Giving synapses a ‘born on’ label

30 06 2008

Memories are thought to be encoded by the patterns of synaptic connections in the brain. Learning can either delete or change the strength of existing synapses, or add new synapses. Following a learning process, how can we tell which synapses were added to encode this new memory?  

One strategy is to make a timelapse movie of the synapses.  In mice, this can be accomplished by installing a cortical window on the skull, and imaging the changes in structure of GFP labelled neurons. However, this is technically demanding, only works with sparsely labeled neurons, and accesses only a small subset of the neurons which may be involved in the learning process.  

Ideally, one could have a tag which can discriminate between synapses existing before learning takes place, and new ones generated after learning has occurred. Whole brain regions could then be examined at a single timepoint to see where new synapses were added. In a large step towards that goal, Michael Lin et. al, from the lab of Roger Tsien, report TimeSTAMP, a genetic label for newly synthesized protein.

The authors engineered the NS3 protease from the hepatitis C virus (HCV) to cleave itself at just the right pace. They then fuse tags (fluorescent proteins or epitopes) before and after the cleavage site. This fusion is then tagged to the end of a protein of interest. Shortly after synthesis, the protein cleaves off the C-terminal tag, but the N-terminal is left on. This cleavage is inhibitable by a variety of small molecule blockers. In the presence of the blocker, the C-terminal tag stays on. By controlling when drug is applied, they can selectively label a set of proteins of a particular age with the tags.

The choice of NS3 protease was very clever, as it is a favorite drug target of biotech and pharma companies.  Many inhibitors of this protein have been synthesized, exhaustively characterized in vitro and in clinical trials. This work is a great example of the standard research flow going in reverse; a basic-science project from an academic lab is actually benefitting from pharma company research. Stability, bioavailablity and toxicity have already been worked out.  One of the biggest impediments is actually getting ahold of these compounds. Companies with their survival hanging on the clinical success of a single small molecule inhibitor are understandably reluctant to hand out stocks for academic research. Note the roller coaster stock price of Vertex following results of its NS3 protease inhibitor (VX-950) trials. 

The authors use PSD-95 tagged to TimeSTAMP as a proxy marker of synaptic age. In neuronal culture, they show that newly synthesized synapses have a C-tag / N-tag ratio of about twice as large as old synapses.

They extend the technique to whole fruit fly brains, showing a very heterogeneous distribution of CaMKII synthesis across Kenyon cells in different areas of the mushroom body.

So far TimeSTAMP has not been shown to work in mice. Mice were not included in the paper due to the long generation time for transgenics. Given the good signal to noise and the large number of possible inhibitor molecules, I think this technique could be quite powerful in mammalian systems. It’s big advantage would be to label large populations of neurons or synapses in diverse brain regions, including those inaccessible to two-photon microscopy. TimeSTAMP’s success in labeling new synapses in the intact brain will be dependent on finding a protein to tag at the synapse with low turnover over the course of a learning experiment. Though PSD-95 appears to be a reasonable marker in culture, others have shown a higher rate of turnover in vivo, making in unsuitable for a synaptic marker. 

Optical imaging of neuronal glutamate release and spillover with GluSnFR

12 03 2008

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!

3D and Multicolor Superresolution Imaging

19 02 2008

Progress in superresolution imaging is still moving very quickly. Here are two more great papers in the field.

First, Huang et al. from Xiaowei Zhuang’s group published a Science paper that moves superresolution imaging into three dimensions. Previously, STORM and PALM techniques were most useful for thin sections where the z-axis depth is well-constrained. Breaking the diffraction limit in the z-dimension was thought to possibly require recording from multiple angles, standing wave TIRF or optical lattice microscopy. Instead, the authors simply inserted a weak cylindrical mirror in between the imaging lens and the objective. This distorted the shape of the point spread function in the x- and y-dimensions, dependent on the z-axis distance from the focal plane. By examining the shape of each photoactivated molecule’s ‘photon cloud’, they were able to unambiguously assign a z-axis depth. This was a simple and clever way to map a third dimension of information on top of the two they were recording.


