Labrigger

January 2, 2012

Use ScanImage? Support it. It’s free.

ScanImage is an excellent software package for controlling 2p scopes. It’s free and open source. It’s been actively developed and released to the public since its inception. RIght now they personnel involved are trying to renew their funding. To help keep this resource actively developed and free, please fill out their survey. It’s very, very short. Don’t take the resource for granted. It takes a lot of salaried time to keep the development going and adding in new features.

By the way, ScanImage 3.8 (new features) and 4.0 (for ThorLabs scopes) are out now (3.5 and 3.6 are no longer supported; 3.7.1 is the current stable release) (link). If you haven’t already tried a new version of ScanImage out, you should. It doesn’t take too long and the feedback is very helpful. Don’t assume that everyone else is already sending in the same feedback.

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December 31, 2011

Optimizing pulse frequency for 2p imaging

The lasers used in multiphoton imaging deliver their photons in pulses. Many commonly used systems pulse at 80 MHz. However, there are good reasons to try different frequencies.

In 2007, Donnert, Eggeling, and Hell published a Nature Methods paper where they used low frequency pulses to get more fluorescence signal out of the preparation. The idea was that many molecules get excited into triplet states that are long-lived. By having a long time between pulses, there is time for the molecules to fall back down into the ground state so that the next pulse will have a large population of molecules available to be excited.

The next year, Ji, Magee, and Betzig published a Nature Methods paper where they used high frequency, low power pulses to get an increase in signal-to-noise ratio with two-photon imaging.

Several people have been confused by these apparently contradictory results. Recently there was a discussion on the Confocal Listserv about this topic, again pointing out the differences between the two papers.

Andrew Ridsdale chimed in with his thoughts (link to post). One of his points is that n different experiments, different factors are limiting the signal.

In Hell’s experiments with low pulse rates, they were imaging cell-free molecules– a very bright signal. Bleaching (triplet-state occupancy) was the limiting factor, rather than damage. Because there weren’t even any cells around to be damaged, other than E. coli cells in the last figure. So allowing for relaxation time and maximum occupancy of the ground state gave the best results. All of the relevant processes were governed by 2p excitation and thus were second order.

In the Ji, Magee, and Betzig experiments, the signals were very dim and the limiting factor was damage to the preparation. Andrew’s point seems to be that in this case, the signal is coming from second order processes and the damage is from higher order processes, maybe even as high as 5th order, though they estimate it to be on average about 2.4 order. So in this case, it’s best to use pulses that are just barely effective for 2p excitation and completely ineffective for higher order processes (damage), and then blast the prep with as many pulses as possible. Since the likelihood of a 2p event is already low, bleaching isn’t as much of a factor.

(btw, Brain Windows did a very nice post on the Ji and Donnert papers)

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December 30, 2011

Optics Planet and Braintree Scientific and New Era syringe pumps: Inexpensive scientific equipment

Optics Planet has a nice selection of inexpensive microscopes and other lab equipment. Such as these chubby, potential Cute Overload stars from Nikon (above, the blue one that is taking a bow is $380).

Braintree Scientific also has a really nice selection of reasonably priced equipment. Tons of very interesting, unique products. Get the catalog and flip through it– the website isn’t so nice to browse. They do custom work too, in case you have something specific in mind. One of their new products is a netbook+syringe pump package, pictured below:

I recognize the syringe pump as one of New Era’s OEM pumps. New Era sells all kinds of syringe pumps, from barebones OEM devices ($500, controlled via RS-232), to digital ($750) and multi-syringe units ($1500). You can use one of the OEM units for things like delivering water rewards in behavior rigs.

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December 23, 2011

Visualizations

Visual.ly recently posted a list of the top visualizations of 2011. This is a map of the world in which Twitter tweets are plotted using their GeoIP info and color-coded based on language. Looks cool, but there is no message. I could have guessed how this map would look. Germans tweet in German, Brazilians tweet in Portuguese, and so on. Big urban centers have a lot of tweets. Developing economic areas and sparsely populated regions don’t. I don’t see anything interesting. And so many of the colors are difficult to distinguish, if there is any new information, it’s difficult to figure out. They should have added more text labels on the map to identify languages near where they are found. Really? This is #1 on the list?

