This is an extremely cool video from Harvard.

the life of the cell

It's Auerbach's plexus

(answer for the Friday what is it)

Here is an H&E stained version of an Auerbach's, or myenteric plexus. Remember that this structure is part of the intrinsic component of the innervation of the intestines. The Auerbach's plexus contains sensory neurons that receive information from nerve endings in the smooth muscle layer regarding the composition of the intestinal content (chemoreceptors) and the degree of expansion of the intestinal wall (mechanoreceptors). Together with the Meissner's plexus found in the submucosa, the Auerbach's plexus is responsible for intestinal contractions.



This bundle of nerves was first described by Leopold Auerbach, a German anatomist, neuropathologist, and histologist. His work was some of the first to define the nervous system via histological staining.

Tissue fixation and processing

Our previous description of tissue cutting and slide preparation dealt with fresh, snap-frozen tissue. In this installment, we'll describe the more common formaldehyde tissue fixation and processing into paraffin. Dr. Rittman has kindly provided us with an excellent summary of the process:

Remember, the function of fixation is to preserve tissue in as lifelike manner as possible for histological examination.

As soon as tissue is removed from the body, pH within cells falls and lysosomal enzymes are released. Tissue rapidly starts to break down in process known as autolysis. In order to examine tissue histologically the process of autolysis need to be stopped as soon as possible after tissue removal. Failure to do so will allow autolysis to progress and will also cause diffusion of substances from their original locations.

There are a variety of methods for fixing tissue but the most common is chemical fixation by immersing pieces of tissue in a chemical solution.There are several thousand fixing solutions (due to the fact that no one fixing solution is ideal for all applications.) Fixation is most often acheived by cross linking the amino acid groups. Therefore, the most commonly used fixative is a buffered formaldehyde solution.

Fixation is generally accomplished by immersion of thin pieces (less than 5 mm thick) in the fixative for various periods of time, typically 24 hours.

At this point, the histologist has a lump of tissue in formaldehyde. That's not going to be very useful for cutting. In order to be able to cut sections thin enough for microscopic examination, tissue must be embedded in a medium (such as paraffin wax) hard enough when solid for such sections to be cut.

Processing requires:
1. The removal of water by use of a dehydrating agent such as graded ethanols from 70% up to 100% ethanol.
2. The use of an intermediary agent that is miscible with both alcohol and paraffin wax. This commonly xylene, a purified form of petroleum. This stage is also called "clearing" as the tissue becomes translucent during this step.
3. Infiltration with molten paraffin wax. This is normally at 58 to 60 degrees Centigrade and results in gradual removal of the xylene and infiltration of paraffin wax into the tissue. The paraffin penetrates into tissue components and even into the cytoplasm of individual cells.
4. Solidification of the tissue in a block of wax. The tissue is oriented for sectioning by placing in a mold and the paraffin wax allowed to solidify.



During fixation and processing most lipids are extracted (this is why adipose tissue looks like chicken wire). Some non proteinaceous substances will be lost in the fixing solution or during the subsequent process. Other substances such as glycogen may be retained as they are associated with proteins. In addition, due to the solvent action and to heat the tissue undergoes a certain amount of shrinkage. With soft tissues the shrinkage is generally in the order of 25 to 30%. Shrinkage often results in separation of components from each other such as separation of muscle fibers and separation of soft from hard tissue at interfaces. By now, you should be noticing this a lot in your example slides in lab!

Here's an example of shrinkage - the keratin layer in this high-power view of skin is separating from the underlying epidermal layers and from itself. The white spaces indicated by the arrows are artifact and not there in real life.





Sectioning of a paraffin-embedded tissue is very similar to frozen tissue sectioning. The solidified block of paraffin wax is fixed in a holder on the chuck on a microtome. The microtome allows a reproducible forward advancement of the block towards the knife that will slice a section from the surface of the block. Typical advancement is in the order of 5 to 10 microns.




As sections are cut, the lower edge of the block melts slightly allowing this edge to join with the trailing edge of the previous section thus forming a ribbon of sections. Such ribbons of sections as seen here may be stored for a short period of time.



During the sectioning a certain amount of compression occurs so that sections have the same width but are smaller from their upper to lower edges than the paraffin block from which they were cut.

