Maybe we'll see this at the next summer Olympics?

I'd give it a 10.

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Extra awesomeness points for the use of pool noodles as mitotic spindles.

It's the small things that matter

Today's topic is presented by Dr. Angel Paredes, also of the Department of Pathology and Laboratory Medicine.

For those dental students reading along, in class yesterday, Dr. Bick discussed transmission electron microscopy (TEM). Cell structures can be imaged using this technique to obtain magnifications of up to the maximum of 25,000,000X in some microscopes. Of course with this kind of power, these magnifications would be like looking at an elephant with a light microscope and that wouldn't make very much sense. Typically magnifications in the range of 2000x to 40,000x are useful for examining things like cells (eukaryotic and prokaryotic), viruses and protein complexes.

For tissue structure, specimens are stained with heavy metals (osmium tetroxide for lipids, Uranyl Acetate for proteins and lipids) and embedded in a resin. The resin is then sectioned thinly and mounted on a metal grid which is then stained again with Uranyl acetate and lead citrate. The grid is placed in the microscope where electrons are passed through the sample where the heavy metal stains interact with the beam giving the image its characteristic contrast.

The way this happens is that electrons hit a structure and change course (because of the heavy metal), some pass through with minimal interference (no heavy metal or just a little). A charged coupled device (CCD or digital camera) camera records the image by measuring the electron density at each pixel, thus creating a black and white EM image like we see in class. Electrons do not have color so images are black and white.

Since samples need to be sectioned for this technique, this doesn't really tell us much about the 3D structure of cells. For that analysis, we need to use a scanning electron microscope. Cells are coated with a very gold palladium in a sputter coater, and the electron beam is bounced off the surface. The resulting image shows only the cell surface.

Recently, however, advances in computing have allowed scientists to further develop electron cryomicroscopy, or cryoEM. This is a technique that is used to image very small biological specimens (< 1 micron) frozen and perfectly preserved in amorphous ice. NOT resin! Why is that important? The specimen that is imaged is perfectly preserved and, in the case of virus, the virus can later be removed from the microscope, thawed from the ice, and used to infect more cells - illustrating that the images recorded were indeed of infectious virus.

The process begins by applying 3-4 microliters of specimen (let's just say we're looking at a virus in this example) in buffer onto a 3 mm copper EM grid coated with a net-like layer of carbon holes. The virus is blotted with filter paper creating a thin layer of buffer in which the specimen is briefly suspended. The EM grid is then quickly plunged into a liquid ethane cup at liquid nitrogen temperature where the virus is flash frozen so quickly ice crystals cannot form and the specimen becomes embedded in a layer of glass-like ice. The virus is then placed into an electron microscope specifically designed to image these frozen specimens. Remember, this is TEM, so electrons are passing through the sample. We get a black and white picture that looks something like this:

These are alphaherpesvirus particles!

For the next step, it is important to understand that for homogenous particles such as viruses, all the particles embedded in the ice are in all possible orientations and can be considered different views of the same object. Using a computer, the digitized images from the microscope and image analysis software, the orientation of each particle is determined relative to the electron beam. Once all the orientations are determined, the computer merges all the different views into one 3D model which then represents the specimen. Because the specimen is reproduced in 3 dimensions, it can be dissected and studied by imaging software. Behold the modeled structure of Sindbis virus (an alphaherpesvirus):




A few words about resolution, from the expert:
"Ok. You would have to ask about resolution. Resolution in the world we live in means essentially the minimum distance that two small elements in an image (dots) are distinguishable as two independent elements or dots.
(Also remember from Dr. Bick's lecture that the distance (d) between points that can be resolved increases as the wavelength of light increases, i.e. the resolving power goes down.)

In cryoEM you relate everything to your pixel size and image size. The pixel is a measurement of a grey value of the specimen you scanned. The pixel size is dependent on the mag. The higher the mag, the smaller the pixel size when you image it with the digital camera on the microscope.

Ok, now you have pixel size and image size. Say you have a pixel size that is 2 angstroms/pixel. This means that the absolute best resolution you can get is 4 angstroms resolution because you cannot achieve a resolution that is equal to your scanning step size or pixel. The best you can do is twice the pixel size.

