UCSD vs. MIT SFN Party Smackdown

3 11 2007

The Society for Neuroscience conference starts today in America’s Finest City (San Diego). The question on everyone’s mind is, who is going to throw the best party? Sure there are plenty of themed mixers and socials, but few really stay interesting for long.

The past few years, the Picower Center for Learning and Memory at MIT has consistently had the biggest bash, really peaking in 2006 at the eye-popping Atlanta mega-club Compound. With a big open bar tab that unfortunately gets drained within an hour, and an open invitation, these are always packed with people early on, go strong till last call, and feature plenty of Neuroscience ‘star power’. This year, the party starts Monday at 9pm at Deco’s on 5th Ave. in the Gaslamp. Get there early, as Deco’s is a relatively small place.

Nature and Neuron each throw lower-key parties, with the best hors d’oeuvres and are definitely the place to do serious science/business networking. Security is pretty loose, as long as you let the door know that you know that the party is for Nature or Neuron. When and where these parties might be in San Diego is under intense investigation by BrainWindows staff.

The most exclusive of all are the mysterious Emory parties, where you better bring the printout of your personalized invitation email if you want to get in.

This year, there is a new group that is trying to dethrone the PCLM as hosts of the biggest event. UCSD Neurosciences is hosting an open-invite, open-bar event this Sunday at Aubergine, at 4th & Island in the Gaslamp. The bar tab opens at 9pm, and if the PCLM parties are any guide, I would get there at 9. Bring friends!

UCSD Neurosciences Party

Who will impress the community the most? PCLM has a five year reputation, and the experience of Earl Miller and Susumu Tonegawa behind it. But UCSD knows San Diego, and it’s grad-student run social committee has held numerous, very successful local events. As a soon-to-be alum of both UCSD and PCLM, I’m looking forward to finding out who does it best. See you there!

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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…
EM
-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
Applications

See you in 2009!





CSHL Meeting Session VI

24 03 2007

Novel Methods to Dissect Neural Circuits – Saturday afternoon

Dmitri Chklovskii, Janelia Farm

Reconstruction of neuronal wiring diagram from automated serial EM. Must be able to track identity of segments between slices, determine synapses and the cells they belong to. Wiring diagram draft was done in c. elegans (~7000 synapses, 279 neurons) in 1986, Mitya’s student finished it in 2006.

How do we do it? Automated alignment of serial sections by translation, slight rotation and elastic stretching. Automated segmentation of color coding, makes a draft that must be reviewed by human editor. State of art is 10x faster than manual tracing, reconstructed complete 10x10x10um^3 volume two man-months. 1000 synapses, 1000 axons, 100 dendrites.

Biological results : If there are an equal # of spines and axons neighboring a single segment of dendrite, no significant wiring rearrangement possible. But connectivity fraction is actually 0.1-0.3 so plenty of room for structural plasticity. For optimal info storage, there should be equal volume of axons and dendrites, which is shown to be true. Axons appear to be concentrated near other axons, dendrites far from other dendrites, but this actually fits random packing of processes.

Questions : Can you see systematic slicing errors from alignment?
A: Only errors from people walking by.
Q: How much shrinkage do you see from fixation?
A: Significant uniform volume shrinkage, but not worried about that. Loss of extracellular space, may effect shape of processes.

Jeff Lichtman, Harvard
Connectomics : Brief definition – Map neural circuits.
Naturally occurring synapse elimination in the developing brain.
Three changes in synaptic connectivity:
1. Decreased axon connectivity
a. Imaging NMJ, decreased convergence with compensatory synaptic takeover by the remaining input
b. Non-monotonic process, appears to be competition
c. Axons are branches, other branches of same axon innervates other targets, these effect the competition
2. Decreased axonal divergance
a. E18 – 80% NMJ innervation. P13 – 4.2% innervation
3. “Synchronization” or rewiring process
a. When two axons compete on multiple terminals, same axon loses in both
b. Is there a deeper hierarchical structure?
Each outcome of synapse elimination causes unique pattern of synapse innervation in each axon.
Automated Tape-collecting lathe ultramicrotome (ATLUM). Grad student made a homemade one with 15uM thickness. Now building 50nM thickness with $200K McKnight. http://www.extremeneuroanatomy.com

Clay Reid, Harvard Med – New tools for imaging the functional anatomy of the visual system.
Originally an electophysiologist only, mapping functional connectivity with electrodes. Now doing functional imaging.
Calcium imaging of the visual cortex. Bulk loading of calcium indications in cerebral cortex, look at 300uM cubes. What is the function of each of these cells? Excite with visual stimuli of anesthetized animal, 2p imaging of 2/3 rat visual cortex. Find orientation selectivity without clustering : salt and pepper. No apparent functional microorginaization. However, in the cat, similar neurons types (horizantal, vertical) cluster together with sharp cutoff between cells in orientation pinwheels. How do they do this?