Section mounting.
Sections are floated onto the surface of a warm water bath (usually 45 to 48 degrees F) where the paraffin section containing the tissue expands.
Sections are then collected and oriented on a glass slide and then allowed to dry.

Staining - it's business time!
Remember, most tissue components are of similar refractive indices and cannot be distinguished from each other unless we use phase contrast microscopy. The better way to differentiate tissue components is by the use of histological stains or histochemistry.

Most stains rely on binding to different components by virtue of some type of chemical bonding. We will deal with hematoxylin and eosin although the same general principals apply to many stains.

Sections on slides must first have the paraffin wax removed by soaking in xylene, followed by graded alcohols from 100% to 70% and finally into distilled water. Shown below are slides in the process of moving through the de-paraffinization process.



Sections are then stained in a solution of hematoxylin where the hematoxylin binds to acidic components. Next, we soak the sections in eosin which binds to basic components. The sections are then dehydrated, cleared in xylene and mounted in a transparent mounting medium. If done correctly, the stained and mounted slides will last for decades (as clearly demonstrated by your lab slide sets).

In a final well-stained preparation, nuclei are blue, and the cytoplasm stains a pale pink, (the cytoplasm of some cells may also be blue due to the presence of acidic components such as RER). Muscle cells stain bright pink, red blood cells bright red. Any traces of mineral will appear dark purple. This is especially noticeable in the partially mineralized enamel of developing tissue.

Decalcification (demineralization).
Embryos (while they do contain some mineral in developing teeth and bone)may be sectioned without removal of the mineral. However, tissues with considerable amounts of mineral such as bone, dentin and cementum must first have the mineral removed before paraffin wax sections can be prepared. This process is known as decalcification or more correctly as demineralization.

The tissue is first adequately fixed and then mineral is removed most often using dilute acids. During this process the tissue may show some changes due to the effects of the acid on components other than the mineral. Further hematoxylin-eosin staining then looks like this:



Bright pink staining of a tissue with very few cells is usually bone.

Friday "What is it?"

This one might be obvious to the dental students who paid attention during Dr. Rittman's lecture on Thursday. Any guesses?

Friday "What is it?"

Today's example is a lovely fluorescent micrograph provided by Dr. Roger Bick. Please contact him for permission to reproduce this image. Any ideas what this is?



Hint, it's a type of connective tissue.

While you're studying for exams

I dare you to try and get as excited about BLOOD as this young gentleman.

(thanks to Lori, Rob, and Mo for the link)

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.

It's a Taste Bud.

Shown below is a lower power section of the tongue, arrows indicate the taste buds. This particular section of tongue is called the foliate papillae, and the taste buds are embedded within the nonkeratinized stratified squamous epithelium.



Taste buds are barrel-shaped, and contain 30-80 spindle shaped cells. Their apical ends terminate just below the epithelial surface (that's the darker red line of cells in the high power view). That's called a taste pit, and the taste pit communicates with the surface through a small opening called a taste pore.

Taste buds are one of the only specialized sensory cells in the oral mucosa. The stimulation of taste seems to be initiated by the material of the taste pits and the microvilli of the cells projecting into those pits. Adsorption of molecules onto the membrane receptors on the surface of the taste bud cells activates a signaling cascade and releases neurotransmitters that then stimulate nerve fibers surrounding the lower half of the taste bud cells.

The first Friday "What is it?"

We plan to make these a lot more complicated in the future, but for now let's put up a relatively simple pic:

Identify the specialized structures at the arrows.



Any students out there have a guess? Answer on Tuesday.

How do you get tissue onto a slide, anyway?

As instructors in the laboratory, we sometimes find ourselves telling students, "well, that's just a bad section." Or, "that's staining artifact". But what does that mean, exactly? And how does it happen?

First, you have to understand how sections are processed and placed onto slides. There are two basic ways histologists do this: paraffin and frozen sections. Pretty much all of the slides in the student lab boxes were prepared from paraffin sections. It works like this: a bit of tissue is dropped into a preservative, usually formalin, then it is embedded into paraffin (a wax-like substance). The paraffin block is then placed into a microtome, and very thin sections are cut from the tissue.

