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Article 4: Histological Techniques: Making paraffin blocks (diagram in spanish)

Valentin Martín 06 / 05 / 2012 Automatic translation (view original)
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The histology is a discipline with more than 100 years old, at this time the histologos have developed lots of tools, techniques and processes in order to visualize the cells and tissues. Describe all these tools, techniques and processes would be reason for a complete collection of books.
The objective of this manual is to introduce the student to the knowledge of the techniques and common instruments in a histology laboratory, with the aim that the student understands how the samples that then it shall examine under the microscope and so improve their learning process have been.
Because of this the main script in this topic we will focus on the techniques and instruments most commonly used, and at the end explain other types of instruments, techniques and protocols, that although they are used in histology make it sporadically for certain and specific studies.

The histology is focused on the study of tissues and cells. Given the small size of these structures, the histologist requires systems increase the image, what we know as microscopes, sinceThese structures are so small that not can be clearly discernible to the naked eye.
Currently, there are multiple types of microscopes, although the microscope optical field course is the basic tool of study and as such the student should sufficiently meet him.

To begin with, there are a number of facts that the student must know:
The human eye is unable to distinguish small things, but like any other optical system has a limit; i.e., there comes a time that is not able to distinguish two points as separate but as a single item. This minimum separation that allows to recognize how separate colon is known as "Resolution limit". This has made the human being has searched systems to enlarge the image.
(B) the development of the first lenses in the 17TH century led to the emergence of the first rudimentary microscopes. Since then, microscopes have evolved to the current. Despite this development, optical microscopes have a limit of resolution limiting the increases and that is determined by the nature of the light.
C nowadays the best microscopes do not exceedthe 1000-1500 increases and there is a limit of 0,2 m (0.0002 mm) resolution.
(D) are called simple microscopes to those who have one, or a single set of lenses. It is colloquially referred to as magnifying glass.
E are called compound microscopes to those who have two lenses or lens sets, some are known as objective lenses and other eye glasses. The current laboratory microscopes are compound microscopes.
F in a compound microscope, it is essential that the sample to be observed is very thin so that light can pass through it and reaching the objective lens.
G light passes through the different tissues of a cut in a similar way so it is almost impossible to distinguish the different components of the Court. That is why it is necessary to dye the cuts in order to distinguish its components.

In addition there are a number of concepts and definitions related to microscopy that the student must also know to understand the microscope and manage it more efficiently.

-Magnification: Is the number of times that a microscope system can increase the size of the image of an object.
-The power of resolutionon: is the ability of an optical system to display two points very near each other as separate elements.
-Resolution limit: is the smallest distance that a microscope can display two points coming as separate elements and not as a single point. This parameter depends on the wavelength of light (energy) and the numerical aperture of the lens used. In practical terms in a conventional optical microscope with a 100 x objective (and eye and 15 x, i.e. to about 1500 increases intermediate lens) is 0.2 m. He is calculated using the formula: LR = /2AN (donde represents the length of wave and AN numerical aperture).
-Numerical aperture: is the ability of the lens to allow light to pass through (mide cone of light that a goal can support). It is unique for each objective.
-Depth of field: the distance between the more separate parts of an object (according to the optical axis of the microscope), that can be seen without changing the focus. This distance is larger in the objectives of small increase and lower in the largest increase.

The optical microscope is an instrumentDepartment of precision formed by a multitude of pieces and parts, which is convenient to the student knows the most important. 1 Histological techniques: How is and how it works the Light Microscope (diagram in spanish)

-Eye: Is the lens Assembly that forms the extended final image that we are witnessing.
-Revolver nosepiece: current microscopes often carried several goals which are arranged in a wheel called revolver. To place the desired objective should move the revolver to the appropriate position. We can find microscopes equipped with 3, 4 or 5 positions revolver.
-Objective: It is the device that contains the set of lenses that capture the light from the sample and generate the first enlargement, there are various magnifications.
-Stage sample: is the surface where the sample is placed to observe.
-System Koeller: enable to focus the beam on the optical axis of the microscope. (Not all microscopes have these systems).
-Condenser: Allows to focus the beam of light in a small area of the sample. (Not visible in the schema).
-Aperture: Allows you to adjust the contrast of the image.
-Control of light intensity: many current microscopes have this light intensity control device.
-Wheels ofdisplacement: are commands that allow you to move the sample to the length and breadth of the deck sample (axes X and and).
-Micrometric approach control: allows you to sample the fine focus by using light displacement (on the Z axis) of the stage.
-Coarse focus control: allows the approximation to the focal plane through major displacements of the deck along the Z axis (vertical) sample.
-Tripod: Is the metal housing that serves as support to the rest of the system. The tripod attaches to the base forming a solid and stable block.

Different sets of lenses and other optical elements define the path of the light microscope.
1. The light necessary for the operation of the microscope is generated by a light bulb.
2. The diameter of the beam is restricted through the use of an opening (a metal plate with a hole).
3. The condensing lens condense the beam on the sample.
4. The objective is a first enlargement of the image. The extension depends on the chosen objective.
5. A Prism changes the direction of the light to make a comfortable observation at a right angle.
6. The eye is the second and final ampthe image liation.

