Doctor Adam Bender from the Medical neurology branch at the National 

Institutes of Health.

And I'm going to be discussing the application of electron 

microscopy to the study of skeletal muscle.

The electron microscope has been largely 

responsible for the precise delineation of muscle 

structure and function over the past two decades.

And when combined with histo chemical techniques,

the electro microscope has more recently allowed a much 

deeper understanding of the basic disease 

processes that affect skeletal muscle.

In this talk,

I'm going to be discussing the contributions of electron 

microscopy to the study of both normal and 

pathological muscle.

First,

I will review the technique of obtaining a 

sample of muscle for a study with the electron 

microscope and then I will discuss normal muscle 

ultra structure and function.

This will be followed by a review of the pathological 

changes in muscle discernible at the ultrastructural 

level.

Both those which are non specific and those which 

can be considered characteristic of certain well defined 

disease processes.

Finally,

I'll mention a few newer applications of electron 

microscopy to the study of 

neuromuscular diseases.

The first thing to realize when obtaining a muscle 

biopsy is that it must be obtained in the 

uncontracted state.

And the easiest way to do this is seen on this 

slide,

we use a clamp to make sure that the muscle is 

not over contracted.

This is the one we use at the NIH any clamp will 

do.

This is actually a triangular shaped 

structure in which the end has been cut off.

And the muscle is obtained between these two jaws.

The muscle is then placed in proper fixatives 

dehydrated and embedded in a special plastic 

material.

And then section for light and electron 

microscopy.

The next slide,

this slide illustrates the importance of proper 

sampling in studying a muscle on the 

electron microscope.

This is the field normally seen under the light 

microscope.

And this little square here represents the size of an 

entire block which is examined on the electron 

scope.

And as you can see,

there are maybe six or seven fibers at most 

which can be examined at one time.

So that let's say that the only abnormal muscle fiber in 

this biopsy was right here and this was the only block 

you looked at.

You might assume that this was typical of this 

biopsy.

It stands to reason that you must look at many blocks before 

coming to a conclusion as to what the abnormality in the 

muscle is.

And likewise,

if there were scattered abnormal fibers throughout 

this sample and this particular 

square had no abnormal fibers,

you might assume that the biopsy were normal.

So it is very important to use proper 

sampling techniques.

Look at many blocks and 

make a proper estimation of the 

prevalence of any particular abnormality.

Next slide,

I'm now going to start reviewing some of the terminology 

as applied to normal skeletal muscle.

First,

here,

the muscle biopsy is taken from a 

skeletal muscle.

This is the deltoid region.

The muscle is 

composed of muscle fasi which 

are bundles of muscle fibers bounded by connective 

tissue,

muscle fasi are composed of 

many single muscle fibers.

And these are the basic histological unit of 

skeletal muscle.

It's actually a sensi,

it's a multi nucleated cylindrical structure.

Most of the interior of the muscle fiber is 

composed of myo fibrils,

which are these small bands 

here,

which are actually made up of repeating 

units called SOS and representing 

this banded pattern.

The SOCO consists of inter 

digit thick and thin 

filaments bounded on each end by a 

dense structure known as the Z disc.

These filaments are composed of specific 

proteins.

The thin filaments are composed of acting 

molecules and you can think of them as being 

two strands of pearls twisted around each other to 

compose one filament.

And it's bounded by the Z disc.

The thick filaments are composed of myosin 

molecules which are long golf cub 

shaped structures having a globular 

head at each end.

These heads protrude from the thick filaments 

as small cross bridges.

And these are very important in muscle 

contraction as we will see later.

So that if you take a cross section at 

this level,

you can see that the thick filament is 

surrounded by an array of six thin 

filaments for every thick,

there are six thin filaments.

And in the next slide,

this illustrates the terminology applied to the 

various bands.

The dense line on either end is known 

as the Z line or the Z band 

where there are only thin filaments present.

There is a light band known as the eye 

band.

The central portion of the SOIR is known as 

the A band is composed entirely of thick 

filaments.

There is a dense line in the center 

of the score known as the M line,

which is responsible for keeping the thick 

filaments in proper register.

The A band remains constant in length.

But when the muscle contracts,

the eye band becomes smaller because during 

contraction,

these filaments actually slide in among the 

thick filaments.

