Tomography

About a month ago I posted an entry about the microscopes in my lab. In this previous post, I briefly described a technique called Electron Tomography. Here is great video that describes in detail the process of making a tomogram. The section used for this was 500 nanometers thick. This is extremely thick for an electron microscope. The data taken for this tomogram was gathered on one of the world's three 1 Million kV transmission electron microscopes! Most microscopes only have the capability to gather images on sections no thicker than 200 nanometers.
About a year ago I was in a class at the New York Structural Biology Center about electron microscopy techniques. The lesson about electron tomography was taught by the person who made this particular tomogram.

Sciatic Nerve

The sciatic nerve is a critical branch of the central nervous system. It connects the legs to the dorsal root nerve, which then connects to the spinal cord. The sciatic nerve is crucial to everyday movement and sensation and is the largest nerve in the body. Here are some highly magnified images from sciatic nerve. This tissue was taken from a mouse, but looks similar in humans and other mammals. The nerve itself is composed of small bundles called Axons (see image right, click to enlarge). Each of these bundles is wrapped a membrane-like material called Myelin, or the Myelin Sheath. The Myelin, made by a specific cell called a Schwan Cell, is crucial to conducting electrical impulses that travel through the nerve (there are two Schwan cells visable on the left side of the right image). The image on the left shows the different layers of myelin as they wrap around the axon (click to enlarge). Multiple Sclerosis (MS) degrades the Myelin Sheath, interfering with signaling. MS can result in a plethora of debilitating neurological problems ranging from muscle weakness to speech problems.
This sample was provided by Hasna Baloui of the Salzer lab in the Smilow Institute of the NYU School of Medicine Neurology Department. The images were gathered on a Philips CM-12 TEM.

Focused Ion Beam H-Bar Technique

Back in school, I learned how to use a Focused Ion Beam (FIB) workstation (taught by Bill Carmichael at MATC Madison). This interesting technology uses a Gallium source to create a beam capable of milling away at very small objects. This machine is used by technology companies such as Intel to aid in the creation of everything from computer chips and data storage devices to LCD displays and C-MOS digital camera detectors.

One technique commonly used by the industry for looking at and editing errors that occurred in the lithography is called the H-Bar technique. The H-bar technique produces an electron transparent cross-section (image top right click to enlarge) of an integrated circuit.
The microchip is polished to an approximate thickness of 20um and mounted to a grid (a 3mm circular piece of metal that can support a sample and is be placed into a Transmission Electron Microscope for examination (image left click to enlarge)). After putting the sample into the FIB, a small Tungsten strip is deposited to protect the circuits and then the sides of the microchip are milled away with the Gallium Ion beam. This results in an H-shaped cross-section of circuits, hence the name, "H-Bar." The sample can then be put into a TEM and the circuits imaged.
The above image was taken on a Hitachi H-800 TEM. The image below (click to enlarge) was taken with an FEI 610 Focused Ion Beam Workstation. Illustrations were made with AppleWorks 6.

Condenser Lens

Here is an interesting piece. This is a condenser lens. It is one of the most important parts of an electron microscope because controls the intensity of the electron beam.My boss gave this to me on one of my first days in the lab and it's one heck of a paperweight. The thing weighs a ton because it is essentially a giant coil of wire. The coil is a big controllable electromagnet and "condenses" the size of the electron beam as it passes through.

More Imge Stitching

As you'll remember, yesterday I posted a guppy eye that had been pieced together from many images into one large image. Here is a more recent example of image stitching. This is a cell (provided by Nicholas Manel of the Littman lab at the NYUMC Skirball Institute) that I took several images of with an electron microscope. You really have to click this one to see all the good stuff:


In this project we were counting HIV viruses. The small dark dots surrounding the cell are the viruses. Notice, some of them are budding from the surface of the cell membrane. The light blob with the double membrane and dark border is the nucleus of the cell. The small round dark gray objects are called mitochondria (there are two at about 1:30 relative to the nucleus), and in about the center of the image there is a beautiful example of a golgi (about 5:00 from the nucleus).
These images taken on a Philips CM-12 Transmission Electron Microscope and were stitched together in Photoshop (I didn't have to spend a long time doing it manually this time). Again, the image size was reduced for the web.

Microscopes in the lab

Here are a couple of pictures I took of the two Transmission Electron Microscopes (click to enlarge) that are currently in our lab. I Photoshoped them a little to cut out the background. On the left is the Philips CM-12. This is our work-horse and the instrument that I use the most. It's source is a Tungsten Filament that can run at 120kV. Most of the TEM images that you will see on this blog were taken using this microscope.
On the right is the Philips CM-200. This microscope is mostly used by the structural biologists in the institute for studying protein crystals and single particles. It's source is a Field Emission Gun that can run at 200kV. The CM-200 has a special cryo-stage for doing cryo-electron microscopy can also be controlled by a computer. This is handy for doing Electron Tomography, a special type of imaging that can make 3D reconstructions. See movie below.

By the way, I can't take credit for this video. Whoever created it likely spent years developing getting the right conditions for the sample and many many hours on image acquisition and processing.

Centrioles!

It can be difficult to find a good pair of centioles. These are two cylindrical shaped organelles arranged perpendicular to each other composed of microtubules. They are located in the centrosome of the cell and are associated with cell division. Finding a complete pair is rare since there are only two of them in a cell and we are looking at a roughly 50 nanometer thick slice of a cell that is 20 microns thick (about 400 times thicker than the section). Click to enlarge.


These were found in Lymphocytes from a mouse Lymph Node. The Lymph Node was processed by Jaime Lladora of the NYU Skirball Institute. The images were gathered on a Philips CM12 Transmission Electron Microscope and a Gatan 4k x 2.67k digital camera.

C. Elegans Vulva

While working on a project where we are looking at the embryos inside the c. elegans worm, we came across this beautiful example of worm anatomy. This is the vulva of the c. elegans. Notice the magnificent smooth muscle. Click to enlarge.


This worm (provided by Ann Wehman of the Nance lab in the NYUMC Skirball Institute) was processed with high pressure freezing (By KD Derr at the NYSBC)and embedded in Epon. Sections were taken with a diamond knife at a thickness of 60 nm on a Leica UC6 ultramicrotome. The image was taken on a Philips CM12 TEM at 120kV with a Gatan 4k x 2.67k camera.


The c. elegans (Caenorhabditis elegans) is a roundworm about a millimeter long that lives in the dirt. They are frequently studied in biological research. NASA is even taking them into space.