GCaMP5 is out

15 11 2011

GCaMP3 is now officially old skool.  GCaMP5 is available at addgene.

Plasmid 31788: pCMV-GCaMP5G, a.k.a. GCaMP3-T302L R303P D380Y

Dimmer baseline F, higher dF/F, picks up significantly more activity in vivo cortex.  Still not holy grail level, but getting closer.

Cortical encoding of somatosensory input

15 11 2011

Wednesday morning, I’m giving the first public presentation of my current work on sensory coding in the barrel cortex. The big picture question is, How does the cortex translate sensory input into perception and recognition of objects and their position?

To study this cortical function, we used a methodology with 3 critical components

1 – Goal-directed discrimination task

The activity of an awake cortex is profoundly different in character from an anesthetized cortex.  There is also gathering evidence that the activity of sensory cortex when attending to and using its sensory input to perform a discrimiation is quite diffent from periods of inattention.  We use a goal-directed discrimination task to enforce attention and to ensure the recorded activity is relevant to the fundamental function of the area.

2 – High speed neural recording techniques

Spikes are the words in the language of cortex. We must record them with high fidelity and temporal precision.  We can then use correlation of neural activity to sensory input to try to understand what the cells are saying.

3 – Precise measurement of sensory input

If we are to use correlational techniques to describe the language of cortex, we must match the millisecond precision of cortical communication with millisecond precision in measuring the exact sensory input.

Within the rodent, no study has yet combined all three components, goal directed discrimination behavior, millisecond precision neuronal recordings and millisecond precision sensory input recordings.

Anyway, if you want to hear more, try to get up in time to make it to the conference by 8:45AM.

Program#/Poster#: 750.04
Presentation Title: Encoding of vibrissal sensory input and task-related variables in the mouse barrel cortex during whisker-based object localization
Location: 152B
Presentation time: Wednesday, Nov 16, 2011, 8:45 AM – 9:00 AM

Useful ChR2 mouse lines

19 08 2011

Guoping Feng’s group has released what looks to be a very useful set of BAC transgenic Channelrhodopsin mice.  The four lines specifically express YFP-tagged ChR2 in GABAergic, cholinergic, serotonergic and parvalbumin-expressing neurons.
They are well characterized and available from Jackson labs.

Journal Club : Classic Single Unit Physiology in Barrel Cortex

29 04 2011

This one is for the aficionados. Here is a little review of four classic single-unit physiology papers investigating the response properties and information flow from whisker through thalamus and into cortex.  It’s quite interesting comparing this data taken from sedated or anesthetized rats to my own in awake, behaving animals. That’s a story for another time and publication venue though :)

Fukushima soil contamination

12 04 2011

I spent the last two nights arguing with a friend about the potential danger of radioactive fallout from Fukushima.  I contended any US fallout was negligible, he disagreed.  This led to reading a Science magazine post outlining the high soil contamination readings near Fukushima, and then to the real raw data, and comparisons to Chernobyl contamination.  We ran the numbers, and still disagree re: the US, but for Japan, they look bad.  If Chernobyl is a valid guide, which is a pretty conservative estimate of the caution of Japan’s government, there will be permanent closure of some towns that are >30km from the plant.

Here is a crude map I whipped up comparing the soil cesium-137 levels that led to restricted access to areas around Chernobyl, with that reported by the Japanese Ministry of Education, Sports, Science and Technology. Here is a directory with the compiled source data, updated daily.  The results are very disturbing.  Anything above 1500 kBq/m^2 was permanently closed near Chernobyl. Anything above 555  kBq/m^2 was permanently restricted.  If my topsoil conversion factors are correct, and they do match the expert’s assumptions in the Science mag story, then there are numerous villages above 555 kBq/m^2 level that are fairly far from the plant.  The 100 kBq/m^2 levels at a distance of 80km are also worryingly high. These aren’t cherry-picked readings to make things look dramatic, they are the rough average of many repeated samples across weeks.  No wonder the government finally upgraded the incident to a level 7. Click the picture for the big view.