Due to increasing point spread widths with greater depth, the localization accuracy decreases with distance from the focal plane. Therefore, they only examined structures within a 500nm window around the focal depth. Z-scanning the focal plane could increase the depth range, though this might waste signal by photobleaching out of focus fluorophores. However, this is less of a concern in the STORM vs. PALM approach as the cyanine dyes used for STORM can be cycled on many times, while the Eos-FP used in PALM permanently bleaches. Of course, if a dye molecule moves position between on-cycles, this will degrade the effective resolution of the STORM approach.

PALM proponents also have a new paper out. Shroff et al. from Eric Betzig’s group show an alternative method of dual-color superresolution imaging. They co-express genes labeled with photoactivatable tandem dimer EosFP and with reversibly photoswitchable Dronpa or PS-CFP. The EosFP-tagged molecules are first photoactivated (405nm illumination), localized (561nm) and bleached. This process photoactivates a signficant population of the Dronpa or PS-CFP molecules. After all EosFP has been bleached, the activated second label is switched back to the dark state (Dronpa), or photobleached (PS-CFP) (488nm). The remaining second label can then be specifically photoactivated, localized and bleached.


A major advantage of this dual-color PALM technique over Zhuang or Hell’s two-color photoswitching approach is that all the fluorescent reagents are genetically encoded rather than antibody labeled. This permits more precise localization of the label to the target of interest. It also allows greater label packing density and more mild fixation. A disadvantage is that genetic overexpression could cause mislocalization of the target or artificial aggregation due to residual dimerization tendencies of the fluorescent tags. However, unnatural aggregation can also be induced with antibody labeling. Perhaps adaptation of Don Arnold’s FP tagged intrabodies could address this concern.

Pulse shaping for 2-photon signal enhancement

18 02 2008

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?

Brainbow mice are out

2 11 2007

Jeff Lichtman‘s Brainbow mouse paper is out! Not that I really need to report that news, as it is, of course, on the cover of Nature. Jean Livet comes up with some really clever genetic strategies involving incompatible, overlapping Lox sites to generate random, combinatorial patterns of multiple fluorescent proteins inside the cell. Around 90 different shades can be discerned by spectral deconvolution.

Besides making pretty covers, why is this so cool?

Well, this technique provides a method for generating high resolution maps of the brain. With a single fluorescent tag, the processes of neighboring cells blur together and became impossible to trace unambiguously. With brainbow, many neighboring axons are clearly resolvable. This is the perfect genetic tool to use for a large-scale, all-out effort for the complete mapping of the circuitry of the mouse brain. It would be a tremendous challenge, but perhaps no more difficult than the human genome project. A large public consortium, or a Celera of the brain can really attack the connectivity problem now.

Of course, there still is the more difficult problem of showing the functional connectivity of the circuit map. Then again, this technique isn’t limited to swapping in static fluorescent tags. The insert cassette could be doped with a single FP functional indicator like G-CaMP2… Would this allow the combination of static circuit mapping with functional testing?

Three quick paper picks

18 10 2007

Here are three papers that are worth reading over. No time for full reviews.

New Single-FP GECIs
The Russian fluorescent protein team has come out with some new single fluorescent protein G-CaMP/pericam-like sensors. They fiddled with the linker sites at the 145 and 148AA insertion points and found a great deal of fluorescence sensitivity to the amino acid composition at those sites. They note two new sensor constructs Case12 and Case16 that have 12-16.5x maximal changes in fluorescence upon calcium binding, a significant improvement over G-CaMP2. The tradeoff appears to be that they are dimmer. They show calcium responses in HeLa, PC-12 and cortical neuron cells, but no direct head-to-head with other sensors in cells.

Multipoint multiphoton microscopy
In this technical paper, an MIT group led by Peter So examines some issues surrounding multipoint excitation multiphoton microscopy. In theory, multipoint excitation will dramatically increase image acquisition rate through parallelization. However, this comes at the expense of large increases in background scattered light, which reduces optical resolution and penetration depth. They present an imaging system using multianode photomultiplier tubes that lets them acquire an 8×8 grid of multiphoton excitiation points. This technique, plus post-hoc deconvolution allows them to approach the resolution and depth of single point multiphoton systems, with a parallel array.

