Several labs have set up their own workflows for imaging the same tissue using both fluorescence and electron microscopy (EM). Zeiss just released a product, called Shuttle-and-Find, to streamline this. It’s pretty simple, just a movable chuck on which the sample is mounted in either machine. The software then finds the ROIs marked in one modality, for imaging with the second modality. (link)
The movable chuck, the “shuttle”.
The automatic software alignment, the “find”.
Structured illumination can be used to increase the resolution of wide field fluorescence imaging. The idea is best motivated by thinking about Moire fringes. In panel (a) below, if you stand away from the screen at some point the two gratings will appear each as gray panels rather than gratings. Thus, this low spatial frequency pattern is revealing information that exists beyond the resolving power. This technique can offer at least a doubling of spatial resolution, and in the presence of nonlinear saturation effects, can theoretically offer unlimited resolution.
We covered a simple lickometer circuit previously. Another useful interface is a capacitive touch sensor. In contrast to light gate sensors, there are no extra photons floating around that could disturb an imaging device, and the touch required gives gentle tactile feedback. In practice, they can be much lower noise as well, in case of a simultaneous electrical recording.
The popular Qprox QT113 (datasheet) makes implementing a capacitive touch sensor dirt simple. If you’d like something more than a simple go-nogo signal, then the AD7746 24-bit Capacitance-to-Digital Converter is for you (datasheet). Using this chip, you can use a capacitance measurement as a proxy for pressure, or some other parameter. For example, if you use a large plate sensor, you can estimate the portion of the animal on the sensor at any one time. For most users, the QT113 will suffice. And its datasheet has some helpful diagrams to get your circuit off the ground.
SensorWiki article on Capacitative Sensing
Tom Igoe’s blog entry on Capacitative Sensing with the QT113 chips (includes microcontroller code)
Need a 1W blue laser for activating channelrhodopsin? This is a very inexpensive and relatively simple project to do yourself. Buy the kit here. The web page is not a stellar example of design, but the video above is easy enough to follow.
Starting with an inexpensive but overbuilt metal flash light, a laser diode and optics are fitted, along with an upgrade to the power supply to source the 1A needed for the diode. The diode itself is about $55, the total price is under $200.
It might not be rock stable, but even the $3000 solid state lasers I’ve used flicker like crazy. For many applications, this is tolerable.
The usual Labrigger disclaimer applies.
(via)
This is just a quick post to highlight a few Mac OSX programs & tips that you might find useful.
Default Folder – This is how all save file dialogs should work. Watch the video. They have a free demo you can try. I bought it and am always happy at all the clicking and navigating it spares me. It also makes it very easy to add tags to saved files so that they will be easier to find using Spotlight. It also shows previews of files as you’re browsing in an open file dialog.
Numbers – An oft forgotten part of iWork. I use it to make up experiment data sheets to fill in, and to organize data. Its big advantage over Excel, as far as I’m concerned, is that it’s fast and easy to make custom page layouts that are clear and fit the different data types.
Postbox – This one is available for Windows and OSX. I like Gmail’s conversation view. Postbox is the closest I’ve found to it in a standalone mail program. It also handles tabs, which helps to reduce window clutter. Postbox lets you browse through images or other files completely separately from the messages they were originally attached to. I like this feature a lot because many times the original message had only an attachment, no subject or message body.
Typinator – I use this to speed up answering emails and other typography characters. For example, when I type “;;so” it is automatically expanded into a full sign off, with my name and affiliation. I also use it for little typographic things, such as Greek letters and the degree symbol. You can also set it up to insert formatted text and even images.
Pages (part of iWork) works with the latest versions Endnote. You don’t have to use Word.
Vector figures from PDF files viewed in Preview can be copied and pasted into Pages documents and they stay vectorized.
The usual color scheme for showing co-localization is to overlay a red image and a green image and have the yellow portions show the sites of co-localization. This is problematic since red-green colorblind people cannot tell what is going on. Following up on the recent post on Daltonization, here’s a colorblind-proof color scheme for showing co-localization. It uses a standard 3-plane RGB scheme. One image only has information in the green channel, the other image has identical information in the red and blue channels. Overlapping portions are white. By using this color scheme, you can ensure that the figure’s information is intact for all three major colorblindness types, as illustrated above.
Here’s the MATLAB code for producing the image:
clear all % initialize variables a=zeros(128); % monochrome image b=a; % monochrome image a_img=zeros([128 128 3]); % RGB color planes b_img=a_img; % RGB color planes % draw square gradients, monochrome from 0-1 a(16:48,48:80)=meshgrid(0.5:0.5/32:1); % gradient a(90:102,32:96)=1; a(16:48,85:90)=1; b(80:112,48:80)=meshgrid(0.5:0.5/32:1); % gradient b(26:38,32:96)=1; b(80:112,85:90)=1; % assign RGB color planes a_img(:,:,2)=a; % just the green color plane is assigned b_img(:,:,1)=b; % both the red and b_img(:,:,3)=b; % blue channels are assigned the same data % display the data imagesc(a_img+b_img) caxis([0 1]) axis image off
It seems like everyone is coming up with new open source PCR machines/thermocyclers. Here’s another project, this time from an outfit called Otyp. What’s unique about this iteration (besides the beautiful prototype photos in this post)? Although they’re short on details, it’s part of a broader initiative to bring better biotech education to the school system. One thing they’re doing right now is leasing traditional PCR machines to schools. Their Cloning a Fluorescent Gene kit is billed as the biotech equivalent of a “Hello World” program. Otyp is looking for funding on Kickstarter.
Jim Pawley upgraded the 39 Steps from a simple MacGuffin to a classic checklist for troubleshooting fluorescent microscopy images. Now 10 years old, it remains very relevant. It covers thirty-nine different parameters to check: from singlet state saturation to embedding medium. It’s a good place to start if you’re puzzled as to why your images don’t look as good as you expected.
Jim Pawley’s The 39 Steps (thanks to CW for the tip)
On a related note, if you’re in London, the stage production of The 39 Steps at the Criterion theater is a great comedy to check out.
BTW, here are the online microscopy and imaging references from the major microscope manufacturers. These can also help in troubleshooting.
Olympus Microscopy Resource Center
Nikon’s Microscopy U
Carl Zeiss MicroImaging Online Campus
Don’t forget catalogs as textbooks!
Shapeways now has a sample pack you can order to help you pick a process and material for your 3D printed object. It’s $30, but comes with a $25 voucher for your next Shapeways order. You can test the materials for rigidity and flexibility, or try tapping a few holes in them to see how it holds a thread.
A new paper in PLoS Computation Biology proposes an elegant formalism for describing neuronal morphology. The reason I’m highlighting it on Labrigger is because the authors have simultaneously released a very extensive, impressive MATLAB toolbox for studying neuronal morphology, including reconstructing neurons from image stacks: the TREES toolbox.
What you’ll find in the TREES toolbox:
o Tools to automatically reconstruct neuronal branching from microscopy image stacks and to generate synthetic axonal and dendritic trees.
o The basic tools to edit, visualize and analyze dendritic and axonal trees.
o Methods for quantitatively comparing branching structures between neurons.
o Tools for exploring how dendritic and axonal branching depends on local optimization of total wiring and conduction distance.
To get an idea of what this toolbox is capable of, look through the extensive manual.