As you can see in the diagram the present in a microscope optical elements are many and varied and we can divide them into two categories: those destined to generate, modulate, focus and condense light (such as diaphragms, condensers, openings, etc...) and others intended to enlarge the image (objectives and eyepieces).
Without a doubt, the most important are objective and eyepiece.
-Objectives: The microscope often have many different goals and positions you gun, the most common are the 4 positions revolvers. The variety of objectives in the market is large, though the 4 most common objectives in laboratory microscopes are usually 4 x, 10 x, 40 x and 100 x, being the last dive.
-Eye: All conventional laboratory microscope has 1 (if monocular) or two eyepieces are the same (if binocular). The most common eyepieces with 10 x (10 x) Although some manufacturers offer, for special jobs, other ocular (15 x, or 5 x) for example.

Taking into account that the total increases of a microscope is the result of the multiplication of the partial objective and eyepiece increases can deduce thatthe range of increases of a conventional optical microscope is 40 x to 1. 000 x.
In binocular microscopes one or the two eyepieces can be graduating, allowing a proper binocular vision even if used glasses, whose optical correction can compensate for graduating the eyepieces. In a monocular microscope is not necessary nor this.

The student must learn to properly operate the microscope. 2 Histological techniques: How it works the optical microscope (diagram in spanish)
The first step is to check that the microscope is connected properly and the light works with the proper intensity.
Firstly, remind the student to use glasses that must adjust the eyepieces.
Although it seems obvious there to place the preparation correctly, i.e. with the coverslip upwards.
The study of any preparation must begin with the smaller goal (in many microscopes objective 4 X). Most common is to study the entire surface of the sample to locate the different structures.
Once located the structure that you want to consider switching to the next objective (10 x) and perform the same operation, even, if necessary, to the more objective (100 x).
No doubt the estudiante will learn the concept of "The diffraction of light", which can be summarized by saying that light slightly changes direction as it passes from one medium to another (for example from the glass into the air). The change of direction occurs at an angle which depends on each one of the means and that can be expressed by a single value for each medium that is known as "refractive index".
Using little power this phenomenon objective barely affects the observation, but it becomes a problem when we use a 100 x objective, mainly because these increases the preparation should be very close to the goal and the change of direction of the light from the glass (slide) air and again to the glass (lens) causes that not can focus correctly the sample. to avoid this place, between the objective lens and the top of the sample a small drop of a special oil (immersion oil), which has a similar to the glass refraction index, so the light does not change direction on this interface, and as a result you can focus without any problems.
If after studying the sample in order to 100 x is necessary to return tostudy with the 40 x objective is necessary to remember that the sample even has a drop of immersion oil that could soil this goal, so it will be clean with a handkerchief or piece of soft paper soaked in cleaning solution.
Once finished the comment removing the food clean, as you have just described, as well as the 100 x objective.
At the end of the observation period should be that both the microscope objectives (deck), as well as the preparations are completely clean.
We will also check that the microscope is properly disconnected and make sure you leave the smaller lens, placed to facilitate the following use of the microscope.

Now that we know that the optical microscope obtains a magnified image to pass light through a sample and a set of lenses, it is clear we can not study an organ or a piece of a body under the microscope, since light could not cross. It is necessary to obtain fine samples the light to pass through and to study under a microscope.
How to make for fine samples?
The answer is obvious, must be dialledAR cuts fine to study body, which is not easy, this it may verify the student if you try to cut a piece of fresh meat with a very sharp knife, you'll find that you can not get thin slices. This is due to the fresh meat does not have consistency nor the hardness required for cuts sufficiently fine, while if we freeze the meat or leave it dry, if it's possible to cut the meat, since this in both processes has increased its hardness and consistency...
As bodies to study have a consistency similar to the piece of fresh meat of the example it is necessary to increase the hardness and consistency of artificial form, there are different methods, the method most used being the inclusion in paraffin. It is not a simple process and involves numerous steps. 3 Histological techniques: How is done to get histological sections, Embedding and sectioning (diagram in spanish)

