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.

Muscle fibers

dailymotion.com has some amazing videos made by a group called Weird_Weird_Science about zooming into objects. They zoom into hair, Aluminum, a tooth, concrete, and many others including this video of lice. In this video, the highlighted feature is muscle tissue.

Here (right)is image that I took on one of our microscopes (the Philips CM-12 TEM) of some muscle tissue from a mouse. This tissue looks better preserved than the lice muscle and may give you a better idea of how the muscle bundles are organized. Click to enlarge.

Pollen Animation

If you've ever wondered about pollen, here's a good one for you. I found this interesting feature while examining a small grass flower in a Scanning Electron Microscope. Click on this link to see the animation. The pollen is sitting on the inside of the petal and as the image rotates, the object that comes in from the top-left and eventually covers up the pollen is the top side of the petal. I apologize if this takes a while to load on your computer. It is a rather large image file. I found that Firefox displays it smoother than Safari.
To produce the animation entailed taking 61 different images with the microscope. After taking each image, I tilted the sample 1 degree in the Z directions and reoriented in the X and Y directions in order to bring the area of interest back to the center. Then, additional focusing a stigmation correction was required in order to keep the image looking sharp. Once all off the data had been collected, the images were adjusted for brightness and contrast with Photoshop and the .gif was made with Image Ready.
These images were taken on a Hitachi 2700 SEM in the Madison Area Technical College Electron Microscopy Lab. The image on the right was colorized in Photoshop.

Colorized Bacteria

This is an SEM image of bacteria infested mouse intestine. It took me several evenings to colorize it with Photoshop. Those tiny bacteria took forever to loop. The sample was provided by Ivalyo Ivanov of the Littman lab in the NYUMC Skirball Insitute. Click to enlarge.
I worked on this sample on a Zeiss Environmental SEM called the EVO but couldn't get very good results. So, we decided to work directly with Zeiss to get improve them. My boss traveled to Harvard and worked with Doug Wei from Zeiss on another Zeiss microscope called the VP Supra SEM. The difference between the EVO and the Supra is that the EVO can image wet samples but has a tungsten filament. The combination produces lower resolution images. The Supra can do variable pressure EM so there is a little more sample prep involved, but you don't have to coat the sample with metal, and this microscope also uses a Field Emission Gun that produces images with much better resolution. We've applied for a grant to purchase this instrument. If it's approved we can get one for our lab and do similar work.
To the right is the original image. It was taken on the Zeiss VP Supra by Doug Wei and colorized by me in Photoshop.

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.

Armadillidiidae gonna eat you

Along the same lines as the guppy teeth, here are a couple of neat images I took of an Armadillidiidae (sometimes called a potato bug or pillbug).
I remember my friend trying to fry one of these guys with a magnifying lens when we were kids. The poor thing rolled up in it's little ball and a minute later we heard it sizzling. But I guess that's no worse than what this guy went though in order for me to be able to put him into the microscope. Sample processing for SEM of biological material consists of fixation with aldehydes and Osmium tetroxide, dehydration with a graded series of alcohol, something called critical point drying, and finally sputter coated with gold. Obviously the insect wasn't alive during all of this. I put him in the refrigerator beforehand. The cold puts them into a sleeping/hibernation mode. Likely he felt no pain.
These images were captured on a Hitachi 2700 SEM.

Microscope Collection

So, last week I talked about an amazing collection of microscopes at the University of Arizona College of Optical Sciences. This week, I decided to share my collection of microscopes with you. Again, they are not so much antique as they are vintage. The smaller one (right)was likely a model for school children in the 1940's or 50's. It came in a neat wooden box containing a few tools, a slide, and an old catalog from the company. Here (left) is an image I took with it of a piece of mica that i found in some potting soil.

The larger one (bottom right) with the odd looking bar is an old Carl Zeiss model (#316645 if you're interested). I consulted with a director of the Zeiss historical society about what it is and what it's function was and he thinks that it was measuring tool designed by Zeiss for use in the Zeiss factory for a quality.

Both of these microscopes were rather affordable. Probably because they were both extremely dirty when I bought them. They were obviously well used, especially the Zeiss. I spent several pleasant evenings in harsh light with a long drawn out whiskey, carefully removing every screw, opening every objective and condenser, polishing every lens, and loving every minute of it before putting it all back together. By the end, they look more or less brand new. In the Zeiss, I found a protractor in the eye-piece that I carefully aligned into the CORRECT position (the factory apparently neglected to do so in manufacturing). I also found a small prism in there.
Click the images to enlarge them.

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.

Guppy Eye

If you couldn't tell by now, I did a big project with guppy embryos when I was in school. Here's another image. Click to enlarge.
This is a 500 nm thick cross section of the eye of a guppy embryo. The lens (bottom part) of the eye was a bit deformed, but I think the rest of it survived the sample processing in tact. It was stained with Polychrome Blue and imaged on an optical light microscope. The interesting thing about this image is how it was constructed. I used a 40x lens and captured about 40 images. They were then manually stitched together in Photoshop (I was using CS1, when there was no option to do it automatically). Once completed, the image was 14,400 x 10,800 pixels (about 155 megapixels) and storage about a half of a gigabyte! I drastically reduced the size in order to publish it here, but I think it still looks pretty crisp. The original version could be zoomed in and viewed at high magnification.

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.

D'na..d'na, d'na, d'na, d'na..wrEEEEEEEE! *jaws theme*

Did you know that guppies have teeth? I didn't 'till i put it in the SEM. This is another one from the guppy embryos back in school.

These images were taken on a Hitachi2700 SEM.

DLP Mirrors

Here's another entry of some images from when I was back in school. One day, my professor (Bill Carmichael at MATC Madison) brought in a DLP chip from a DLP projection television. The concept of how a DLP television works is quite unique. The chip is set up as an array of tiny little mirrors. Each one moves individually to project tiny bits of the image. With most DLPs, each mirror is responsible for one pixel.


Due to a mishap in the sample preparation of the chip, some of the mirrors fell off of their mechanical posts. This turned out to be a happy accident as it exposed the machinery so that more than just an array of mirrors could be seen. I captured these images on a Hitachi 2700 Scanning Electron Microscope. Click on each image to enlarge them.

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.

A few pics

Let's add some fun pics. I've been in the EM field for a few years now. Here are a few images from back in school. All can be clicked to view them larger. More current work will follow.
This is a diatome. In the background are broken up ones. This was gathered from some diatomaceuos earth that we bought to keep slugs away from the veggies we were growing. The image was gathered with a Hitachi 2700 SEM and colorized in Photoshop.


This is some moss I found growing between two pieces of sidewalk. The image was gathered with a Hitachi 2700 SEM and colorized in Photoshop.


Here are a few images of some gills from a guppy embryo. Some were freeze fractured with liquid nitrogen to expose the contents. The images were gathered on a JEOL 840 SEM. One was colorized in Photoshop.