vivo showdown between various GECIs and a synthetic dye. Their paper, Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro, is very interesting and deserves a full write-up. I will present a detailed analysis of the paper in a future update.  For now, check the abstract.

Recent advance in the design of genetically encoded calcium indicators (GECIs) has further increased their potential fordirect measurements of activity in intact neural circuits. However, a quantitative analysis of their fluorescence changes (F) in vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration ([Ca²⁺]_i) has not been given. We used two-photon microscopy, microinjection of synthetic Ca²⁺ dyes and in vivocalibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca²⁺]_i at rest and [Ca²⁺]_i at different action potential frequencies in presynaptic motoneuron boutons of transgenic Drosophila larvae. We calibrated F of eight different GECIs in vivo to neural activity, [Ca²⁺]_i, and F of purified GECI protein at similar [Ca²⁺] in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum F compared with YC3.3 and TN-L15 in vivo. Maximum F of GCaMP2 and GCaMP1.6 were almost identical. Small [Ca²⁺]_i were reported best by YC3.60, D3cpv, and YC2.60. The kinetics of [Ca²⁺]_i was massively distorted by all GECIs, with YC2.60 showing the slowest kinetics, whereas TN-XL exhibited the fastest decay. Single spikes were only reported by OGB-1; all GECIswere blind for [Ca²⁺]_i associated with single action potentials. YC3.60 and D3cpv tentatively reported spike doublets. In vivo, the K_D(dissociation constant) of all GECIs was shifted toward lower values, the Hill coefficient was changed, and the maximum F was reduced. The latter could be attributed to resting [Ca²⁺]_i and the optical filters of the equipment. These results suggest increased sensitivity of new GECIs but still slow on rates for calcium binding.

Sensing salty currents with Mermaids

A new genetically-encoded voltage sensor paper is out from a friend and former mentor of mine, Atsushi Miyawaki. One memorable moment when working in his lab during the RIKEN summer program of 2002 was when Atsushi took me into his office and whipped out a custom green laser pointer. These had been banned in Japan, as fans would shine their powerful light into the eyes of pitchers and batters at baseball games. Atsushi was really proud of his. He smiled and then started sweeping the light point over the rocks in his fishtank. Each ‘rock’ was actually coral his lab had collected from fluorescent protein hunting trips, and each glowed a different color when the green light hit it. He has been putting these novel discoveries to good use.

In Improving membrane voltage measurements using FRET with new fluorescent proteins, Tsutsui et. al take two fluorescent proteins discovered and 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 transfer which is voltage dependent.  They get 40% dR/R per 100mV with a 2 component association rate of around 10 and 200ms. Unsurprisingly, the kinetics speed up at physiological temperatures to 5-20ms on and off.  They are able to pick up single pseudo-action potentials in Neuro2A cells, though the response is highly filtered. They are also able to see very clear spontaneous waves of potential change in cardiomyocytes (23% dR/R) and single spikes in cultured neurons (2% dR/R for 1AP). They dub this voltage sensor “Mermaid”.

The authors state that they used the new FPs due to their improved photostability and especially pH resistance. 

Additionally, because Aequorea GFP variants are pH-sensitive, and neuronal activity causes considerable acidification, the responses of sensors to depolarization in intact neurons may be overwhelmed by sustained changes resulting from acidification.

Granted that mOrange2 is pretty pH-sensitive, but I’m not sure this is a real issue, or a potential issue to justify using their new FPs.  From the spectra of mUKG vs. EGFP, it would seem that EGFP’s 10nm further redshifted emission would be a superior FRET pair for mKOk.  It smells like there may be a bit of bundling of various independent projects into this paper.  However, they do make a good point that this pair will have a different preferred dipole orientation than existing FRET pairs, which could lead to improved performance in some constructs.  

Things I’m still wondering :

• Have they tried using the improved VSFP3.1 scaffold? This was shown to be much faster than 2.1.  I suspect the mUKG is not as tolerant to C-terminal truncation than CFP and GFP.  
• What about using EGFP as the donor?  Could you then use the VSFP3.1 scaffold?
• Is there a rapid non-FRET quenching of the donor upon depolarization as seen in VSFP3.1?
• Why is the single wavelength fluorescence increasing in both channels in figure 2d?  Is there some photoactivation going on?
• I’d love to see a head to head comparison of VSFP3.1 and Mermaid under identical conditions. Also responses in brain slice at physiological temperatures.

Voltage sensitive imaging powering up

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…

Optical imaging of neuronal glutamate release and spillover with GluSnFR

This post is difficult to craft. I’ve been struggling with whether to write an epic post describing the history of glutamate imaging, the major advances and players in the field and where I fit into it, or a simple post focused on my new paper. Since glutamate imaging is my field, I’ve got tons to say about it, but also there is probably no way to avoid significant personal bias in my account. So, I’ll go with the short form. For those interested in further reading, please check out these earlier reports, including our brief mention of neuronal glutamate measurements with GluSnFR prototypes, neuronal glutamate measurement with FLIPE and the optimization of FLIPE constructs from Wolf Frommer’s group, and the use of FLIPE’s in brain slice to look at broad patterns of glutamate release from the Huguenard group.