Effects of linker length and stiffness on FRET
This paper from Harold Erickson’s group carefully examines the role of linker length and stiffness in determining the amount of FRET transfer between CFP and YFP derivatives. They show that intrinsically unstructured domains of identical amino acid length produce significant differences in FRET between tethered FPs. They propose a formula for estimating the stiffness of linkers from the degree of FRET, which corroborates a 2006 study by Evers et al. They also demonstrate that the reported enhancement of FRET between the CyPet and YPet pair is due to an enhanced tendency for the proteins to dimerize. This reinforces my thinking that the most needed and generalizable improvement to two-FP FRET systems is an enhanced photostability of the FRET acceptor. Somebody please screen for a bleach-resistant YFP!  Props to Michael Lin for pointing out the paper.

Breakthrough in Far-field Optical Nanoscopy

8 10 2007

Its thesis crunch time for me, so I have had limited time to do ‘extracurricular’ reading and reporting for Brainwindows. However, there have been some very exciting developments in the field of superresolution fluorescence imaging that deserve a mention.

First, let’s take a look at this excellent review of far-field superresolution imaging techniques by Stefan Hell. I was almost able to understand the basics of the current techniques after reading it. Hopefully my summary doesn’t contain too many errors ☺.

Axial resolution is particularly bad in conventional superresolution techniques. Confocal imaging and 2 photon imaging provides ~450 nm resolution at best, while 4Pi microscopy with immersion lenses above and below the sample has delivered ~100nm axial resolution images of fixed and live cells. Lens aperture is the limiting factor in 4Pi and I5M imaging systems. These systems do not break the diffraction limit; they just push it.

In the absence of bleaching, fluorescent molecules can be localized to arbitrary precision (1nm) provided there are no other spectrally identical molecules within <lamda/2n. How do we constrain the coordinates of excited molecules? The new far-field superresolution techniques rely on sequential recording of fluorescent markers in a light state that is switchable with a dark state. Hell’s approach of stimulated emission depletion (STED) illuminates a sample with two beams, a short-wavelength excitation beam surrounded by a longer-wavelength, donut-shaped beam. The donut beam produces stimulated emission that drives the fluorophores in the excited (S1) state back to the ground (S0) state by photon emission of identical wavelength. These long wavelength photons are discarded. This process competes with excitation from the short-wavelength laser. Only in the very center of the donut does the S1 excited state, driven by absorption of the short-wavelength excitation beam, last long enough to decay with a photon of wavelength between the excitation beam and the stimulated emission beam. This donut is scanned across the sample and the intermediate wavelength emission photons are collected. In practice, STEM has yielded axial resolution of 100nm with a single lens and 33-60nm in conjunction with 4Pi imaging. STED is expected to reach 10nm resolution with more advanced 4Pi setups.

Other STED like systems include:

Ground state depletion (GSD) – The depletion donut is produced by pumping the dye to metastable triplet state, which decays much more slowly than the S1 state. This requires far less laser power (100kW/cm2) than STED and only single-wavelength illumination, but has been practically limited by photobleaching in the triplet state.

Saturated structured illumination microscopy (SSIM/SPEM) – Sample is illuminated with structured sharp lines between saturated S1 states and dark S0 states. The illumination pattern is shifted and rotated. Superresolution images can be computationally reconstructed from the results. Demonstrated lateral resolution of 50nm with beads.

RESOLFT – Photochemical rather than photophysical state transitions using photoswitchable fluorescent proteins such as Dronpa and asFP595. Can be done with ultralow laser power (10W/cm2).

The practical limitation with these methods is that the rate of fluorophore bleaching is dependent on the number of state transitions it makes. All of these methods induce a large number of cycles per usable photon coming out. Therefore, the power of these techniques should be improved with more bleach-resistant dyes and fluorescent proteins.

Two competing methods that are not as sensitive to cycling induced bleaching are photoactivatable localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). Rather than precisely define the location of fluorescence emission as in STED, these techniques use an ultradim laser to stochastically activate a constellation of well-spaced fluorophores throughout the sample. In PALM, these are then repeatedly excited with a different wavelength laser till bleached and their centroids are determined. The process is repeated and computationally summed till a superresolution (2-25nm) Seurat-like image is composed. A major benefit of this approach is that the fluorophores are efficiently used. Each fluorophore is switched on only once, and a maximal amount of photons is collected from it until bleaching. A limitation of the approach is that the sequential integration of images requires long imaging periods (hours), so far making it useful only for immobile proteins or fixed tissues. If background noise can be reduced enough to permit wide-field camera-based recording rather than laser-scanning, the acquisition rate should be greatly enhanced.