The field of data visualization is not well defined. The comeliness of a visualization is important, but if there is nothing revealed by the visualization– no story told– then it fails. There seems to be quite a number of people who enjoy studying data visualization who are almost purely interested in the aesthetics. This makes it difficult to really get good advice. A recent book review in Science by Robert Kosara seems to be wrestling with this issue. Two books are covered, Yau’s Visualize This, and Lima’s Visual Complexity. The former is a practical guide to creating effective visualizations, the latter is a collection of breath takingly beautiful works of art with no apparent message. Kosara notes that Lima “never attempts to explain what viewers can learn from any of the examples.”

Tufte has already said much of what needs to be said about data visualization. For example: “Have a message.” “No chart junk.” “Maximize the data-ink ratio.” Tufte himself said of Lima’s book, “One useful question to ask of each image is: What did I learn from this, in addition to seeing an elegant architecture?”

Yau does an excellent job following Tufte’s principles. He has a website that is worth checking out, flowingdata.com. And don’t forget Tufte’s own website. Junk Charts is good too. It just posted this holiday Venn diagram:

1 comments

December 11, 2011

Teaching MATLAB

In a previous post, I gave the two line MATLAB code to generate a Gabor patch. At the other end of the spectrum, this web page spends an entire MATLAB tutorial on making a Gabor patch. It’s a great neuroscience example for someone learning the methods of MATLAB programming.

If you’re interested in stuff like this, it’s from UCL’s Institute of Cognitive Neuroscience’s MATLAB tutorial class. A lot of their class materials are online. (link) Also, this book isn’t a bad place for a beginner to start.

4 comments

December 5, 2011

Raster plots and MATLAB

A colleague once told me: “in MATLAB, drawing a raster plot is a trivial, one line command”. True, but then you spend another 10 lines of code trying to make it not look like complete crap. Which is both time consuming and futile. In the end, you know you’re going to be spending half a day with Adobe Illustrator to fix it. But it does all start with one line of code.

Here’s some code to draw simple, one line raster plots:

function raster(in)
if size(in,1) > size(in,2)
    in=in';
end
axis([0 max(in)+1 -1 2])
plot([in;in],[ones(size(in));zeros(size(in))],'k-')
set(gca,'TickDir','out') % draw the tick marks on the outside
set(gca,'YTick', []) % don't draw y-axis ticks
set(gca,'PlotBoxAspectRatio',[1 0.05 1]) % short and wide
set(gca,'Color',get(gcf,'Color')) % match figure background
set(gca,'YColor',get(gcf,'Color')) % hide the y axis
box off

As an aside…

Looking for a wry take on MATLAB’s shortcomings from an anonymous neuroscientist? Try Abandon MATLAB. This blog’s posts include these gems:
Matlab doesn’t know how to draw one ball out of an urn containing one ball
The Mathworks don’t even know how to look up functions in their own global namespace
And this image (source):

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Tycho Brahe

Like the Mayan astronomers over 600 years before him, Tycho Brahe was a data factory. A data factory in the same vein as the Human Genome Project. Or as the Allen Institute for Brain Science is today.

In most formulations of the scientific method, the hypothesis is generated somewhere in the middle. What comes first is careful observation. What comes last are the hypothesis-testing experiments and controls. Often these individual steps are handled by different scientists and groups. For example, the Human Genome Project’s primary goal was one of observation, not hypothesis testing. Perhaps the same is true for the Mayans who observed the movement of Venus across the sky.

Tycho Brahe did what the Mayans did, 600 years later and in even more detail. He had the best primary data for the positions of celestial objects in the sky at the time. It was this high quality data that enabled Kepler to work out elliptical orbits for the planets.

A new website, Neurotycho.org, is set out on a similar mission. There, you can download data from primate experiments and reanalyze it. The setup seems as if they’ll accept data from other people at some point, but so far, it’s a one lab show. That one lab is Naotaka Fujii’s RIKEN lab.

There are similar efforts elsewhere in neuroscience. What’s unique about Neurotycho is that they seem to be reaching out to a very general audience. They also have a wiki with more details.

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November 30, 2011

An Arch-based voltage sensor

The newest voltage sensor is from Adam Cohen’s lab and based on a microbial rhodopsin, a class of light sensitive proteins already being put to use in optogenetics.

In this case, they’ve take Archaerhodopsin 3 (a related protein to Arch, which has been used to hyperpolarize neurons and silence activity) and removed it’s ion fluxing ability. But since it still senses voltage and light, its optical characteristics change with voltage.

How does it compare with existing voltage sensors? It has a relatively large change in fluorescence over a physiological voltage range (about 50%), but it’s very slow (41 ms onset). They can nicely see APs in single sweeps in cultured cells. In slices and in vivo, the signals will be smaller. One of the best voltage imaging schemes around, DiO+DPA, has a similar fluorescence change and is way faster (<1 ms). Note that signals from Arch(D95N) in culture (top picture below) don’t look massively better than DiO+DPA in slice (bottom picture below). Hopefully Arch(D95N)’s signals will still be usable in slices and in vivo.