Biological material, from the time of death, undergoes a process of degradation, known as rot, due both to endogenous causes (autolysis) or exogenous (bacterial attacks). It is clear that this degradation makes progressively more difficult (more time to more degradation) the study of biological structures to the mycroscopio.
To avoid this degradation is necessary to stabilize the structures and make them unavailable to such degradation, so used chemicals known as "clips". The chemical nature of the clips is varied but they tend to be molecules with several active groups that bind to different molecules of the cell creating an interconnected molecular network that attacks bacterial and enzymatic are unsuccessful, thus keeping the cell structure.
For conventional optical microscopy techniques are often used based on formaldehyde, fixatives in buffer solutions or mixed with other chemical species (as with picric acid in Bouin's fixative) either.
How to manage the post to the sample depends on the circumstances of collection.
-Well, in human (and animal) specimens obtained post-mortem (necropsy) or obtained by direct extraction (biopsies), the fixing method is simple: immerses the sample in a container filled with the binding substance. The fastener must spread tissues to perform its action. At times, and depending on the nature of tissues this penetration is not completeand there is a gradient of fixing, still better fixed the worst fixation, the central areas and peripheral areas.
-In the case of animals for experimentation and to avoid the effect of gradient of fixing is commonly used method of perfusion. The idea is simple: it is injecting fixative liquid in the cardiovascular system so this circulate throughout the body and thus the fixation is homogeneous in all tissues. Typically is injecting liquid fixative to the height of the heart ventricle or aortic artery and at the height of the Atrium blood must be drained to prevent overpressurization of the system causing rupture of capillaries.
In one case or the other left a long piece to ensure that the locking fluid concluded its effect. At this point the piece is inserted into a cassette sample (a small plastic box holes). This sample allows easy change from samples of a few containers others maintaining the integrity of the piece. Fixing time piece should cleaned with frequent water baths, first tap water and then distilled water to remove all traces of fijador which could react with chemical substances that must be used later.

Inclusion is the process by which will increase the hardness and consistency of the piece to allow his court. This is achieved including the piece in a substance which hardness and proper consistency. The paraffin is usually used in this process. The challenge is to replace the water that is in the inside and outside of cells by paraffin.
Paraffin is a substance that is liquid and solidified below this temperature over the Garcia, this facilitates dissemination of paraffin by tissues when liquid, however another problem that must be overcome is the fact that the paraffin is highly hydrophobic, i.e. cannot be mixed with water or substances in aqueous media. Therefore the next step that must suffer the samples is the removal of water from the sample: dehydration.
The dehydration of the samples is achieved by a gradual replacement of the water by ethanol. To get it undergoes successive baths of gradation growing ethanol parts, starting with 500 or 700 ethanol and concluding withdifferent baths of absolute ethanol (1000), passing by ethanol 960 baths.
The piece, already dehydrated, yet not can be passed to paraffin since ethanol is miscible with paraffin. An intermediary agent, i.e. a substance which is miscible with ethanol as the paraffin is used. The commonly used intermediary is xylene, in which the piece suffers several baths to completely replace the ethanol.
With the workpiece in xylene, usually through a bath of a mixture of Xileno-Parafina 50% to favour the penetration of paraffin. Subsequent to this bathroom occur several bathrooms in paraffin pure until paraffin has gone completely in the entire piece. All these bathrooms that include paraffin are Garcia stove to keep the liquid paraffin. In some laboratories throughout this process is automated using a device (robot), changing a fluid samples to another using a preset program.
Once past the time that paraffin penetrates the tissues, the question is to perform a block with all of this, which can be used to get cuts on microtome. 4 Histological Techniques: Making paraffin blocks (diagram in spanish)
The easiest way is to usea mold in which the paraffin is poured and which introduces the sample processed and let it cool for to solidify the set. A station is used for this purpose in many laboratories. These stations have a tank of liquid paraffin (Garcia), from which can be dispensed paraffin on the mold through a tap; There is also a camera where you store different molds to Garcia and a hot plate (Garcia) and other cold (40 c). Dynamics is simple, fits the mold under the tap of paraffin and is filled with liquid paraffin, the workpiece is placed and oriented, finally by the base of the cassette of inclusion. Set above the cold plate moves with care to achieve a rapid solidification not forming crystals and solidified once removed the mold getting a block ready to cut, as shown in the interactive diagram.

The microtome (Greek, "small" micros and volumes "section/part") is the appropriate instrument for fine cuts of paraffin-embedded biological material.
In essence, the microtome consists of a fixed blade and a mobile arm, which anticipatesand rises and low displays, so it falls on the blade and get cuts. This type of microtomes are called "Minot or rotary microtome". 5 Histological techniques how it is and how it works the Microtome (diagram in spanish)
The arm is capable of advancing the sample very small distances (usually 5 to 10 m) with a precision mechanical system based on a very fine thread pitch screw.
At the end of the arm, there are anoint clip in which fit the cassette bases used to form the block. Arm moves with the block in high position, once you have the desired amount of Micron advanced arm low block strikes the edge of the blade and the court settles on it, when the arm reaches its lowest position, goes up and begins a new cycle of court. The process is controlled by a handle circular, that to be operated concluded a cutting cycle for each lap.
Microtomy blades are very sharp to achieve homogeneous and fine cuts, most laboratories often use interchangeable blades, which are replaced when they lose sharpness.
Affect the block on the edge of the blade, by friction, increases the temperature enough that thethe new edge slightly cut its cover and will join with the previous cut, thus making several cuts, these form a bead on the surface of the blade.

To see the cuts to the microscope needed mounted on a thin sheet of glass (slide). 6 Histological techniques: Handling histological sections (diagram in spanish)
To do this, first separate cuts one by one or in small strips that can be mounted on the slide.
The cuts come out wrinkled of the microtome due to friction between the blade and the block, so it becomes necessary to stretch the cuts to its correct observation.
To achieve this the cuts made to float in a bath of water, lukewarm (about 35-360C). Due to the heat, the paraffin expands by stretching the cut until it is completely smooth. When the cuts are stretched, they simply fish with the slides. Previously on the slide surface extends a thin layer of a magnetic substance that secures the cut to slide and prevents the cut comes off in the subsequent processes. The steps - lysine as adherent substance is used in many laboratories.
When we have the cuts above the slide left to dry (usually in an oven to)(35-360C).