Are functionally similar groups of cells:
Spontaneously co-active?
Correlated with cell type?
Anatomically/functionally connected?
What is the wiring diagram?
Tracing individual connections with viruses
Tracing many/all connections with serial electron microscopy
Use conventional sectioning and imaging with high throughput camera array.
Large volumes up to 500uM cubes at 5nm x-y resolution
Large datasets of 10-100 terabytes
Record everything but analyze only a bit, a very relevant bit
Showing preliminary data of automated serial em collection and analysis

Andre Fiala – Optophysiological techniques for the dissection of neuronal circuits underlying learning and memory in Drosophila
[Great talk content, but my notes are poor.]
In vivo monitoring of neural activity
Glue fly under coverslip. 1p DualView with Cameleon expressed in dopaminergic neurons, which have extensive innervation throughout brain. Following 8 training sessions, dopamine neurons show prolonged activity that persists thru conditioned stimulus, suggesting predictive abilities.
Expresses ChR2 in fly larva and can control contraction on larva with light. Can substitute light stimulation in octopamine neurons for appetitive odor stimulus in learning paradigm. Substitute ChR2 dopamine light stimulation for aversive stimulus. Express ChR2 in gustatory neurons, flash light, proboscis extends. “The light tastes sweet.”

Tamily Weissman, Harvard – Mapping neural circuitry in the cerebellum using multicolor fluorescent “Brainbow” mice
Gain neuronal identity in labeling by using combinations of fluorescent proteins “Technicolor Golgi stain”.
Thy-1 promoter-L1-L2-RFP-L1-mYFP-L2-mCFP with incompatible Lox sites. PreCre get RFP, Post Cre get YFP or CFP. Since multiple copies per cell, get blends of colors. [I doubt there is any FRET since they are using monomeric (A206K) mutants of C/YFP.] How many colors? Hard to say, conservative estimate for 100% confidence by eye is 78 colors eye can descriminate. [Why limit by eye? What is the limit using spectral deconvolution?] Limiting 20% mossy fiber, 5% granual cell and can do total reconstruction of this fairly dense labeling. Appears there is some convergence in circuit of mossy fibers onto granual cells by looking at ratio of filled terminals. Granual cells sometimes innervate same presynaptic mossy fiber at two distinct terminals on different dendrites.

Wei Chen – In vivo two photon imaging of firing and wiring of local neuronal circuits.
[Speaker is the lead author on the in vivo electroporation paper we recently covered, see the paper for more details.] Understanding the brain depends on sparse labeling of neurons. Konnerth, Reid using bulk loading, but this obscures fine neuronal structures. Tried bulk loading, G-CaMP2 mouse, now trying local electroporation. Following electroporation, only very small change in field recording. Hey but aren’t only a small proportion of the neurons electroporated? Hmm….

Ian Wickersham, Salk – Transcomplemented transsynaptic tracing : mediation by helper viruses
[I was planning on covering this work in the recent publication in Neuron, but will just do it here.] How do we determine what cell is monosynaptically connected to other cell types? Classic transsynaptic tracers pass at different rates due to connection strength and can move through strong polysynaptic connection steps. Enter transcomplemented tracing.

Component 1 – Deletion mutant tracing virus
Component 2 – Complement of the deletion, activates virus.

Rabies virus, RNA virus (can’t use Cre recombinase)
Replace the glycoprotein of rabies virus with GFP. Virus can replicate core but cannot cross membrane. Pseudotype virus with coat glycoprotein to avian ASLV’s membrane protein. Express gene of ASLV receptor, dsRed and native virus coat protein complementation gene in single neuron in the brain. Then pseudotyped virus infects that single cell and can cross 1 step. But, since complementation gene only exists in single cell, virus stops crossing after 1 step.

Day 1 : shoot in triple gene coated particles with genegun
Day 2 : Apply pseudotyped rabies virus
Get 1 red cell, and many sparse green cells that are monosynaptically connected.

Aravinthan Samuel, Harvard – Brain and behavior in freely moving worms
Thermotaxis exhibits long-term plasticity. Thermosensation occurs at tip of nose. Side to side wiggles and net forward movements could contribute to perception of thermogradients. Express cameleon in AFD neuron using cell-specific promoters.

Worm wants 2 pieces of info:
Is temp higher than it likes?
Is temp rising or falling?

Immobilized worm subjected to defined thermosensory inputs. Increasing T in a linear rate with wiggle induces a phased locked ratio change to the wiggle that starts above about 18C. Getting 150% dR with YC3.60 in response to wiggles. Ratio in AFD is directly correlated to T in tail fixed worms moving head around on a temp gradient. Turning off gradient kills correlation, reversing grad reverses side correlation.
AFD detects the temp variations driven by self-movement in a spatial gradient.