And in the next slide,

this is a high power electron micrograph of 

insect flight muscle.

To show the filaments in cross section,

you see the thick filaments surrounded by an 

array of six thin filaments every 

in this very nice repeating pattern,

almost like a crystalline pattern.

The next slide,

this illustrates the membranous 

structures within the muscle fiber.

Each muscle fiber in its entirety is 

surrounded by a membrane known as the 

sarcolemma and at various points 

along the muscle fiber.

This membrane actually invaginated to 

form T tubules.

These are T tubules here are actually invagination 

on the outside membrane into the muscle 

fiber and are responsible for conduction of 

impulses transmitted along the muscle fiber.

In among the myo five brill.

This is actually cardiac muscle showing that there is 

only one T tubule per sarco here in 

regular skeletal muscle that actually there are two T 

tubes per score.

One on either side of the band in 

between two sets of T tubules is a 

complex membranous structure known as 

the sarcoplasm reticulum.

The sarcoplasm reticulum is in close 

contact with the T tubule on each end 

via a dilated portion known as the terminal 

cistern or the lateral sac.

There is no actual anatomical contact here.

These are merely in close opposition and this 

structure contains calcium which is 

released during contraction 

toward the center of the score.

The sarcoplasm reticulum assumes a complex 

cisternal pattern and is broken up into many 

small CIA.

As you see here in the next 

slide at the 

region where the nerve and the muscle join.

A very specialized structure 

is found.

This is known as the neuromuscular junction or the motor 

end plate.

You see here,

a nerve axon coming in and forming 

contact with the muscle at these three points 

in the terminal part of the axon.

There are small round vesicles which contain the 

neurotransmitter,

acetylcholine.

The muscle membrane or the postsynaptic 

membrane is characteristically thrown into a series 

of complex folds which you see here 

when a nerve impulse comes down the axon,

it is thought to cause the release of acetylcholine from these 

small vesicles or packets.

The Cetalol then diffuses across this 

membranous cleft and interacts with an acetylcholine 

receptor on the muscle fiber 

and the muscle end.

Now,

in the next slide,

once the nerve impulse is initiated 

or initiates an action potential at the 

neuromuscular junction,

the action potential is then conducted 

along the sarcolemma membrane 

and down into the T tubule.

As you see here,

the pluses change to minuses.

The nerve impulse comes down causes 

the sarcoplasm reticulum to release 

calcium.

In among the myo fibri,

the release of calcium then causes the relaxed 

muscle to contract.

As you see here,

the thin filaments have slid in among the thick 

filaments in accordion like fashion.

Next slide.

This is a diagram of how the calcium is thought to 

initiate contraction.

This is the thick filament,

the myosin.

And you see the cross bridge here,

those little globular golf color heads protruding from the 

molecule on the thin filaments.

There is an active site which is normally covered by a 

Tropomyosin troponin 

structure.

As you see here,

protein.

And as soon as calcium comes in 

these squares represent 

calcium,

this active site becomes exposed as this 

structure moves over.

Once the active site is exposed,

the cross bridge is then attracted to 

this site and results in contraction.

The energy for this process is 

obtained from A T P and 

there is some A T P A molecule located right on 

the cross bridge.

As you see here,

these two dots representing A T P A.

And in the next slide,

this shows how this attraction of the cross bridge.

The thin filament results in contraction.

This is the muscle in its relaxed state with the thin 

filaments,

the thick filaments and the little cross bridges protruding as 

little discs.

Here,

calcium comes in,

the little cross bridge is attracted to the thin 

filament.

As you see here,

it then pushes or actually pulls 

the thin filament in almost like the action 

of caterpillar feet or a 

ratchet actually.

And this Z band as you see here,

normally here starts to move in 

and then once this is accomplished,

the cross bridge,

then lets go and comes back again and attaches 

once more so that it's a constant joining and 

releasing.

So that eventually where thin filaments are 

separated,

they come together in this fashion and contraction 

results once the muscle is 

contracted,

calcium is actively taken up by the sarcoplasm 

reticulum and relaxation then occurs.

He slide.

I'm going to show you some electron micrograph of 

some of the normal structures that I've been mentioning.