Fine cut biological material is basically transparent, so it cannot distinguish anything to observe under the microscope.
This is why that it is necessary to stain samples to distinguish the cells and tissues.
Staining protocols are performed by sequential baths of various dyes and reagents. There are different ways of doing this:
-Can be slides onto a cooling rack placed in a tray and using a pipette will placing the different dyes and reagents.
Several slides placed in special baskets of glass which go from a bucket to another, containing each bucket-can stain or dye reagent relevant.
-The process of staining in racks can be through an automated using a robot that automatically changes the sieve tray by a preset times.
Whatever the method used, the (sequence of steps) to use staining Protocol will depend on what you want to display the processed tissue.
Staining protocols (techniques), numerous books have been written since they are very numerous and varied. At the endThis topic we will make a summary of the most common techniques used in histology.
Among all staining techniques is one that stands out above the others since it is by far the most widely used around the world, it is the technique of the hematoxylin-eosin. 7 Simple columnar epithelial tissue
Hematoxylin is a dye mixture (there are different variations) that is basic in nature so it binds to acidic substances. In the cells, the more acidic area is the core since it is full of nucleic acids (DNA), by which the core turn with an azul-violaceo color hematoxylin.
Eosin, a dye colour is rosa-rojo which is dissolved in ethanol and has an acidic, so it binds to the Basic (high pH) structures of the tissues. Structures with higher pH of the tissue are proteins, because the bridges of sulphur and nitrogen have. It is that in samples processed with this technique, stained pink, preferably, the cytoplasm and the extracellular matrix, both rich in protein structures.
All staining process can be divided into three phases:
-The first phase withsystem in the dewaxing and hydration of the cuts. For desparafinar cuts with xylene, which dissolves the paraffin bathe and hydration is a sequence of steps reverse the dehydration.
-With the sample in an aqueous medium is with the chosen staining Protocol. The Protocol is usually consist of a sequence of baths with dyes, cleaners and reagents, which is typical of each technique.
-The third stage aims the permanent mounting of specimens for observation under a microscope. Mounting medium is placed on top of them to fit and protect cuts: a liquid substance that crystallizes with air (polymerizes) and a very thin sheet of glass (coverslip), they form a whole stable and durable. The mounting medium is usually a hydrophobic substance, by what it cuts prior to be mounted have to be dehydrated, following a protocol similar to that used during the inclusion.
The mounted preparation is left to dry for a few hours and kept in dark to prevent light to degrade the colors.

With everything explained so far the student can be a clear idea of the process that isperforms until the preparations to study under a microscope.
Now, it is desirable that the student should take into account some necessary concepts for the study of samples under a microscope. 8 Histological techniques: Levels and depth of the histological section (diagram in spanish)
-Firstly, the student has to understand that the biological structures are usually complex and three-dimensional structures of which, under the microscope, you can only see flats (in two dimensions). Thus, for example, the kidney tubules are cylindrical tubes, with what could be expected that, under the microscope these tubules were like rings (the section of a cylinder). The issue is that these tubes are not straight and have many turns (like other tubes in the body, e.g. the gut), so in a same cut and a same tubule many sections can be viewed as shown in the diagram. I.e. the student should follow the same structure cut into different orientation can display different sections.
-Abound in this topic of the three-dimensionality, another fact that must be taken into account is the depth of the cut. This problem is perfectly illustrated in the diagram where you take as an example an egg (which in essence is u)(na cell). This still cut egg in the same plane can display different sections depending on the depth to which the cut has been made.
It is important that students understand both concepts to interpret correctly the structures observed under the microscope.
On the other hand the student must take a correct dynamic observation of samples for the maximum utilization of each study session.
Our advice is:
-Start by observing the preparation with the naked eye by placing it on a white surface. In many cases the student can locate the most notable anatomical elements of the body to study.
-Secondly, placing the preparation on the deck and observe it with the smaller lens (4 x) and browse through all the preparation by locating the areas of interest and singular elements.
-Finally, and with areas of interest located, go progressively using the objectives of greater increase in each of these areas, to distinguish elements structural, tissues and cell types characteristic of the studied sample.

So far we have described procedures, instruments and more usual staining processesin the conventional study of Anatomy, we will now give a brief review to other types of tools, processes of inclusion and staining protocols, which are also often used in histology.