Another way to prepare tissue for cutting is by freezing. Frozen sections are the method of choice for most immunohistochemistry and immunofluorescent applications, since the cross-linking of surface antigens by formalin fixation can cause trouble with specific antigen binding by antibodies. Instead, the tissue is isolated and placed directly into a tray containing a special glycerol based compound called O.C.T. (optimum cutting temperature) compound.

Once the tissue is immersed in the O.C.T., it's quick frozen, usually on dry ice, or in an ice/isopropanol slurry. Quick freezing is the key - freeze too slowly and you'll get all kinds of tissue damage and distortion. You don't want bubbles in your O.C.T. either, that will also mess up the architecture of the tissue and make the block very difficult to cut. Obviously, the freezing compound is a big component of the success as well - if we froze the tissue directly, we'd end up with ice crystals all over and inside our tissue - again pretty much destroying the cellular structures.

OK, so, once you've got your tissue frozen, you get a little block that looks like this:

This example is rat abdominal muscle, in case you were wondering.

The next step is to mount the block onto a tissue chuck. This is the holder that will be placed into the microtome. The histologist will remove the plastic backing, then use some more OCT to stick the block to the chuck (this step is usually performed on a block of dry ice - it has to freeze on there). It's important not to get any bubbles in the O.C.T. - this results in the dreaded "chatter" when you cut the tissue, leading to uneven sections. Trim the block a bit, and you're ready to move to the microtome. Shown below is a cryotome, or cryostat - since we're cutting frozen sections, we need to keep everything cold, hence, there's a refrigeration unit surrounding the microtome. If this was a paraffin section, the microtome wouldn't need all that infrastructure. That can cut at room temp.

Next we have a closer shot of the innards of the cryotome. Our friendly neighborhood histologist, Brian Poindexter from Dr. Roger Bick's lab, is holding the tissue chuck with the muscle sample mounted to it. The square structure behind his hand is the microtome itself, and the chuck will be placed into that black holder in the center.

The chuck (+ block of tissue) is placed into the holder, adjusted so that the block is parallel to the knife blade and now the process of cutting really begins. That black part of the microtome you see there? That's controlled by a handle - every turn of the handle inches the microtome closer to the knife by a pre-set distance (this is adjusted on the machine). Imagine the butcher at the deli thin-slicing meat, and you have the idea. First, the histologist slices away the extra O.C.T. from the tissue, and once he reaches the tissue itself he starts to make really thin slices. Below, you can see the muscle tissue clearly, and there is a section in the process of being cut.

The section will peel off of the main block like onion skin. This one is just starting to separate. You should be able to make out the (very sharp) knife blade at the block. Incidentally, this blogger had a very nasty experience in grad school involving a cryotome knife that resulted in a lot of blood all over the cryotome and 5 stitches in her fingertip. Not recommended.

The microtome operator continues to slice the section free in a smooth motion, and should wind up with something like this:

Most sections are cut at 5 - 10 MICROmeters, so you don't want to sneeze while you're in the process of slicing! (Also something your blogger has done whilst in grad school). An unstable block, or unstable speed of cutting can lead to thick/thin portions on a section, or tearing of the tissue before it peels off all the way.

Now it's time to get the tissue onto the slide. This is probably the point in the procedure where bad things are most likely to happen. The histologist uses a brush to gently coax the thin tissue + O.C.T. onto a slide, like so:

If you're not careful, you can double up the section on the slide, or get a big wrinkle in it, or tear it. Ever wonder why some example slides have tissue that looks like it's been folded in parts? This is usually the cause. You can also "stretch out" the tissue, pulling it apart and spreading it out on the slide, again leading to a lot of artifact and distortion.

Finally, the histologist moves the slide out of the cold of the cryotome into room temperature air, the O.C.T. melts, and voila! Tissue sections on a slide!

Obviously, some more prep has to go into getting the sections ready to stain, but we'll leave that for another post.

Big thanks to Brian Poindexter, M.S., with the Imaging Core Lab, Department of Pathology and Laboratory Medicine.

Welcome to the HistoloBlog. This blog is run by Dr. Keri C. Smith, from the Department of Pathology and Laboratory Medicine at the University of Texas Health Science Center at Houston. Its primary purpose will be to provide more detail on the study of histology and microscopy for the dental and medical students at our institution. I hope to develop it into a repository for images, for student discussion, and hopefully, as an appreciation for the history of histology.