Now, let’s look at the new paper.

A major thrust of the PALM/STORM groups has been to develop multi-color labeling methods, so the interaction between two or more proteins can be studied. Xiaowei Zhuang’s group has recently demonstrated a system of photoactivatable dye pairs that theoretically allow up to nine color imaging. Long-wavelength ‘reporter’ cyanine dyes (Cy5, Cy5.5 or Cy7) are paired with shorter-wavelength ‘activator’ dyes (A405, Cy2, Cy3) on a single antibody. The reporter section of dye pairs can be selectively activated by laser pulses at 405, 457, or 532 nm, dependent on the activator section. Illumination with a red laser then produces a short period of fluorescence, whose emission wavelength is dependent on the reporter dye. Following this fluorescence, the reporter transitions to a dark state, which can be reactivated with another short-wavelength pulse.

Using dim activation pulses similar to PALM, the authors demonstrate three-color imaging of immobilized DNA molecules and two-color imaging of antibody-stained proteins in fixed cells with ~25nm resolution. The typical image used TIRF illumination and consisted of 2000-100,000 passes of recorded at 19Hz on a CCD camera. Thus, high quality images required as little as 2 minutes to record. There was some spectral crosstalk and false activation of the dye pairs that they were able to statistically correct. The spread in the localization of the antibody on a protein is now a significant contributor to the optical resolution limit.

These super-resolution techniques are getting very close to being usable in living samples, with both PALM and STORM making very quick progress. Be sure to check out the beautiful pictures in the paper figs.

An update on new FRET Biosensor software

2 07 2007

Apologies for the long delay between updates. I’ve been writing up and editing my work, also spent the last 3 weeks at the CSHL Ion Channel Physiology course. Jeff Diamond, Mark Farrant, Kenton Swartz and Michael Hausser ran a very informative and entertaining program. So, what’s new in FRET sensor land?

Producing useful FRET sensors requires some structural insight, theoretical knowledge, patience to screen many variants, and luck. Making a new sensor that works marginally well is often not that difficult. However, making a reversible sensor with high speed and S/N takes a lot of time and effort. How does a researcher select the protein substrate, linker sequences and fluorescent proteins from the vast space available? A new paper and software from Kevin Truong’s group aims to help guide the researcher towards good candidates.

In the paper, the authors present a simple computational strategy for determining optimal sensor constituants. A PDB file of the sensor substrate is fused to two FP structures via fully flexible linkers. A large number of possible conformations is then sampled, with predicted FRET efficiency calculated for each, then averaged. The authors compared the simulated mean emission ratios between several variations of genetically encoded calcium sensors. In general, there was poor correlation between the simulated emission ratios and experimentally determined ratios across different classes of sensor. However, within sensors grouped by substrate, the change in simulated ratio between variations was qualitatively predictive of the difference in experimentally determined ratios.

They also investigate the effects of various cpVenus substituations into YC2.1. These change the relative orientation of the chromophores and can lead to improved sensors. Again, the changes in simulated ratios were qualitatively predictive of experimentally determined ratios. Interestingly, the simulated ratios only changed from 0.25% to 10%, while experimentally, they varied from 0 to 210%. Although the simulations match the rank order of experimental response, why is there such a disconnect in the magnitude of the change? Perhaps this is due to the flexibility in the modeled linkers.

How useful will this FPMOD software be to developers of FRET sensors? It doesn’t (yet) incorporate linker rigidity, FP dimerization tendency, interactions with endogenous proteins, conformational kinetics or bleaching. Without these factors, will it be useful in predicting those rare “super-responders” like YC3.60, not to mention the current concerns with optimization of in vivo performance? How well does it work for ligands other than calcium?