Apart from the optical microscope field course, that it is the most used in any laboratory of histology, there are other types of microscopes results (images) the student will probably used during his apprenticeship, although it is unlikely that you use it directly since they tend to be scarce, expensive and complex to use. 9 Histological techniques: Examples of different types of microscopes (diagram in spanish)

The transmission electron microscope is an instrument of study that uses the same conceptual structure than a conventional optical microscope, but it uses a beam of electrons rather than a beam of photons (light beam). 10 Histological techniques: How is and how works the MET (transmission electron microscope) (diagram in spanish)
The wavelength of the electron is less than 1nm, i.e. approximately 500 times smaller than the wavelength of light (approx. 400-600 nm), so with a transmission electron microscope can get approximately 500,000 of increases.
Using electrons in a microscope involves a series of problemas:

-First, should take into account that the electrons are electrically charged so if these electrons, in his career, found with atoms, by electrostatic, clouds of electrons from the atoms forwarded to the electrons in the beam, making it impossible to obtain a coherent image. Why microscopes inside should be the most complete vacuum.
-Secondly, we must bear in mind that to make a microscope behave as such, it is necessary to have some lenses that change the path of the beam. The problem is that the optical lenses do not serve for this purpose, so you have to give the electron microscope lenses otherwise. How to change a beam of electrons is through the use of a magnetic field, this is why an electron microscope lenses are electromagnetic coils which generate these magnetic fields.
-Third, there is the thickness of the cut. If we use a cut used for optical microscopy (from 5 to 10 m of thickness), the amount of biological material within this thickness is such that the image would be incomprehensible. This is whythe thickness of the cuts for this type of microscopy should be much more fine, ranging between 40 and 50 nm. (0.04-0.05 m). Nor can also be used glass slides (for very fine whatever) since the electron beam not traversed it, is why is often used as slides a thin metal grille (copper), the Court rests on the filaments of the grid being suspended in the spaces between filaments.
-Then we must solve the problem of contrast (staining). Biological samples present a contrast against the electrons very similar between if and in general very low, so it becomes necessary to increase it. In electron microscopy are useless dyes used in optical microscopy (just offer contrast to electrons), so it is necessary to use other contrastadoras substances. Uranium acetate is used basically, this molecule binds chemically to the biological material and with the large amount of electrons from the atom of uranium, makes that there where there is an accumulation of biological material (with uranium chemically bonded acetate) electrons do not traverse the sample to be diverted by the electron cloud todayso there where there is a low concentration of biological material, the electrons will pass through the sample easier. This causes the final image is composed by the presence or absence of electrons. IMAGES FROM MICROSCOPES ARE MONOCHROME (BLACK AND WHITE).
-The last problem that has to be solved is to obtain the image. The human eye is not able to see electrons, so it is necessary to devise a system to obtain a visible image. The system used is a plate of phosphorus. Phosphorus has the property that to be reached by an electron releases a photon, so you get the picture. Besides, the electrons can also impress a conventional photographic plate, and even with the help of a digital sensor of electrons, an image on a computer monitor can be obtained.

Processing of samples for the MET
Processing of the samples to the MET in essence is similar to that used in optical microscopy, conventional, taking into account the peculiar characteristic of the MET.
-In the first place must be borne in mind that to see more increases it is necessary that the conservation of structures biologiCAs is much more faithful and accurate for optical microscopy. It is why are often used far more powerful fasteners, such as the Glutardialdehido, and even usually do a postfijacion with tetroxide of osmium (OSO4), which has four active groups that form much more dense networks of biological material in the sample.
-Then have to take into account, as we have said the Court has to be much thinner (40-50 nm = 0.04 - 0.05 m), are referred to as ultrafine cuts. So it is obvious that we need a higher precision (ultramicrotome) microtome. In addition parts must be in a material harder that the paraffin to get such thick cuts. The most commonly used materials are synthetic resins poli-componente, requiring a processing similar to the paraffin, with the difference that these resins are liquid at room temperature and that polymerizes at certain temperatures.
-The ultramicrotome works essentially as the Minot Rotary microtome with the peculiarity that its mechanics is much more precise, allowing movements so small that they require to obtain ultrafine cuts. Another peculiarity of this type of microtomia lies in the nature of the blades, which have to be of a material extremely hard to cut blocks of resin. These blades are usually of a specially treated glass that allows obtaining a series of 30 or 40 cuts, after which the blade has to be replaced since he has lost the edge. On certain occasions, you can use blades with edge of diamond, which means as the student are unusual given its high price. Attaches the blade with a piece of plastic at the end of which forms a cavity which is full of water where float cuts, once made, and where they are captured with sample grids.
-Racks with attached cuts through the reagents required to perform contrast (uranium acetate and others), then left to dry and can already be observed to the MET. Unlike the processing with paraffin, this technique does not remove the mounting (resin) medium.

The ultramicrotome with thicker cuts can also be obtained (1-2 mm) to his studio in conventional optical microscopy (of bright field), these cuts are known as "semi-fine".

The electron microscope (scanning), also uses a beam of electrons and uses electro-magnetic coils such as lenses, but there the similarities end. 11 Histological techniques. How is and how works the SEM (scanning electron microscope) (diagram in sapnish)
This microscope is used for the study of surfaces, not to the study of the intimate components of tissues and cells.
So the electron beam does not pass through the sample, but rather a deflector coils made a sweep of the surface of the sample.