In this PNAS paper, Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters, by Hires et al. from the group of Roger Tsien, the authors report on the optimization of GluSnFR, a genetically-encoded Glutamate Sensitive Fluorescent Reporter, and its application to the study of glutamate spillover. A cyan and yellow fluorescent protein bracket a glutamate periplasmic binding protein. Glutamate binding to the PBP causes a conformational change and a reduction in the amount of FRET between the fluorescent proteins. Glutamate concentration can be quantitatively determined by observing the ratio of the blue to yellow fluorescence. When fused to an extra-cellular membrane targeting motif and expressed in neurons, optical responses to synaptic glutamate release were detected.

FRET constructs are very fickle, with their response being very sensitive to how the sensor components are fused together. This paper clearly demonstrates this, as 176 linker combinations were screened for maximal ratio change and only one was far superior to all others. The optimized SuperGluSnFR showed a 44% ratio change between zero and saturating glutamate levels in Ringer’s solution, a 6.2-fold improvement over the original prototype. Importantly, the screen took place in a system, HEK cells with surface displayed GluSnFRs, that was physiologically similar to the neuronal system where the sensor was ultimately used. This ensured that the screen discovered useful improvements, rather than ones that worked great in the screening system, but did not express or respond well when expressed in neurons. Previous glutamate sensor optimization in bacteria lead to large responses in vitro that did not translate well when ported to surface-displayed plasmids. Note though that this optimized sensor, FLI⁸¹PE, has found use when bath applied to brain slice.

SuperGluSnFR was used to address questions about glutamate spillover. Under what conditions might glutamate spill beyond the synaptic cleft? How long does this spillover last, and what effects might it have? The paper makes the first direct, quantitative measurements of the timecourse of glutamate spillover. It shows that, at least in cell culture, spillover following burst stimulation can cause a significant glutamate transient along the entire dendritic surface, not just at the synaptic active zone. After a single action potential, spillover is insufficient to activate any extrasynaptic glutamate receptors. But, after a burst of stimulation, sub-micromolar glutamate levels persist long enough to activate extrasynaptic NMDA receptors. This could have a tremendous impact on dendritic computation, synaptic independence and heterosynaptic long term potentiation or depression.

There are two significant limitations to the conclusions of this paper. First is that the experiments were done in dissociated hippocampal culture at room temperature. Glutamate transporters are faster at physiological temperatures, and the geometry of the neuropil in vivo might reduce the impact of spillover. Secondly, there is no electrophysiology to directly support the NMDAR activation assertion. Hopefully, some other group will pick up this thread and do more rigorous testing. GluSnFR imaging in acute brain slice should be easy enough using in utero electroporation techniques, and many, many labs have the electrophysiology experience needed. I’m tempted to do the experiments myself, but there is simply no time!

If anyone would like to try SuperGluSnFR for their own work, send me an email and I’ll be happy to send out an aliquot!

Pulse shaping for 2-photon signal enhancement

Gains in signal to noise ratios of organic dyes and genetically encoded indicators often come in modest steps following screening of large numbers of compounds or clones. Improvements are usually specific to individual chromophores, leading to the pigeonholing of development efforts on a small handful of indicators that have already undergone systemic optimization (i.e. cameleons, G-CaMP and troponin-based GECIs). Indicator photobleaching imposes strict limits on the amount of information which can be extracted by optical indicators. Improvement of specific indicators and their constituents is a worthy and necessary goal, but more generalizable improvements can be made by changing the nature of the illumination source. A series of papers from a variety of groups has shown that careful manipulation of the structure of pulse laser illumination can produce dramatic improvements in signal/noise and photobleaching during non-linear (two-photon) imaging. This is generalizable to numerous optical indicators. Reduction of photobleaching and photoinduced tissue damage will be essential for continuous optical monitoring of sparse neural activity.

 My first encounter with these techniques came in 2003 during a lab presentation by Atsushi Miyawaki, who showed intriguing results with two-photon illumination of GFP. Kawano et al. shined ultra-short (28 femtosecond) pulses from a Ti-Sapphire laser on a plate of immobilized GFP. Due to the uncertainty principal, these ultra-short pulse durations cause a broad spread (~100nm) in the frequency of the laser pulse. They then actively modulated the phase of different frequency bands of the pulse. The interesting part is that they coupled this modulation to a feedback genetic algorithm that sought to increase the ratio of the GFP fluorescence to the intensity of the laser input. Over several hundred iterations of modulation, the system learned how to dramatically increase the output fluorescence over input power by tuning the phase of the frequency components. Using these optimally shaped pulses, they reduced the photobleaching rate by a factor of four! This was an impressive result, but it is unclear how useful this technique would be to live samples, in their heterogeneous aqueous environments. The tuning parameters might be less stable across a non-uniform sample.