This is a normal muscle fiber,

you see here that it is bound by a membrane.

The circle lema membrane here and the membrane 

actually has two components to it.

This outer membrane which looks like a little fuzz on 

the outside is known as the basement membrane.

And is composed of mucopolysaccharide 

substance.

Underneath,

this is a thin more well defined membrane 

known as the plasma membrane.

And this is composed of lipo protein 

structures.

Nuclei in the muscle fiber typically lie at the 

periphery of the cell just underneath the scala 

membrane.

And there is typically chromatin clumped 

around the edge of the nucleus.

As you see here,

these small little black dots 

which are present just about in every 

space available are glycogen particles 

which are useful in the energy 

for contraction.

In the next slide,

this is a muscle fiber down here and up here 

is actually a separate structure.

This is a satellite cell.

It is not part of the muscle.

As you see there is this membrane separating 

or actually two membranes separating this 

cell from the muscle cell.

And yet this cell is still underneath the basement 

membrane which envelops the muscle fiber.

These cells are important in regeneration 

because once a muscle fiber is injured,

these satellite cells which are usually dormant,

become active and actually participate 

in the regeneration of muscle fibers,

they are thought to become MYOB blasts and actually 

form new muscle cells.

Once the,

once the fiber is injured,

you see here,

these structures in the Iban region which are 

mitochondria typically located in the Iban 

region and these are responsible for energy 

derived from oxidated metabolism.

The next slide,

this is what a neuromuscular junction or motor end 

plate looks like.

And you see here the axon tip,

mitochondria.

And if you look carefully,

you can see the small round vesicles 

representing the packets of Acela cole.

Here,

this is the nucleus of a Schwann cell covering the axon 

tip.

Then the muscle fiber down here with its 

membrane thrown into a series of 

complex folds.

And this is the typical appearance of the neuromuscular junction.

The next slide,

this shows you what the appearance of the 

T tubules and the sarcoplasm reticulum look 

like.

The combination of a T tubule and two 

lateral sacks of the sarcoplasm reticulum is 

known as a triad.

And as you can see,

there's no contact between the sarcoplasm reticulum 

and the T tubule in the center.

Remember,

there are two triads per 

next,

I'm going to be discussing abnormal muscle fibers 

in discussing the ultras structural changes seen in 

pathological muscle.

A few basic points must first be emphasized.

First of all,

most changes that can be seen are rather non 

specific specific in their nature and they can be 

seen in any process in which skeletal muscle 

fibers are injured.

Secondly,

there are certain rare conditions in which a 

single otherwise non specific 

pathological change is prominent throughout 

the biopsy in these instances.

Such a specific change can become 

diagnostic but only by virtue of its 

prevalence within the biopsy.

This is especially true in conditions producing 

congenital non progressive weakness.

Thirdly,

the electron microscope is best used to evaluate 

changes within individual muscle fibers.

The prevalence of a particular abnormality that is the 

number of fibers involved must be first 

determined by light microscopy.

Fourth,

in just about every abnormality seen with the electron 

microscope,

you can first suspect this abnormality with a light 

microscope.

It is therefore only necessary to do electron 

microscopy.

If some unusual change is first noted with a light 

microscope,

a sample for electron microscopy should always be 

taken at every biopsy but may be 

discarded if his a chemical evaluation does not 

reveal any atypical features 

in discussing the pathological changes seen in 

muscle biopsies on the ultrastructural 

level.

I'm first going to show a light micrograph 

stained with histo chemical techniques 

to show a particular abnormality.

This will be followed by an electron micrograph showing the 

ultrastructural equivalent.

And in this first light micrograph,

the first disease I'm going to be discussing is 

denervation.

There are two characteristic changes in 

denervation seen on a light microscope 

level.

These are the appearance of small 

angular fibers as you see here in this light 

micrograph and the presence of target 

fibers which I'll show you in the next 

picture.

But first,

I'm going to concentrate on these small angular fibers you 

see here the normal appearing fibers and 

these small angular fibers.

This is AD P N H tetrazolium reductase 

stain which stains these angular fibers 

darkly.

The electron microscopic equivalent of this 

fiber is seen in this electron 

micrograph.

This is a small angular fiber.