Processing of samples for the MEB
The sample for this type of microscopy, is not a fine cut but it is a sample full (or almost full), so with this technique can be studied small whole organisms, like for example, insects.
The preparation of the sample is also different to that is done to the M.E.T. For the M.E.B. There are cuts but the piece, (a portion of tissue, an insect, etc...) It is dehydrated and covered with a thin layer (monomolecular) of a conductive metal (usually gold).

Bombarding the sample with a beam of electrons (primary electrons) the metal layer reacts by emitting an electron for each electron that receives. These electronecast s (which are called secondary electrons) have the feature that are emitted at an angle that depends on the angle of incidence of the primary electron with respect to the surface of the sample.
The secondary electrons are collected by a collector of electrons divided into an array of cells, then a computer system is responsible for generating an image on a monitor, on the basis of: a point of light for each detected electron.
As the secondary electrons may indexed in the matrix in different numbers in each cell, due to the angle in which they are generated, the image shows light and dark, reflecting the three-dimensional surface of the sample, giving additional information of tissues to those that can be obtained with a conventional optical microscope or even a MET.

In this section we will give a brief review of other microscopes optical (which use light), used in the histological, although in certain circumstances.

Phase contrast microscope:
This type of microscope is based on the optical of a beam of light phase shift property to traverse an object composed of different in materialsHe says of refraction. Using this technique you can see materials unstained and is especially useful for the study of living material (cell cultures, etc...)

Dark-field device:
This device does not fall perpendicularly to the sample, but tangentially, so is refracted by the sample toward the goal, in areas in which there is no material is all black, hence the name of "dark field".

Fluorescence microscope:
This microscope uses ultraviolet light instead of white light. This light causes fluorescence in certain substances (fluoro-baseball cards) that are used as dyes. In this type of microscope, the background is black and chrome-fluoro-labeled structures emit its own light (which is which is).

Confocal microscope:
The confocal microscope is an optical microscope of special features and recent emergence.
This microscope benefits are unique, so for example, you can see thick specimens, from which the microscope obtains images not of all the thickness of the sample, but sections of small thickness (in the style of the computerized axial tomography) as luego, by computer programs, can rebuild in a three-dimensional structure that is displayed on a computer screen.
It also allows the study of live samples (cell culture) over time, which makes it ideal for the understanding of certain biological processes.
It is a microscope of recent onset, not by the optical complexity of their construction, which was already in operation in the 1950s, but by the complexity of hardware and computer software necessary.
The confocal microscope has several transmitters laser, which are the sources of light used.
Each one of those lasers is different wavelength and incident on the sample where excited to foto-cromos (which are "dyes") that respond, each at a given wavelength, allowing for multiple marks on the same sample, revealing different structures in different colors.

In the section "STAINING" commented the technique of hematoxylin-eosin, which is, without doubt, the most used in histology technique, but obviously there are many more staining techniques. In this sectionWe will do the review of other techniques, among many, that are also used in histology, albeit with much less frequently than the H and always to display specific features of tissue and/or cell types. 12 Histological techniques: Examples of staining techniques (diagram in spànish)

These techniques are intended to show the General characteristics, especially the topography of tissues and organs. The base of these techniques is a chemical (acid-base, redox) reaction between colours and the structural elements of the tissues.
These techniques are many and varied, that can be classified according to the number of colours, in: monocromicas, bicromicas and tricromicas.
In this section we include the technique of the (bicromica) h & E explained above.

Monocromicas techniques
These techniques use only dye tints like all tissues and differentiation is achieved thanks to the different nature of the tissues, thus an epithelium formed by a continuous layer of cells is dyed more intensely than the connective tissue where live fibres (which stained less) withsome cells.
These techniques include:
-Aniline blue
-Toluidine blue
-Hematoxylin Hedienhain
-Neutral Red
These techniques are often performed on cuts of between 5 and 10 m paraffin.

Tricromicas techniques
As its name indicates, these techniques use a combination of three colours. A characteristic of these techniques is that tend to stain the connective tissue of differential form, since one of the dyes tend to have affinity for fibers (collagen) of the extracellular matrix of this tissue.
These techniques are often used, as well as the hematoxylin-eosin, for the topographic study of bodies, with the added value of the ease of recognition of the connective tissue.
Tricromicas most commonly used techniques are:
-Masson trichrome
-Mallory trichrome
These techniques are often performed on cuts of between 5 and 10 m paraffin.

Included in this section those techniques that are based on a conventional chemical reaction between a dye and a specific element of cell types, extracellular fibres, etc...
Below are a few of these techniques that are commonly used in histology laboratories.

PAS-H technique
This technique is based on the combined use of the periodic acid along with the Schiff reagent.
This combination stained selectively mucopolysaccharides. These parts are in the basal plates of epithelia, mucous secretions, or the glycocalyx of microvilli, so these items are selectively stained rosa-rojo color.
Usually combined with hematoxylin, which stains nuclei blue-violet color and allows better locate items stained with PAS.
This technique is usually performed on cuts of between 5 and 10 m paraffin.

