Microscopy Techniques

Fundamentals 1 – 10:00 – 11:00 lecture

Dr. Cotlin, August 13, 2008

Microscopy Techniques

Histology

There are at least 200 types of specialized cells. All of these types of cells make up the tissues of the body.
There are about four basic classes of tissues in the body. Amongst all the cells and all the extracellular tissue in the body, all of that can be classified as epithelium, connective tissue, muscle or nerve tissue. Every type of tissue in the body can be classified as one of these. Organ systems are made up of all of these types just in a different arrangement.
Pulmonary has the same type of tissue as the GI system, it just a different complement of cells expressing different proteins, a different arrangement of tissues, but they are all going to contain some epithelium, some connective tissue, some muscle and nerve tissue.
When we talk about epithelium and connective tissue, I will be talking about epithelium just in it’s generalized version and all the different types. I won’t expect you to know where these things are found, but I will use them as examples (i.e. this thing is found here, and this, here). Until we get to the organ systems, you won’t have to know where these things are found.

Slide 3 – this will be the general range that we will be observing in basic light microscopy (fingerprints, stratified squamous epithelial, cell level, mitochondrial level, the cytoplasmic level and down to the atomic level). Light microscopy deals with these levels.

Slide 4 – Here is a scale of light microscopy (note: animal cells)

Slide 5 – (Read directly off the slide). All of these are different ways of playing with the light to get a different image.

Slide 6 – Electron Microscopy: This is a more sensitive level that deals electrons and their respective beams, not visible light.

Transmission EM: When I say your “basic EM” I am really talking about transmission EM and this is similar, but instead we are dealing with electron beams.
Scanning EM: Tissue is coated with metals. Electrons are beamed on the tissue and beamed off and you get a 3-D version of what that tissue is like.
Freeze Fracture: Chemical fixations that we crack open cells/tissues and are able to visualize the surface of the chemical makeup by electron microscopy.
I am not going to ask you any details about scope, but this is just to remind you of the features.

Slide 7 – Three types of microscopes: Upright light microscope, Transmission EM, and Scanning EM.

Slide 8 – Just to show you some of the different types of light microscopy with the same cell.

Notice that Phase Contrast Microscopy and Differential Interference-Contrast Microscopy have a 3-D feel to them.
Dark-field Microscopy looks like a negative.

Slide 9 – Scanning EM

You can really see the 3-D version of this tissue here compared to just a regular Transmission EM or Interference-Contrast Microscopy.

Slide 10 – Basic EM

Notice the nucleus, euchromatin, condensed hetero-chromatin and rough endoplasmic reticulum.

Slide 11 – Scanning EM

Left: classic “doughnut” shape to red blood cells
Right: large white blood cell with a good 3-D appearance

Slide 12 – Specimen Preparation. How do you get this material?

First you need to do is “fix the tissue”, which is cross-linking the molecules so the cells of the tissue are locked into place.
On a cellular level, we are 70% water. On the organ level we are 70% water. The earth is about 70% water. Pattern?
We remove the water by passing it through different alcohols.
Then embed it with some type of embedding media, usually a type of wax mixture. It will depend on exactly what you are doing. Basically you will embed it in something.
Then, section it and stain it selectively. We can stain it based on what we want to look for.
A basic stain that you will see often is hemotoxylineosine (H&E)
This is going to stain acidic material purple and eosine will stain basic material red or pink.
Another common one is called Periodic Acid Schiff (PAS) and this stains carbohydrates, so if we are looking at materials that are heavily glycosylated, then we can use PAS and nice carbohydrate structures will show up.
MAIN POINT: we can stain for what we want to see

Slide 13 – I just told you all of this, and here it is just presented in sentences form.

Slide 14 - Immunohistochemistry

Immunohistochemistry is using antibodies in the staining process. The H&E and PAS mentioned above generally stain tissues altogether, but if we are trying to localize a specific protein (e.g. LDL receptor on the cell surface…we know that protein and we have antibodies to that protein) we can use a technique and label that protein with fluorescent label and look at just the proteins we would like to look at. It is really a powerful tool if you have the antibodies for the antigen.
Immunohistochemistry is basically the use of antibodies to stain tissues.
Immunocytochemistry deals with looking at individual cells.

Slide 15 – Stained with H&E

The nucleus is going to stain this purplish-red.
These pictures are of collecting ducts in the kidney.
Would most of the molecules in the nucleus be acidic or basic in nature?
Acidic, because full of nucleic acids, so it will react with the basic dyes and become purple
H&E always very characteristically stains the nucleus in this pattern.

Slide 16 – Stained with H&E

These are fibroblasts that are infected with Trypanosoma cruzi
Note the large nuclei of the fibroblasts and the nuclei of the parasites.
It doesn’t matter what kind of tissue we are looking at, they are going to have the same patterns of staining if a nucleus is present.
They all have a nucleus, they all are going to stain the same way.
All eukaryotic organisms have the same type of nucleus filled with nucleic acids so it makes sense that they would all stain the same way.
These stains are not tissue specific, but it is just based upon the properties of the cell (i.e. presence of nucleus)

Slide 17 – Primary oocyte stained with H&E

You can see the large oocytes, which are one of the largest cells in the female body. Notice the prominent nucleus and nucleolus (small dark purple dot).
(These are all accessory supporting cells).
MAIN POINT: H&E is your basic stain and the nucleus is your focal point.