Notice that the membrane,

the atom membrane has a folded appearance 

that the nuclei no 

longer have chromatin at the periphery but 

have a dense chromatin throughout giving the 

nuclei a PG appearance of seen in the light 

microscope.

And you can see that the my fibrils are 

basically intact,

they're slightly disorganized,

but they are still present.

Uh Typically there is increase in the,

in the membranous components within these muscle fibers.

And if this fiber continued to get 

even smaller,

eventually,

it would get to be just,

it would look as if it were just nuclei and it would 

appear on the light microscope as a PG nuclear 

clump.

And in the next uh light micrograph,

I'm now going to show a target fiber.

I want you to concentrate on this particular 

section here which is an oxidative enzyme stain 

and shows you a typical appearing target fiber which is 

characteristic of denervation.

There is a central clear zone 

surrounded by a zone of increased 

staining and then an outer zone of 

relatively normal staining.

So this is a target fiber rather 

characteristic of denervation.

The electron microscope in this 

slide here shows us why the 

fiber has this target appearance.

The central portion of the muscle fiber 

has totally absent mitochondria,

membranous components and the Z band as you see 

here is not normal but has a 

streamed appearance.

This is in the center portion of the target,

the intermediate zone where you remember there is 

increased oxidative enzyme staining has actually 

increased numbers of mitochondria and increased 

membranous components forming a collar around the 

central zone and the outer zone seen 

here is relatively normal appearing.

So this is the target fiber characteristic of 

denervation.

The next fiber I'm going to show is more 

characteristic of so called 

myopathic conditions or conditions in 

which the major change is thought to be in the 

muscle fiber rather than in the nerve.

And what you see here are two abnormal 

fibers among some relatively normal appearing 

fibers.

This is these fibers would be termed 

degenerating fibers.

And at the same time as these fibers are 

degenerating,

they may be regenerating.

Hence,

the two processes may be going on simultaneously 

degeneration and regeneration.

And we sometimes call these de regen 

fibers.

And in this electron micrograph,

this is an example of one of these degen regen 

fibers.

Typically,

the nuclei are slightly larger than 

normal and they have a vesicular appearance because the 

clumped chromatin which is normally present is 

no longer present.

The nucleus has a paler appearance as compared to the pg 

appearance in denervation.

The nuclei are as you see 

internal in location rather than being 

peripheral.

Now,

they actually in this particular muscle fiber 

degeneration is going on this portion and 

regeneration is going on.

In this portion,

you see the degenerating fibr where this 

bundle here has actually been cut off right 

here,

there's increase in membranes components.

And over here you see newly forming 

bundles of myo fibers.

So this is a typical degenerating regenerating 

fiber.

What you see on the periphery here is 

probably a a satellite 

cell which is becoming a 

myoblast.

In other words,

the injury to the muscle fiber has produced 

a change in this fiber.

You see that the endoplasm reticulum,

the fiber,

this cell is becoming activated to 

produce new muscle fibers.

The next micrograph 

shows what we call a ring 

fiber.

This is a rather non 

specific abnormality and it's 

most typically seen in 

myotonic atrophy or myotonic dystrophy.

Instead of the my 

fibros are running from the screen out 

in this manner,

the myo fibros are disrupted so that they 

form a ring around the muscle fiber.

And you see here this ring and if you look carefully,

you can see that there are actually striations within this 

ring.

This is a curious abnormality.

Again,

it is non specific but is most common in my 

atomic atrophy and an electron 

micrograph.

You see again,

this ring fiber,

the central portion of the muscle fiber in cross 

section shows the typical 

appearance of a cross section of a muscle.

And in the peripheral portion,

you can see the muscle fibers running actually 

perpendicular.

Uh This is the ring around the muscle fiber 

on the outer membrane located here.

Z bands,

et cetera.

The next group of diseases I'm 

going to be discussing are the uh 

the so called congenital myopathies or 

diseases in which there is muscle weakness at 

birth,

which is relatively non progressive.

And the first one is this,

which is rod disease also called nele 

myopathy.

Typically,

these muscle 

biopsies on a light microscope.

This is a modified trichrome stain.

You see the appearance of many of these small 

red rod shaped structures which form 

clumps within the muscle fiber.