Luckily, the Truong group has made the software binaries freely available for all of us to download and play with. It would be great to publish the source code to see how it all works. Perhaps as a group, the scientific community could dramatically improve the functionality of the package. Although the GUI front end is clean and functional, I’d also like to see documentation of each parameter, as it is confusing for a newbie like me to figure out how to run the package properly. Just a few more hours work on documentation would yield a great increase in usefulness of the software. Even with these caveats, FPMOD looks like a significant step forward towards rational design of FRET sensors. Any serious FRET sensor developer would be remiss to not carefully read the paper and test how well the software works in his or her system.

CSHL Meeting – Session VII – Super-Resolution Optical Techniques

25 03 2007

Jean-Louis Bessereau – Ultrastructural mapping of functional domains of synapse at the synapse using high pressure imaging
High pressure freezing instantaneously converts up to 0.3mm thick water into amorphous ice. C Elegans only .1mm thick at maximum. HPF entire worm to obtain EM of ‘living’ synapses. Vesicle priming occurs within 100nm of presynaptic density, directly across from post receptors. However, vesicle recycling occurs only at sites >150nm lateral from presynaptic release sites.

Mark Ellisman – Multiscale light and electron microscopic imaging of the nervous system
Two-color correlated light and EM microscopy using FlAsH and ReAsH. Quantum dot immunohistochemistry for multicolor correlated light and EM. QDs of different wavelength are differently sized and can be distinguished on EM.

Eric Betzig – Superresolution optical location of single proteins.
Sparsely photoactivate PA-GFP tagged proteins with very weak laser pulse. Then image with high laser power to collect light from many point sources. Determine point source centers, then repeat process many times to find protein locations to 2-25nm resolution. See the science 2006 paper.

Winfried Denk – Automated circuit reconstruction
EM :
When doing automated serial EM, individual sections can get lost or crumpled before being imaged. Instead image the block face then shave off a section. Resolution is significantly degraded, because lower power 3keV to limit z-penetration. Penetration goes as E^(5/3). Monte Carlo simulation of backscatter at 3 vs 10keV to determine point spreads and depth. Do 30nm sections, which is very [pun alert] cutting edge. Circuit reconstruction fidelity is limited by the dimension of least resolution, so usually Z section thickness. Block face EM actually used in the 1940s. Can still identify synaptic densities. Use backscatter to see heavy things. Constructing whole c. elegans. Demonstrate by hand reconstruction of some axons, dendrites and synapses as proof of principal. Using ion gun to acquire without need of a vacuum.

Staining Technology :
Colloidal lanthanum greatly enhances contrast of extracellular space but poor tissue penetration. Kevin Briggman and Denk like using extracellular HRP currently. Good membrane contrast. Moritz Helmstadter working on automated segmentation, collaborating with Sebastian Seung @ MIT for neural network based methods. Neural network method looks pretty good.

Lukyanov – New fluorescent proteins
Cloned the first RFP from coral, DsRed. DsRed tends to aggregate due to tetramer nature. Comparison of DsRed2, TurboRFP, TagRFP and mCherry. Brightness by e*QY. 100,172,134,44 respectively. TagRFP has shorter emission wavelength (<600nm) than mCherry (618nm). Can distinguish TagRFP from mCherry by the significantly difference in lifetime. [What about mCherry2?]

Redshifted FPs.
Katusha excitation 588, em 635. QY 0.34 e 45,000. Not quite as redshifted as mPlum but significantly brighter. But not monomeric. Made mKate which has similar brightness but is monomeric.

Cyan to green photoswitchable PS-CFP. Also made Dendra, monomeric green to red photoconvertible FP. [How much bleaching to red occurs to get 90% conversion? In our hands no photoswitchable protein allows total conversion without significant bleaching.]

Visualization of targent protein degredation in real time at single cell level using Dendra2. Zhang Biotechniques Apr 2007. IkBa-Dendra2 degredation down to 20% at 5hr with cycloheximide treatment. Fluoresence stays fixed with proteasome inhibitor. 20min protein halflife following PMA treatment. Hydrogen peroxide sensor HyPer of cpYFP inserted into OxyR-RD. OxyR transcription factor forms reversible S-S bonds in bacteria. Around 2-fold ratio change 490/410 between 0 and 250nM H2O2. Showed some small responses in cells to EGF stimulation. [Is this response reversible? Never showed a recovery trace.] cpCitrine145 with m13 on N, calmodulin on C term makes a GCaMP like sensor, 12-16x maximum ratio change. [How EXACTLY is this sensor different from GCaMP2?]