Slide 18 – Different Staining of H&E

Depiction of how different the tissue can look based on the staining.
Depending on what we stain and what we look for, the tissue is going to look very different. Be aware of what you’re looking for and what the stain should look like

Slide 19 – PAS staining

Stains carbohydrates. Will stain a very dense pink.
The epithelial cells in the lining of our GI tract have cell surface structures, microvilli and lots of glycoproteins and glycolipids that make up the glycocalix (“the sugar coat”) and this is such a dense coating on cells that when we stain you can see the bright pink line and this is the staining of the sugars.
The sugars are so concentrated it shows up as a dense pink. If you see a PAS, you know you are going to try to locate some carbohydrates.

Slide 20 – Glomerulus PAS

Another hotspot for glycoproteins and glycolipids is the basement membrane, which is enriched in carbohydrates.
The dense line in the picture is the basement membrane and the epithelial cells are sitting on the basement membrane. All of the dark heavy pink lines here are the basement membrane.
This is an example of me not asking you “What structure is this?” or “What organ is this?” I might point and ask you “What is this (pointing to the basement membrane)?” and I would expect you to know. You wouldn’t be expected to know that this is a glomerulus.

Slide 21 – Staining to detect particular ions

Besides your basic stains, we can treat to detect specific ions (ex: calcium) in things such as bone development. This slide is stained to look at calcium deposits and you can see the dense regions of calcium.

Slide 22 – Back to Immunocytochemistry…

Direct and Indirect methods
Overview: The antigen is the structure for which we have the antibody against. Here we are trying to detect the antigen pictured and we have an antibody to detect it. Tagging this antibody with the florescent molecule, so that when we incubate this tissue with the antibody, the antibody will bind and when a UV shines you can locate the protein.
The indirect method is used to preserve precious antibodies in which small amounts are used of the primary antibody to detect the antigen and then use a secondary antibody that is fluorescently tagged. This secondary antibody can bind to other things sometimes, which can add difficulty.
The direct method is ideal if you know you have a specific antibody that will detect only that specific antigen.
You know you are detecting something in the presence of an antibody anytime you see “Immunocytochemistry”

Slide 23 – Examples of Immunohistochemistry fluorescent labeling

Some diving cells: tubulin, actin and DNA (looking at chromosomes)
MAIN POINT: we can use this to detect almost anything

Slide 24 – Bacterial infected cell

Fibroblast picture: the green is the actin which really shows the actin structure that we discussed yesterday (8/12/08) concerning that all plasma membranes have an actin network underneath to help maintain the structure.
You can see the bacteria that have actin as well and actin is very conserved across species. We can stain in mammalian cells just as well as we can in a bacterial cell.

Slide 25 – Neuron picture

Cell body with processes going out and you can see the actin network of all these processes going outward.

Slide 26 – Confocal Microscopy

This is the last type of microscopy I will mention.
Deals with using a tubing system and gives you a more 3-D image.

Slide 27 – Conventional Light vs. Confocal Florescence

Diagram: Left - Conventional, Right - Confocal
Conventional tends to blow up the image because of lighting differences and you can’t see fine detail.
The Confocal has an array of laser beams going through the channels and focuses in much better detail
You can see much finer detail with Confocal Fluorescence Microscopy

Slide 28 – Marking Calcium Ions

Another technique using fluorescent markers that will congregate to ions and in this case it is localized to calcium.
Red: high concentrations of calcium
Blue: low concentrations of calcium
MAIN POINT: You can use fluorescents for locating other things in the cell besides proteins.

Slide 29 - Autoradiography

Using radioactive molecules that will show very electron dense.
In this case, they injected radioactive hydrogen and then look at the cell taking up this material and observe its flow.
These dots represent the electron dense molecules.
We can use radioisotopes as well to look at things in the tissue

Slide 30 – Autoradiography: Salivary Gland

Injected with radioactive hydrogen sugars, and you can see all the dots that represent where in that cell the material has gone after incubation.
Using radioisotopes is not the preferred method, but it is a good method because it is easy to see and count in the lab.
We need to limit our exposure to these radioisotopes.
However, this is a very handy tool to detect small molecules such as ions and sugars.

Slide 31 – Freeze Fracture Technique

Take the tissue, submerge it in liquid nitrogen, which will freeze the tissue and when you warm it up it will crack.
Cells will tend to break apart at the membranes, which leads to a nice profile of the membranes (see picture). It is almost like you are looking at the details within the cell, and this is a really good technique.

Slide 32 – Profile shot of membrane

The divots in the picture are where vesicles probably were. This diagram really allows you to see the profile of what all is happening on membranes.
In the same way, you freeze fracture the tissue, coat it with metals and perform Scanning EM.