And you see here also many little rods almost 

looks like tubercle,

bacillus stained with an acid fast stain.

We use the electron microscope to give us an 

idea of what these rods are composed of.

And you see here several rods under the electron 

microscope.

And if you look down here,

you see that the rods seem to arise from the 

Z disc.

This is a normal Z disk and here is what 

is probably the beginning of a rod.

Here is one which is slightly larger and then these 

may be considered more mature rods,

these larger structures.

So the electron microscope tells us that these rods 

probably arise for some reason 

out of the Z band.

The ideology of this disease is at this point 

quite obscure.

You see these black dots representing glycogen out 

here.

Next change which is rather 

characteristic is this 

here,

which is central core disease.

And again,

I want you to concentrate on this 

this stain here,

which is the oxidative enzyme stain.

And you see that typically in central core,

there is almost a punched out region in the center 

of the muscle fibers.

And this is rather characteristic.

And an occasional lesion like this within a 

muscle biopsy is not significant.

However,

in central core disease,

this is an extremely prominent finding.

It is present in the vast majority 

of specifically the type one 

muscle fibers which are dark with 

the D P tetrazolium Duta and 

light with the regular A P stain.

This is a reverse a stain.

Now what is responsible for the appearance of these 

cores?

If you look at the electron micrograph,

you can see again an appearance which is actually 

sort of similar to that of the target fiber,

the central portion of the muscle fiber,

as you see here is a punch out abnormal 

region with streaming of the Z band.

And if you look carefully,

there are no mitochondria again within this region,

explaining the absence of oxidative enzyme 

staining slightly different from the 

target fiber is that there usually is no 

intermediate zone of increased 

staining.

So in other words,

it's a two zone affair.

This central core would run the entire 

length of a muscle fiber.

Whereas in a target fiber,

this might be shorter in length and not run the 

entire length of muscle fiber,

the periphery of the muscle fiber is normal as you see 

here.

The next light micrograph shows 

what may be a variation of central cord 

disease.

This has been called several things.

It has been called multi core disease.

It has been called focal loss of cross tris.

And what you see here is instead of a central 

core of abnormality,

there actually is a perpendicular 

core running in this direction rather than in this 

direction.

And these are may be multiple within the muscle 

fiber.

And hence the name multi core.

And typically there may be disruption of the 

normal cross durational pattern.

Hence the name focal loss of cross striations 

in these lesions.

There are frequently nuclei seen at their 

periphery.

And this is an electron micrograph of what is 

probably a variant of this multi core condition.

What I want you to notice here is the difference between this 

side of the slide and this side of the slide.

If you look from here over there are large numbers of 

mitochondria at present and large amounts of 

membranous components and the Z band is relatively 

intact.

And if you go from here on,

there's not one single mitochondria in present.

And as soon as the mitochondria go away,

the Z band has this streamed 

appearance,

which is typical when mitochondria are no longer there.

Hence,

this has a similar appearance to a 

core,

but you can think of it as perpendicular to the 

plane of the usual core and these would be multiple 

within a single muscle fiber.

Next,

light micrograph shows another congenital 

condition producing muscle weakness.

And this we have called type one fiber 

smallness or hypotrophy with central nuclei.

Another name for this condition is 

myo tubular myopathy and the 

reason people used to call this myo tubular 

myopathy is that these small muscle 

fibers with single central 

nuclei have a striking resemblance 

to muscle fiber in its embryonic state in 

which there are small fibers with 

many nuclei lined up in rows,

giving a central tube appearance or myo 

tubular.

It is thought that this is possibly a defect in muscle 

maturation.

And that that's why these myo tubes 

persist.

And an electron micrograph 

of a small fiber with internal 

nuclei.

Seen here,

you can see that the outer part of the fiber is relatively 

normal.

Actually,

the mitochondria are sparser out here than 

they are in the center,

which is also typical of myo tubes.

And in the center of the fiber are many nuclei 

usually lined up in rows.

There are,

this condition has also been called 

central nuclear myopathy by some.

And if you notice the central portion of the 

fiber also has a degenerated appearance.

And hence some people even call this 

per central nuclear myopathy 

indicating that the damage is around the 

nuclei.