So, what does resolution mean? In cryoEM resolution is quantifiable. You have an image. The image has noise and you refine the data to achieve the best resolution and that is when the noise and signal equal each other and the ration is 1. You cannot get better than that. With the image and pixel size you know exactly at what resolution this occurs. For my newest virus image, I get to 13 ansgtroms resolution. That does not mean that I can see two dots no closer than 13 angstroms. In fact I can see detail that is smaller than 13 angstroms. The resolution in this case is a quantifiable measure of how far out I can signal from the data in terms of resolution with the maximum being 2 times the pixel size.

I know it's complicated but YOU ASKED."

When Neutrophils attack

Today in class the dental students learned about blood, and the cells contained within. I promised to post a cool video of a neutrophil "chasing" a bacteria, so here it is:

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Poor bacteria never stood a chance.

There are a lot of interesting processes going on here. The first, obviously, is that the neutrophil appears to "see" the bacteria. Since we all got a good look at cell structure last week, we know that cells don't have eyes! So just how does this neutrophil know to attack?

The neutrophil is most likely sensing chemical signals coming from the bacteria. So-called "quorum sensing signals" are basically messages to other bacteria to come and join the infection party. It is likely that our neutrophil has picked up on these quorum sensing molecules and is using them to sniff out the bacteria. In addition, we know that neutrophils, like other phagocytes, express what are called "Toll-like receptors",or TLRs, on their cell surface. These receptors, important for innate immunity, are designed to recognize bacteria-specific sugars and DNA sequences.

So the neutrophil has identified the target, the target is escaping - how does the neutrophil move? This involves a whole lot of specialized cell signaling. First, the neutrophil is exhibiting "chemotaxis", or movement along a chemical gradient. It wants to go where the chemical signal from the bacteria is most concentrated. So, receptors on the cell surface bind the bacterial proteins, then trigger changes in the cell.

One thing that's happening is that our neutrophil has developed polarity -now it has a front end and a back end. We're not quite sure how this happens, but it seems likely that the cell is probably sending out random pseudopodia in all directions. Whichever psuedopod gets positive reinforcement (e.g. lots of binding to bacterial protein receptors) gets to stay and grow, while the other pseudopodia which don't encounter their target are pulled back into the cell.

To move, the neutrophil needs to rearrange some cellular structures. This involves polymerization of actin and myosin, and probably some growth of microtubules as well.

Once the neutrophil catches up to the bacteria, the process of phagocytosis begins. The bacteria is "grabbed" by specific receptors and pulled into an endosome. That endosome will eventually fuse with a lysosome, and the resulting phagosome is where the bacteria ultimately meets its end.

This is an extremely cool video from Harvard.

the life of the cell

Freaky pathology of the day

Dr. Bick was kind enough to bring this to my attention. Thankfully, it was before lunch hour: Coral man has "shells" cut from his body.



Based on the reporting, it seems this is a similar condition to the "tree man of Indonesia"

So what's going on here? This is referred to by pathologists as "generalized verrucosis" - lots and lots of warts. Warts on the skin are caused by Human Papilloma Virus (HPV) infection. HPV has gotten a lot of press lately as it is also the cause of genital warts, and there is a new HPV vaccine on the market.

To put it simply, HPV causes proliferation of zones of cells in the epidermis. This is what results in the elevated lesions that are warts on skin. Usually these warts are self-limiting and can regress on their own. So what happened to the coral man and the tree man?

In the case of the "tree man", he had a corresponding immune deficiency, so the HPV infection ceased to be self limiting. His warts proliferated and proliferated, and those growths are the result of dysplastic changes in the skin which are associated with the build-up of excess keratin. These lesions, also called "actinic keratoses" produced so much keratin that they developed into "cutaneous horns" - which, if they get bad enough, can make the victim look like a coral reef or part human, part tree.

The coral man has undergone surgery and radiotherapy, and as you can see, he looks much better.

The tree man appears to have a tougher time of it, though the specialist thinks that regular doses of Vitamin A might help to overcome the immune deficiency.