Killer Red, genetically encoded photosensitizer. Screened different natural FPs for phototoxic proteins that kill bacterial colonies under light. Most FPs non-toxic, 2-5fold increase in bacterial phototoxicity, but 1 FP had 1000x increase. In mammlian cells, expression in cytosol is not enough to cause sufficient oxidative stress to kill cell with light. But target to mitochondria and can kill cells with light. Targeted to cell membrane, blebs occur in 3 minutes. Use to kill off specific muscle cell with light in zebrafish. CALI of phospholipase C1-d PH domain by fusion to killer red. However it is still dimeric, so doesn’t fuse well to some things.

Karel Svoboda – Meeting Summary
Many spatial and timescales of neural questions require development of variety techniques. Interface of new imaging techs and genetically targeted probes making lots of progress on addressing these questions. The developmental talks were some of the best applications of the new technologies. These meetings will be measured by the crystallization of new, unexpected directions in research. Of course GECIs have been very exciting, but in the last 2 years also seen big progress on…
-sectioning and data collection, segmentation and reconstruction, EM on targeted neurons.
Light based approaches
- PALM, STED –EM type resolution in far field optical microscope, spectral multi-plexing, synapse specific tracing.
Optical remote control with light.
ChR2 – The silver bullet
HR – hyperpolarlize
Rhodopsin – seconds time-scale modulation of plasticity
Optical switches without transgenes

See you in 2009!

CSHL Meeting – Richard Axel, Keynote Speaker

23 03 2007

Keynote Speaker
Richard Axel, Columbia

Difference between representation and reality.
1300 ORN types random distribution in epithilium and converge on single distinct glomeruli. Additive activation of glomeruli to scents. 2p imaging in piriform cortex. Little overlap between cells in pirifom cortex to 2 different scents. Fly olfactory system 10^5 numerically simpler in fly. 80 OR types in fly, not homologous to mammlian system. Single OR per neuron + 1 fixed chaperone. ->protocerebrum. Convergence of common org between mam and fly. Individual ORN types project to single glomeruli. Axons that project from same glomeruli project to very similar arborizations in MB and protocerebrum, but different glomeruli projects are very different. Highly stereotyped circuit.

1. Is the map functional?
Express GCaMP line, take out brain, put in agar, puff odors for up to 6hr. Conserved patterns between animals. Some interneurons connect to every glomerulus. Excitation of PNs is specific. There is not rich interconnectivity between PNs. KO of Or43b specifically knocks out activity in a single glomerulus even though other glomeruli are activated by odors that normally activate both, shown with Ca and electrophysiology.

2. Can we correlate spatial patterns of neural activity with spatial outputs?
Stress flies, other flies will avoid the air from the tube of stressed flies (1960 Benzer).
dSO Avoidance is Mediated by OR42b and OR21a. One component of stress air is CO2, activates single V glomerulus. Very sensitive. Silencing Gr21a ORNs eliminates avoidance behavior. ChR2 light stim of Gr21a sensory neurons elicit avoidance. Necessary and sufficient. Glomerulus specific. Is this a special case or similar to other glomerular/activity?
Sexual behavior. Fruitless controls sexual behavior. Sex splice variants. Glomerulus larger in males. 67d neurons respond to male specific odor. KO males court males. KO females are less receptive to courtship. cVA Activates the DA1 Glomerulus. Use UV photoactivatable PA-GFP. Activate 1 glomerulus, see 6 PNs and the axon bundle. Instead, weakly activate glomerulus then strongly activate 1 slightly activated PN. Single DA1 PN responds to cVA. DA1 PNs Exhibit sexually dimorphic projections, and it is under control of fruitless gene. Express the opposite sex form of fruitless and the projection shifts to opposite sex.

3. Can we drive behavior by altering the structure of neuronal projections?
Now they ‘feminize’ 5 DA1 neurons in the brain with a DA specific opposite sex fruitless line. Now males court males. However, size does not matter. Mutant males also have the extra large ‘male sized’ DA glomerulus.
With associative behaviors, the complex interdigitation between projections in the protocerebrum may allow more complex integration…


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