The next uh disorder which you see on the on 

the light micrograph 

is periodic paralysis.

Now here a very characteristic change 

takes place which is almost diagnostic 

for this condition.

And this is the appearance of many single 

central vacuoles.

These pale regions within the muscle 

fiber.

These vacuoles occur in both the 

hypo and the hyperkalemic forms of 

periodic paralysis but are much more commonly seen in 

the hypokalemic form.

They can occur either during or in 

between attacks of weakness,

which are characteristic of this disease.

What are these vacuoles composed 

of?

If you look here at the electron micrograph,

you see one of these vacuoles in the center of the muscle 

fiber.

And what you see here is a T tubule 

actually leading up to this vacuole and 

actually having anatomical communication with it.

So that these vacuoles are thought to be 

actually dili locations of normal 

membranous structures within the muscle fiber and are therefore 

reversible.

When this dili 

decreases.

The next light micrograph 

shows an example of one of the glycogen 

storage diseases in 

glycogen storage disease.

The characteristic finding is that of many 

vacuoles within the muscle fiber.

These vacuoles typically are located just 

underneath the saral membrane.

The vacuoles are the result of glycogen 

accumulation.

This is the modified 

trichrome stain and here is the P S stain which stains 

glycogen and you see the red material 

representing glycogen.

This again is Myla deficiency or 

Mac's disease characteristically,

there are accumulations of glycogen.

So that when you see vacuoles on a muscle 

fiber like this and when they stain 

positively with the P S stain,

if you look at the electron micrograph of such a 

fiber,

you can see the large accumulations of 

glycogen material within the muscle fiber actually 

displacing the normal myo fibular 

structures.

Here,

he's the muscle membrane with a subir 

accumulation of glycogen.

This is phosphorylase deficiency 

in the next light micrograph.

You see the most one of the most severe forms of 

glycogen stores,

disease,

namely acid maltase deficiency.

This is the juvenile form which affects a large 

majority of the fibers.

And you can see that the muscle fibers have almost a Swiss cheese appearance.

They're almost totally replaced with glycogen and the 

infantile form of this disease.

All of the muscle fibers in the biopsy will have this appearance 

and it will sometimes be mistaken for a tissue 

other than muscle.

It will look so bizarre.

And this is a an electron micrograph 

of a case of infantile acid 

maltase deficiency.

And you can see the massive accumulation of 

glycogen within this biopsy.

Displacing the normal my fibro 

structures.

You see here,

this is all glycogen in here underneath the 

membrane.

And in the uh 

next light micrograph,

you see the appearance of what a 

biopsy looks like on a light microscope 

level when one of the so called 

mitochondrial myopathies is present.

These are conditions in which the primary 

abnormality appears to be within 

the mitochondria.

And when you stain such a biopsy with a 

modified trichrome stain,

you see that there is a clumping of red 

material around the periphery of the fiber.

And in addition,

the central portion of the muscle fiber has a rather 

ragged appearance due to the presence of 

excessive numbers of mitochondria as well as 

lipid and glycogen 

structures.

Hence,

we call these ragged red fibers.

And these are typical of diseases in which there 

are increased numbers of mitochondria.

And if you look at these such a fiber on the electron 

microscope,

you see here that there are large numbers of 

mitochondria toward the periphery of the fiber 

beneath the muscle membrane 

here and near the 

nucleus here.

These are abnormal in number 

but normal in size.

Uh in the uh 

next uh electron micrograph.

This is another example of uh 

such an abnormal biopsy 

with abnormal mitochondria.

This is a higher power and you see here uh 

this is one single large 

mitochondria and within it is this 

long crystalline structure.

And these also are typically seen in 

patients with ragged red fibers that the mitochondria are not 

only increased in number and size but 

typically contain large crystalline 

structures within them.

The ideology of these conditions is at this moment 

very obscure.

And in the next electron micrograph,

you see another one of these abnormal mitochondria.

Uh This one has many little squares of 

crystals within it.

The mitochondria has this bizarre uh round 

shape with concentric circles around it.

And the next slide 

you see here,

another variation of this,

this patient had large numbers of of 

mitochondria.

The mitochondria were enlarged and distorted,

appearing.