Slide 33 – In-situ Hybridization

Molecules hybridizing (or coming together) using RNA probes or DNA probes can be done in-situ
Let’s say you wanted to look at protein expression and you didn’t have antibodies to the proteins, so there was no way of detecting if that protein was there, but you knew the gene sequence. You say if this cell is making this protein, then there should be lots of messenger RNA, so you can use a complimentary molecule (nucleic acid) that will bind to the RNA and we can use radioisotopes of fluorescent markers to show it.
This is just another way of looking at protein expression when you don’t have a protein. This technique is used a lot to see patterns and development.
This picture is of a Drosophila larva where a protein is trying to be detected. But you can take the whole tissue and treat it the same and you can see all of these regions that may be expressing that protein.
Nice method when you don’t have antibodies, but do know gene sequence. This is used quite often.

Slide 34

When we are looking at images (EM images, light microscopy, normal tissue, etc.) you have to use your imagination.
Here is a depiction of a blood vessel. We think of it as a hollow tube and if you crossed it, you will see a nice little ring. You have to keep in mind that things run in all sorts of direction (e.g. blood vessels) and we are not always going to slice a blood vessel immediately in a cross section appearance, it may be at an angle and will look like an oval. Sometimes we will just skim it and will look like a flat band of cells and won’t even look like a tube. Sometimes we will catch it longitudinally and will see a slice of a blood vessel from the side.

Slide 35 – Orange and glomerulus

This is showing you that just like the orange, the glomerulus will change shape depending on where you slice it, and it will have a different look with each cut.
(Dr. Cotlin went on for a while on this slide about all of the different angles that an orange can be cut, but the main point is that you have to use your imagination when looking at these cross-section images)

Slide 36 – Atomic Force Microscopy

Uses a laser beam that acts like a finger tip as you run your finger along and gives you the outline of the structure. This is a DNA molecule on the diagram and the laser just traces the outline of this structure.
(Microphone went out at 29:18 into the lecture. Dr. Cotlin was just talking off the record about the efficiency of microscopy and that accidents sometimes happen and big gapping holes form as a result. Last two slides of this lecture did not get recorded. Lecture resumed at 32:13 with Epithelium section. See next page for continued notes)

Epithelium and Glands

Epithelium is one of four basic tissue types. Glands are an extension of epithelium…structures of invaginated epithelium that has developed into a gland structure.

Slide 2 - Functions

Epithelium has lots of functions relative to let’s say muscle which contracts or nerves that conduct.
When we hear epithelium a lot of us usually think about our epidermis (a specialized epithelium), our skin and that is probably it’s ultimate function—protection.
There is also absorption, where epithelial cells line the GI tract and mediate the take up of all these nutrient molecules during digestion.
Also, secretion (e.g. sweat glands, salivary glands).
We have secretion going on all over and glands are derived from epithelial tissue. Secretion is a major component.
Excretory and filtration purposes that goes on in the kidney.
Lubrication where our organs are lined with epithelium on the outside with a thin layer and they secrete this lubricant so everything moves properly (like a type of WD-40 for our organs so everything moves along smoothly).
Sensory function: olfactory cells are actually nerve cells in the eye. There is specialized epithelium in the eye that will be discussed later.
Our taste buds are modified epithelium that detect taste as sensory cells.
Finally, reproductive function where the germinal epithelium in male testis is a basic epithelium. Sperm is generated in this modified epithelial environment.
There is a huge array of functions for the epithelium depending on where it is located.

Slide 3 – Structural Features

Epithelium is going to cover every free or exposed surface being lined with epithelium (e.g. our epidermis, entire GI tract, respiratory tract, male and female tracts.)
Epithelium is a main barrier between inside and outside as well as a barrier between environments within the body.
Your blood vessels are lined with epithelium, keeping blood and extracellular fluid separated.
The kidney when it produces urine is lined with epithelium so we can keep the urine filtrate away from everything else
Epithelium is going to line every kind of cavity in which we need to keep things separated. All free and exposed surfaces will be lined with epithelium.
Epithelium always sits on the basement membrane and it has to be attached to be to the connective tissue underneath it.
If it is going to be this ultimate barrier, the basement membrane is even more of a barrier because the epithelium always sits on the basement membrane, which separates it from the connective tissue underneath.
It is important to know that all epithelium is avascular, meaning there are no blood vessels, no capillary networks that extend through the basement membrane into epithelial tissue. This makes sense because the epithelium is that barrier. We want to keep things out of the blood, which is a pure environment. That basement membrane is that barrier. The epithelium is on the outside so we don’t want the blood vessels being exposed to that outside world.
These are normally polarized, which means that there are two distinct regions and basically they all are going to sit on the basement membrane. So, in the simplest layer (a single layer of epithelial cells), we have one side of the cell that sits on the basement membrane and we have one side of the cell that is exposed to the outside. As such they are oriented and regarded as polarized tissue.
We also see specialized cell junctions, which is a way of keeping all of these cells together. If they serve as a barrier, then they need to be really tight to their neighbor so we don’t have movement through them.
Form a boundary between external environment and organs as stated earlier. These are derived from all three germ layers ectoderm, mesoderm and endoderm.

Slide 4 - Terms