Arch(D95N) in culture

DiO+DPA in slice

But of course, having a genetically targettable construct is a huge advantage. So a fairer comparison would be to other voltage sensing proteins. In that case, Arch(D95N)’s fluorescence change is excellent, but its slow speed is a handicap and makes that big fluorescence change less useful. Perhaps the biggest problem with Arch(D95N) is that it’s dim. The quantum yield is 9e-4 and an EMCCD was required for these experiments. For comparison, many GFP based proteins have quantum yields over 5e-1. It’s extiction coefficient is decent– it absorbs light– it’s just really unlikely to emit a fluorescence photon.

Maybe the protein engineers will follow up and make a brighter and faster version, the authors themselves report that they’re actively working on speed. For what it’s worth, the native protein (with it’s fluxing capability intact) is much faster, so there’s hope on that front.

Knopfel has taken perhaps the most rigorous and deliberate approach to voltage sensing protein design. The first big problem is that the sensor has to be at the membrane, so unlike calcium sensors, the signal-generating ROI is pretty small. The second big problem is that the sensor adds an unnatural capacitance to the membrane, so when expression levels increase to improve S:N, the capacitance also increases and messes up excitability. They have a nice analysis in this paper.

Nevertheless, there’s a lot to do with voltage sensors and I don’t think people have really started to fully explore what they’re capable of. These systems don’t need to be perfect, there is a huge amount of physiology to explore even using the existing voltage sensors.

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Arrests likely in Olympus case

If you live in Japan, then you’ve already been updated by the nightly news over the past few weeks. For those of you for whom this has slipped under the radar: Olympus, the 92-year-old company which includes the microscope business, is likely going to be dismantled due to massive fraud that may involve upcoming arrests.

Olympus, Tokyo Police, and Japan’s version of the SEC have all worked to piece together what former management did to cover losses in the 90s and 00s. It’s at least on the order of 100s of millions. Currently there is talk that yakuza may be involved in some of the shady financial dealings. Olympus is probably going to be delisted, but could still avoid that fate.

The short version is that Olympus hid losses in the 90s, then covered it up for quite a while. Recently they hired a new CEO, that guy questioned the former dealings, Olympus fired him, the scandal broke, the stock plummetted, and so they fired some more people and are trying to come clean. The stock rebounded a bit now that it seems that Olympus is trying to sort stuff out. But they’re still in a heap of trouble and could be ripe for a takeover.

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November 27, 2011

Thorlabs’ B-scope

Thorlabs’ scope pieces and kits have been mentioned in these pages before. At SfN, they had their new B-scope on display. This is like the Sutter MOM and the UCLA scope, in that the microscope rotates in one plane in addition to x-y-z movement. A few differences with the Thorlabs scope:

1. The objective rotates around the focal plane, and the rotation is motorized.
With the Sutter/UCLA style scopes, the objective rotates about an axis along the scan path, so the focus point changes a ton when rotating. The rotation can really only be changed before there is a prep on there, because the objective swings a big arc whenever the rotation changes and it ends up pointing at a completely different point in space.

By contrast, the Thorlabs scope is set up to rotate about an axis that is in the plane of focus. So you can be looking at a cell and then, while imaging, rotate the scope (since it’s motorized) and still keep looking at the same thing, just from a different angle.

This is why they have the crazy periscope you can see to the right in the photo below.

I remember seeing a scope with this same feature (rotation around an axis in the image plane) at a conference at least 2 years ago. I think it was a group based out of Switzerland. Can anyone fill in the details for me?

2. No conventional scanners, just the Thorlabs conventional scanners.
This might not be true for long. Thorlabs has their own conventional scanners, but they’re not as fast as Cambridge Technologies (CTI) scanners. This is probably why they opted to put their resonant scanners in the system.

I’m guessing that they’ll help out buyers if they want to fit the scope with a set of conventional scanners from CTI. I say this because Thorlabs told more than one person at SfN that they would help them fit the Thorlabs resonant scanner kit to their Sutter MOM scope. This was news to Sutter.

Use ScanImage? Support it. It’s free.

Optimizing pulse frequency for 2p imaging

Optics Planet and Braintree Scientific and New Era syringe pumps: Inexpensive scientific equipment

Visualizations

Teaching MATLAB