And also there were many of these 

small light core dense 

particles which were very prominent within 

mitochondria.

So this is another variation of the conditions in 

which large numbers of mitochondria may be 

seen 

in the next slide.

Now,

I'm going to be discussing one of the newer techniques 

applied to muscle biopsies.

What I've shown you now up until now are just 

electron micrograph of normal and 

abnormal muscle biopsies.

The more recent methods have 

involved specifically staining 

these muscle fibers for 

specific reactions.

And one of the ways to do this at the ultrastructural 

level is to use the immuno peroxidase 

technique,

which is illustrated here.

What we have been interested in doing at the NIH is 

localizing the acetylcholine receptor 

on muscle fibers.

It is known that the acetylcholine receptor 

specifically and irreversibly reacts with 

a snake venom known as alpha BGA 

toxin represented by this snake here.

So that if you put bunga toxin onto a muscle 

biopsy sample,

you specifically bind it to the acetycholine 

receptor region.

This is representing the Nplate here.

If you then take an antibody to bungo toxin made 

up in a rabbit and follow that by a goat antibody to 

the rabbit.

And if you attach a enzyme marker to this goat 

antibody known as peroxidase and then 

react it with a hydrogen 

peroxide uh dino benzine solution,

you get a brown stain and this brown 

stain then specifically localizes the 

receptor because what you've done is you've actually produced a 

chain through sequential antibodies 

which is attached to Broin and 

which stains its localization to the cey Coline 

receptor.

And in the next slide,

you can see the application of this technique.

This is a normal human electron 

normal human muscle biopsy,

an electron micrograph of the of the neuromuscular 

junction region.

These are two ax on tips and this is the muscle 

down here.

These are the junctional folds.

And you see this black staining representing 

the localization of bogo toxin binding or the 

receptor.

And you can see that it's specifically on the peaks 

of the post junctional fold of the muscle membrane.

And if you do a higher magnification,

you can see that in addition to being here on the muscle,

there is a slight amount of staining on the 

postsynaptic Exxon membrane as well.

So that we have localized through this 

technique,

the silicon receptor 

on skeletal muscle.

It's actually sort of a histo chemical technique applied to the outs 

structure level.

One of the curious things about the Acela coin receptor 

is that in a normal state,

it's localized only at the neuromuscular junction.

But following denervation,

the receptor can be found not only here on the 

junctional region but on the entire sarcolemma 

membrane out here,

this is normal.

So it's unstained.

And if you look at this picture down 

here,

you can see these small angular fibers representing 

denervated fibers as I've shown you previously and they have a 

rim of black staining around them.

This is a normal fiber here which you see is completely 

unstained and this is an electron micrograph 

of such a situation.

This is the denervated fiber showing the extra junctional staining 

and the normal fiber with no staining of the 

receptor.

So this is one of the trends of electron 

microscopy now to 

apply such techniques as the immuno 

peroxidase to specifically localize 

certain antigens on the muscle 

fiber.

Other techniques such as auto radiography can be 

used in which a radioactive 

substance is used 

rather than a enzyme marker.

And when the radioactive decay takes 

place,

a small silver grain can be seen 

to be deposited in the region of the molecule that you're 

looking for.

And you can actually see these in electron microscope.

And this is another way to localize things at the 

structural level.

So in conclusion,

when properly applied,

the electron microscope is a very important tool in the 

evaluation of skeletal muscle disease.

It is not meant as a routine procedure to be 

applied to every muscle biopsy but is best 

used as an adjunct to histo chemical 

evaluation.

Only when some very unusual change is seen.

Caution must be taken to avoid sampling 

errors in interpreting 

electromyographs and to avoid 

assigning significance to artifacts which may be 

common,

any change in order to be significant.

And I have to re emphasize this that any 

change that you see in the electron microscope 

must be very prominent throughout the biopsy in order to be 

considered significant.

Anything,

any one of the changes that I showed you,

if you see it in one or two fibers means nothing.

It's only when it's prevalent throughout the biopsy that it's 

important 

newer techniques such as the localization of 

specific cellular antigens with the immuno 

peroxidase and auto radiographic methods 

promised to reveal a considerable amount of 

information concerning the basic disease processes 

which affect skeletal muscle.

Thank you.