Comparison to the whole of Europe is also illuminating, the numbers are the same scale as above.

Commentary : #tigerblood vs. Tiger Mother

3 03 2011

This is straying pretty far from the usual blog posts, but its topical. Listening to Bill Simmons’s podcast with Chuck Klosterman while driving home last night got me thinking about the role of confidence in society.  Would be interested in hearing feedback on it.

A common reaction to periods of high anxiety, stress (or hangovers), is negative self-talk.  Muttering things like “I hate myself, I’m worthless” etc.  These reflexive utterances, though not fully contemplated, produce a running monologue that reinforces negative thought processes during the period of anxiety.  With the amount of stress, hangovers and therapy that Charlie Sheen has been through, I suspect that he has trained himself to reflexively substitute negative self-talk phrases with positive phrases, “I’m winning.” “I have tiger blood.”  Does this lead to a better outcome?

Certainly, it seems that he has exerted more control of the public narrative than other celebrity implosions (Mel Gibson, John Galliano). It is difficult for the public to reach a consensus. Is Sheen on drugs?  Is he in a manic episode?  Or is this Sheen’s “new normal”, after his reward circuits have been twisted by heavy stimulant use paired with his conscious effort to train his mind to project a positive attitude? His hyper-confidence, coupled with his success in certain metrics (money, fame, sexual conquest), and his self-promotion through traditional (TV interviews) and social media (1M twitter followers in 24 hours, #winning and #tigerblood hash-tags), enable him to continue his behavior and promote its acceptance.

Compare Sheen’s #tigerblood approach to that of the Tiger Mother, Amy Chua.  Chua used relentless criticism on her children to drill them into technical mastery of academics and musical performance.  Her kids achieved by her metrics, though felt terrible about their perceived failures. She then cleverly tapped into America’s faltering confidence in the face of rising Chinese competition to drive an incredible volume of analysis and publicity for her book on the Tiger Mother approach to parenting.  The most telling quote is from Elizabeth Kolbert’s analysis in the New Yorker

Just about the only category in which American students outperform the competition is self-regard. Researchers at the Brookings Institution, in one of their frequent studies of education policy, compared students’ assessments of their abilities in math with their scores on a standardized test. Nearly forty per cent of American eighth graders agreed “a lot” with the statement “I usually do well in mathematics,” even though only seven per cent of American students actually got enough correct answers on the test to qualify as advanced. Among Singaporean students, eighteen per cent said they usually did well in math; forty-four per cent qualified as advanced.

Implicit in this quote is that academic achievement is of greater value than self-regard.  But as Larry Summers pointed out to Chua at Davos

Which two freshmen at Harvard have arguably been most transformative of the world in the last 25 years?  You can make a reasonable case for Bill Gates and Mark Zuckerberg, neither of whom graduated.

Both of these people had extreme self-confidence, though also significant intellectual candlepower. Charlie can’t match on the brains front, but he possesses a certain charisma that is perhaps the Hollywood equivalent of book smarts.

The real fascination with Charlie Sheen saga is what it says about the role of confidence in society. Sheen represents an extreme test case to the question, “Is confidence the key to success?”

Rapid warping of two-photon illumination wavefronts

16 02 2011

A short paper in Optics Express looks interesting.  In A high speed wavefront determination method based on spatial frequency modulations for focusing light through random scattering media, Meng Cui presents a method for rapidly determining the optimal wavefront to ‘cancel out’ the scattering when 785nm light passes through turbid media.  In his example, a glass diffuser was used, but the clear goal for this work is to replace the glass with a brain.