The above paper raises intriguing questions on the nature of two-photon excited states and photobleaching. GFP and other fluorescent proteins have multiple bleaching modes, some permanent, some dark or UV-reversible. A more clear understanding of the photochemistry of bleaching could lead to improved illumination pulse design that could keep the chromophore away from these undesired states.

Early last year, Stenfan Hell’s group demonstrated a dramatic reduction in one and two-photon photobleaching by avoiding recurrent excitation of the GFP chromophore when it was in a dark absorbing state.Standard two-photon imaging procedure is to illuminate with a Ti-Sapphire laser pulsed at 80MHz, with a interpulse gap of 12.5ns. This gap is five times longer then the 2.4ns fluorescent lifetime of EGFP, giving the chromophore plenty of time to emit a photon and decay from the excited singlet S1 state. But is the singlet state the precursor to most photobleaching? Donnert et al. varied the pulse rate from 40 to 0.5MHz and discovered that photobleaching was dramatically reduced at the lower pulse rates, especially below 1MHz (1us interpulse interval). Under one photon illumination, total photons extracted from GFP before bleaching was increased 20-fold, while the rhodamine dye Atto532 increased by 8-fold. This suggests that the primary precursor to photobleaching is not the S1 state but is due to photon absorbtion during a dark triplet state T1, which has a relatively long lifetime of ~1us. Don’t illuminate during this state, and prevent most photobleaching! Under two photon illumination (800nm), even greater reductions in photobleaching of 25 and 20-fold respectively took place. This is particularly important because the much higher illumination power used in 2p excitation normally causes a dramatic, non-linear increase in the rate of photobleaching over 1p imaging in the region of focus.

What is special about the T1 triplet state that makes it more prone to causing photobleaching? Is it simply the longer lifetime gives a greater opportunity for an additional photon to hit, jumping to T2 and inducing a photochemical breakdown of the chromophore or the surrounding residues? Does T1 have a broader range of vibrational energies that can more easily engage the variety of photobleaching reactions than S1? How does the photobleaching rate of S2 compare to T2?

Despite the impressive reduction in photobleaching, and hence the greater S/N for a given bleach rate, Hell’s approach has a major drawback. Slowing the pulse train down ~100-fold also slows acquisition time down 100-fold. Therefore this technique is most useful for fixed specimens. Real-time, high resolution imaging of dynamic processes would be seriously degraded. There are work-arounds, such as wide-field pulsed illumination, rapid laser sweeping or multipoint parallel illumination, but these require additional technical development to make them feasible for the average, or even well-above average investigator.

Is there any related solution that can be easily applied to imaging live, dynamic cells? In this month’s Nature Methods, Na et al. from Eric Betzig’s group present an ‘exciting’ approach. They note that with conventional two photon illumination, most of the available laser power is wasted, intentionally blocked to reduce photodamage. As alluded to above, photodamage increases non-linearly with illumination intensity, making two-photon illumination methods particularly harmful. The authors demonstrate that this damage increases proportional to intensity to the ~2.4 power. To try to attenuate this effect, they used a series of mirrors to split the single ultra-short intense pulse (140 femtosecond) in half, and half again and again and again… They end up with 128 pulses of 1/128 the full intensity nearly evenly spaced every 37 picoseconds. This entire pulse group has a duration of 12ns, and with a 80MHz pulse cycle (12.5ns inter pulse interval) illuminates the chromophore with a steady stream of relatively dim pulsed light.

This split pulse illumination dramatically enhances acquisition speed and signal/noise. Images acquired at a rate of 0.4us/pixel with splitting look far more clear than those acquired at 25.6us/pixel without splitting. Photobleaching is reduced by a factor of four and acute photodamage is also reduced. Additional splitting may be possible and further improve the photobleaching attenuation. Importantly, they demonstrate this technique with GFP in fixed brain slices and in live worms and imagine dynamic responses with a calcium dye in living hippocampal slices. This technique appears to let you eat your cake and have it too. The implementation of the pulse splitting is modular, appears relatively simple to those with customizable two-photon instruments and works with existing Ti-Sapphire lasers. I anticipate rapid adoption by serious imaging labs.

Each of the above advances attacks the problem of indicator photobleaching by a different approach, and each focuses on a different aspect of the photochemistry. Theoretically, one could even combine all three for maximum photon collection efficiency before photobleaching, though this would also require combining the drawbacks of each. Photobleaching will continue to be a major concern in the imaging of dynamic processes, particularly when the signal is not synchronized with the onset of image acquisition. These techniques show substantial progress towards alleviating this concern, and I’m heartened to see a number of excellent labs are focusing so much energy on it.

A final question : How will each of these techniques affect acceptor photobleaching (I’m looking at you Citrine and Venus) in FRET imaging experiments? Do the same processes apply when the excitation is coming from a FRET donor?