Alcian blue - PAS technique
This technique combines the reaction of the PAS with the alcian blue. The alcian blue stains specifically mucopolysaccharides of acidic in contrast of the PAS that stains mucopolysaccharides of neutral character.
Why this technique is preferably used in the study of mucous secretions of the digestive tract mucous secretions neutral (rosa-rojo) to differentiate from acidic mucous secretions (blue).
This technique is usually performed on cuts of between 5 and 10 m paraffin.

Technique of Orcein Picro-Indigo-Carmine
This technique is especially recommended for the study of the cardiovascular system, since it dyed stiff elastic fibres (e.g. of elastic arteries or the handset endocardium), while tints of blue-green pale Collagen fibers (for example those of the adventitious arterial or venous or of the heart epicardium).
These technique is usually performed on cuts of between 5 and 10 m paraffin.

Technique of Gordon-Sweets
It is technique is based on the use of silver salts, which in combination with other reagents used in this technique selectively stained black the reticular fibers of the connective tissue.
This technique is usually performed on cuts of between 5 and 10 m paraffin.

Sudan IV technique
Sudan IV is a fat-soluble dye, so it is very suitable for dyeing fatty elements, such as adipocytes.
To perform this technique, during the processing of tissues should not be used any solvent orgonly, because that would dissolve fatty elements that are intended to dye; This makes it impossible to include samples in paraffin, so samples are usually cut by freezing in a thickness of approximately 30 m made with the cryostat.

These types of staining are not based on basic chemical reactions but more specific reactions such as the enzymatic or immunological activity.
The kinds of reactions that are leveraged as these techniques are varied.
Thus we can find reactions of high specificity between molecules such as the technique of tomato lectin.

Technique of tomato Lectin
Lectins are proteins that bind selectively to certain sugars, tomato lectin is combined with N-acetylglucosamine, that in the liver, for example, found in the macrophage (Kupffer cells) and endothelial walls of vessels (sinusoids).
The tomato lectin combined with a marker molecule (such as biotin) is used in histology. Biotin can then be put of manifest in a process of developing. The process is simple: place thelectin marked over the cut, expected one sufficient time so that the lectin is a sugar and then reveals that you see under a microscope.
This technique can be, both on cuts of about 30 m, made by freezing in the cryostat, both cuts of 5 to 10 m in paraffin.

The Histochemical (Histoenzimaticas) techniques based on enzymatic reactions of molecules (enzymes) present in the tissues of the sample.
General mechanics is based on placing a substrate adapted to the enzyme to study over histological cut, so that the enzyme reaction and later to detect any of the products of that reaction.
An example of this type of techniques is the NDPasa techniques.

The NDPasa technique
The NDPasa is an enzyme which is, among others in the Central nervous system microglia cells and the wall of the blood vessels.
To reveal structures that contain this enzyme in histological sections, what you do is place a product this enzyme degrades, in this case Inositol-phosphate on the Court. Stops time so enzi reactionMatica whose result is the formation of precipitate in the place where the reaction takes place. Marking through a treatment of the precipitate with sulfide and silver salts, is subsequently amplified which provides you with a very dark brown. A contrast with toluidine blue is done to locate the unmarked cells.
Marked cells (microglia) appear under the microscope of a dark brown colour, as well as blood vessels, while other cell types are tinged blue.
This technique is usually performed on cuts of around of 30 m, made by freezing in the cryostat.

Immuno-Histochemical techniques are based on the natural phenomenon of the specific recognition of a molecule (Antigen) by another (antibody), this is known as immune reaction. If we use an antibody marked the result is that the mark will be there where the Antigen (given the specificity of the Antigen-antibody reaction).
Now the bioindustry offers lots of marked antibodies against a wide variety of antigens.

Technique of the GFAP
The GFAP is a protein that isfound exclusively in astrocytes and cells of the same lineage.
In this technical immuno-Histochemical what is done is to place over the cut a solution of the labeled antibody to antibody locate and selectively attach to the GFAP. Later it is revealed, so get that only appear colored cells containing such protein, is this case the astrocytes.
This technique is usually performed on cuts of around of 30 m, made by freezing in the cryostat.

Neurons (along with the glial cells), are cells of the nervous system, and as the student is known, have a body or cell soma of the starting, both dendrites and axon. This peculiarity makes the nervous tissue to present a special structure in which intermingle dendrites and axons (nerve parenchyma / neuropil) and in which are located the neuronal somas.
This peculiar feature along with the interest that the study of the nervous system has led to the development of many of this tissue staining techniques. In this section, we discuss some of the technical mused as.

Technique of Nissl
This technique is commonly used in the study of the nervous tissue rather than the hematoxylin-eosin. That is a topographic technique that shows the distribution of the neuronal somas in the neuropil.
The dye used in this technique is the toluidine blue, that applies after pre-processing in potassium dichromate. Violet Cresilo used as dye in some variants.
Neuronal somas are stained dark blue while the parenchyma appears almost white. Glial cells (their cell bodies) are also stained in dark blue and can be to distinguish the different types by its shape and location. Cuts the sufficiently fine (up 5-7 m of thickness), to large increases (400-1,000 x) are seen in the cytoplasm of the neuronal body (soma) some accumulations that are known as Nissl bodies, which correspond to stacks of cisterns of rough endoplasmic reticulum.
This technique is usually performed on cuts of between 5 and 10 m in paraffin.

