Journal Scan – Transynaptic tracing, fly olfaction, fast super-resolution, localization of perception

8 05 2009

Here’s a group of four recent papers that are worth checking out but I don’t have the time to cover.  The first provides a set of tools for neuronal circuit tracing. The second pushes super-resolution imaging into fast, live-cell imaging.  The third, by a friend from graduate school, uses G-CaMP to make strong claims about olfactory coding in fruit flies. The last reports remarkable data pointing to the distributed nature of conscious perception in humans, which would have been a great data set to reference in my recent talk on free will.

Genetically timed, activity-sensor and rainbow transsynaptic viral tools 

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.

Super-resolution video microscopy of live cells by structured illumination

Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.

Select Drosophila glomeruli mediate innate olfactory attraction and aversion.

Fruitflies show robust attraction to food odours, which usually excite several glomeruli. To understand how the representation of such odours leads to behaviour, we used genetic tools to dissect the contribution of each activated glomerulus. Apple cider vinegar triggers robust innate attraction at a relatively low concentration, which activates six glomeruli. By silencing individual glomeruli, here we show that the absence of activity in two glomeruli, DM1 and VA2, markedly reduces attraction. Conversely, when each of these two glomeruli was selectively activated, flies showed as robust an attraction to vinegar as wild-type flies. Notably, a higher concentration of vinegar excites an additional glomerulus and is less attractive to flies. We show that activation of the extra glomerulus is necessary and sufficient to mediate the behavioural switch. Together, these results indicate that individual glomeruli, rather than the entire pattern of active glomeruli, mediate innate behavioural output.

Movement Intention After Parietal Cortex Stimulation in Humans

Parietal and premotor cortex regions are serious contenders for bringing motor intentions and motor responses into awareness. We used electrical stimulation in seven patients undergoing awake brain surgery. Stimulating the right inferior parietal regions triggered a strong intention and desire to move the contralateral hand, arm, or foot, whereas stimulating the left inferior parietal region provoked the intention to move the lips and to talk. When stimulation intensity was increased in parietal areas, participants believed they had really performed these movements, although no electromyographic activity was detected. Stimulation of the premotor region triggered overt mouth and contralateral limb movements. Yet, patients firmly denied that they had moved. Conscious intention and motor awareness thus arise from increased parietal activity before movement execution.

Previews : sCRACM, Red PA-FPs, ATP sensor, Deep tissue PALM

4 02 2009

Here is a quick list of papers Brain Windows will be covering in greater depth the next two weeks.

The subcellular organization of neocortical excitatory connections : A new technique, subcellular ChR2-assisted circuit mapping (sCRACM), is used to map neuronal circuitry.  

A genetically encoded fluorescent reporter of ATP:ADP ratio : A new single-FP indicator that reports the relative concentration of ATP to ADP in a cells. 

A bright and photostable photoconvertible fluorescent protein : Evolution of monomeric Eos into a fluorescent protein with better properties for super-resolution imaging.

Photoactivatable mCherry for high-resolution two-color fluorescence microscopy : Evolution of mCherry into a photoactivatable fluorescent protein with better properties for super-resolution imaging.

Multilayer three-dimensional super resolution imaging of thick biological samples : Advanced laser pulse shaping techniques to achieve super-resolution imaging in thick samples.

Method of the year to Super-resolution microscopy

17 12 2008

Super-resolution microscopy has seen a flurry of advances since 2006 and has won Nature Methods award for Method of the Year. Super-resolution imaging was the subject of one of the first posts on Brain Windows, with four additional articles detailing more recent progress. Nature Methods has a special feature covering this and other recent breakthroughs. This includes a nice video feature where you can hear a basic introduction to the technology from inventors of the competing techniques. How did they miss putting the obvious “super” selection joke when asked to select the video resolution??

Unfortunately, Eric Betzig, who gives one of the most entertaining talks in science, isn’t interviewed in the video. Also included in the special feature is a bunch of runner-up methods, including high-throughput imagingplane illumination in thick tissues, and optogenetic neuronal control. When will “optogenetics” make it into the OED?

Xiaowei Zhuang's laser rig for STORM imaging

Xiaowei Zhuang's laser rig for STORM imaging

UPDATE : Superresolution imaging

20 06 2008

A couple of awesome new papers out on superresolution imaging.  

The first one is on PALM imaging in live tissues. Despite Eric’s promise that he’s “getting out of PALM imaging”, the Betzig lab continues to pump out papers on the topic. In a May Nature Methods paper, they do the logical extention of their previous work, demonstrating PALM imaging on live tissue. Frame rates of up to 1/25 Hz and spatial resolution of 60nm. Clearly the resolution is not optimal at these speeds, but they are now able to see dynamic processes. Improved photoconvertable fluorescent proteins could dramatically increase the speed. Don’t miss Mats Gustafsson’s informative commentary on the work. We ♥ this Mats, not this Mats.

Next up is a collaboration between Heinrich Leonhardt, John Sedat, and Gustafsson’s groups. Using 3D structured illumination (3D-SIM), they do multicolor 3D superresolution imaging in fixed tissue. SIM works by repeatedly illuminating a sample with gratings of interfering light, rotating the angle of the illumination pattern. The resulting dataset can be used to reconstruct the sample beyond the diffraction limit. Here the authors add an illumination pattern that varies in the z-dimension, allowing them to image with superresolution in all three axes. 3D-SIM seems to be a bit behind competing techniques on the typically achievable spatial resolution. However, it has a distinct advantage over PALM, STORM and STED. It uses standard fluorescent dyes, making it well suited for multicolor acquisition and compatible with the huge library of existing labels.

Finally, Stefan Hell’s group has managed to extend the STED technique into 3D. Using a 4Pi objective configuration (objective on the top and the bottom) with STED, they were able to sculpt their excitation spot into a 45nm sphere. Sweeping this across the sample allowed two-color 3D reconstruction of mitochondria morphology @ 40nm resolution in fixed mammalian cells. The excitation volume can theoretically be continuously tuned to arbitrary size.

Blog Roundup

27 02 2008

Here’s a quick overview of some posts that got my attention in the last month…

Neurodudes has a brief writeup of video-rate superresolution imaging from Stefan Hell’s group. I don’t have access to Science Express PDF’s through our institutional subscription (how much must they be charging for that?), making a full writeup impossible. But you can at least check out the abstract and supporting info here. Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement (Westphal et al.). The optical resolution isn’t quite as good as PALM or STORM, but the speed of acquisition is fantastic, permitting its use on dynamic living processes.

Eric Thomson from Neurochannels has posted a detailed Journal Club style review at Nature Network of the paper Spatiotemporal Dynamics of Cortical Sensorimotor Integration in Behaving Mice (Ferezou et al.) from Carl Petersen’s group. Using voltage-sensitive dyes they show the timing and spreading of activity from the sensory to motor cortex, following whisker stimulation in awake mice.

Biosingularity reports on results from Susumu Tonegawa’s group published in Science Express as Transgenic Inhibition of Synaptic Transmission Reveals Role of CA3 Output in Hippocampal Learning (Nakashiba et al.). They use a novel method, doxycycline-inhibited circuit exocytosis-knockdown (DICE-K), to transiently and selectively shut down the tri-synaptic pathway of the hippocampus (ER->DG->CA3->CA1->ER), while leaving the monosynaptic pathway (ER->CA1->ER) intact. These mice can still learn incrementally, but one-trial contextual learning and pattern completion recall is wiped out.

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.

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.