To understand why this is so important for in vivo two-photon imaging, let’s review how 2-p imaging works. Light from a laser is focused to a point and swept across the field in a raster. The resulting fluorescence is of a different wavelength and can thus be filtered out from the excitation light. For each voxel, all the fluorescence that re-enters the objective is collected, regardless of its source.  The total amount of fluorescence collected for that timepoint in the sweep is assigned as the brightness of that voxel. Since the user knows where the laser was being aimed, scattering of fluorescence emission may reduce the brightness but will not blur the image.  However, scattering of the excitation light can dramatically reduce the excitation at the target voxel while increasing the off-target excitation of its neighbors. This causes a rapid increase in background fluorescence and blur at increasing brain depth.

The vasculature was labeled by injecting flourescein dextran into the circulatory stream. The light source was a regenerative amplifier. ‘‘0 mm’’ corresponds to the top of the brain. Left, XZ projection. Right, examples of XY projections. Note the increase in background fluo- rescence deeper than 600 mm in the brain due to out-of-focus 2PE. (Theer et al., 2003)

Previous reports work has shown that one can use adaptive optics to adjust the phase of the wavefront of the excitation light to correct for the scattering of the excitation.  However, determination of the optimal wavefront for a field of view took minutes, which could be problematic for imaging in an awake animal.  Any changes in the precise position of the brain might change the optimal wavefront.  Ideally, one would want a system that could optimize the wavefront every second, or even before every frame of acquisition (typically 4-8 Hz in a raster scan in vivo experiment)

Scattering in the brain warps two-photon excitation light, but adaptive optics can correct this.

I’ll let Meng Cui explain the technique in his own words

Elastic scattering is the dominant factor limiting the optical imaging depth in tissues. Take gray matter as an example, at 800 nm the scattering coefficient is 77 /cm and the absorption coefficient is 0.2 / cm. If there is a way to suppress scattering, the optical imaging depth could be greatly improved. Despite the apparent randomness, scattering is a deterministic process. A properly engineered wave can propagate inside scattering media and form a focus, a well understood phenomenon in the time reversal and optical phase conjugation (OPC) studies…

For applications on biological tissues, acquisition time on the order of one millisecond (ms) per degree of freedom is desired. Deformable mirrors can provide a high modulation speed. However the degrees of freedom are rather limited. A phase-only SLM can provide about one million degrees of freedom at a much lower modulation speed. In this work, I present a novel method, capable of providing as many degrees of freedom as a SLM with a data acquisition time of one ms per degree of freedom. The method was employed to focus light through a random scattering medium with a 400 ms total data acquisition time, ~three orders of magnitude faster than the previous report [25].

The essence of a COAT system is to phase modulate different input spatial modes while detecting the output signal from the target. To greatly improve the operation speed, the experiment requires a device that can provide fast phase modulation and can access a large number of spatial modes very quickly. To meet these two requirements, a pair of scanning Galvanometer mirrors was used to quickly visit different modes in the spatial frequency domain or k space, and a frequency shifted reference beam was provided for a heterodyne detection. The wavefront profile was first determined in k space and then transformed to the spatial domain. The spatial phase profile was displayed on a SLM to focus light onto the target. In such a design, the number of degrees of freedom is limited by the number of pixels on the SLM and the experiment speed is determined by the scanning mirror speed…

Compared to existing techniques, the reported method can provide both a high operation speed and a large number of degrees of freedom. In the current design, the operation speed is limited by the scanning mirror speed and the maximum number of degrees of freedom is limited by the SLM pixel number. In this demonstration, 400 spatial modes in k space were visited and the determined phase profile was displayed on the SLM. Depending on the scattering property of the media, more (up to 1920 x 1080) or less number of degrees of freedom can be used to optimize the focus quality and the operation speed.

Using a stepwise position scanning, the method achieves an operation speed of one ms (400 μs transition time + 600 μs recording time) per spatial mode, ~three orders of magnitude faster than the previous report. Using a continuous position scanning and a faster position scanner such as resonant scanning mirrors, polygon mirror scanners, or acousto-optic deflectors, the operation speed can be potentially increased by at least one order of magnitude. It is anticipated that the reported technique will find a broad range of applications in biomedical deep tissue imaging.


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