Technique of Klüver - Barrera
The Klüver-Barrera technique uses apara the estuHe gave topographic nervous system of both the somas neural of fibres (axons) mielinicos packages.
In this technique, in addition to the blue of Toluidine (for staining of the neuronal somas), used the Luxol fast blue, which selectively stains the myelin surrounding certain axons (mielinicos axons).
This technique is usually performed on cuts of between 5 and 10 m in paraffin.

Technique of reduced silver of Cajal
This technique is used primarily for the study of axons packages (not myelinated) amielinicos.
This technique is based on an impregnation silver of nervous tissue and subsequent chemical reduction. This process dyed dark brown neurofilament and neurotubulos, so it highlights axons amielinicos (in the mielinicos silver nitrate not can pass through the myelin sheath), dendrites and cell body, not the kernel (which no stains).
This stain is done in block, i.e. stains the entire brain (or a portion), and once stained, tissue includes paraffin, cut (at a thickness of 10 m), and is mounted on the slide.

TechniqueGolgi apparatus
The Golgi technique is perhaps the most paradigmatic of the staining techniques in the study of the nervous system.
This technique aims to be able to study full neurons, including soma, dendrites, and axon. As the student is known, the neurons are cells with extensions (dendrites and axon) forested dealing with a large volume of tissue, a polyhedron that includes a "type" neuron can have 100-200 m x m-100-200 x 100-1000 m edges.
This involves various circumstances to be taken into account:
-Firstly it is obvious that complete neurons in a cut of 5, 10 or 30 m of thickness may not be seen, and will need to be thicker cuts (100-150 m).
-Secondly, should only stain a small proportion of neurons, since if they tiñeran all, staining all their elements, some neurons of others could not be distinguished.
Both circumstances are given in the technique of Golgi, where nervous tissue with a solution of silver nitrate, is impregnated into the block after an induration with potassium dichromate. This process manages to impregnate one very smallENA proportion of total of neurons (approximately 0.03%). Today, is still under discussion the mechanism by which some neurons are stained and others do not.
The Golgi technique is done in block, i.e. with the complete brain (or a portion of it). The cuts have to be thick (100-150 m), the piece is not included in paraffin or cut with a rotary microtome. The piece impregnated with the Golgi technique may include in celloidin (purified Collodion), or simply snap into place gelatine or even paraffin. For cutting are often used other less sophisticated devices, such as a vibratomo, a sliding microtome or even a simple Barber on a hand microtome blade.
As a result of this technique, a microscope so observed complete neurons that can follow the winding course of its dendrites, using the movement of the deck and the focus knob control. As the silver nitrate can not cross the myelin sheath, will be only the axons not myelinated.
You can also view cell glial and blood vessels.

As it has aimor in the preceding paragraphs, in histology, as well as the explained, using other instruments, protocols and procedures, depending on the outcome that is intended to obtain.
So for example a variety of apparatus is used to cut: 13 Histological techniques: types of microtome

In addition to Rotary microtome or Minot for paraffin cutting, as already explained, in routine practice for the development of specific techniques in histology.

In essence this instrument continues the mechanical model of the Rotary microtome, but with a considerably greater precision, to get much more fine cuts (40-50 nm) to be used in transmission electron microscopy.
Another type of harder blades, made of specially treated as even with the edge of diamond glass are used in this microtome.

The cryostat is, in essence, a rotary microtome inside a refrigerated food locker. Obviously used to cut samples at low temperatures, for techniques that require the maintenance of biological activity in samples, for example, the technical histoenzimaticas and in the Immunohistochemistry.
Frozen samplesan, after treatment with a cryoprotectant, and cut, riding the cuts on slide, which is usually done the treatment Histochemistry and Immunohistochemistry.

Vibratomo, as its name suggests, is based on a resonant arm that is not too thin cuts (between 25 and 500 m). Typically used for technical histoenzimaticas and immunohistochemical since it is not necessary to include paraffin parts, although currently the cryostat is used more for these techniques.
Occasionally, the vibratomo has been used to make cuts with the Golgi technique.

Hand microtome
It is the simplest of the microtomes and simply is a worm that raises the sample on a surface flat, on which slides a sharp trimmer blade for cuts.
HIS court rank ranges from 20 to 150 m and is widely used in plant histology. Animal histology is used for thicker cuts in the Golgi technique.

As the student has seen the technique is complex and involves many steps, so it can happen that in any of these steps areproduce distortion or small bug, which then found during the study of the sample under a microscope, is what is known as artifacts of staining. 14 Histological techniques. Staining artifacts
In themselves the artifacts have no histological value, but you can get to the found was them during the study of preparations the student to confuse them with elements of the fabric which makes it convenient to the student aware of its existence.
Most appliances produce microscopic alterations that are completely invisible to the naked eye so it is very difficult the histologist can avoid them.
In the following pages the student will find different examples of the most common artifacts that can be found in the study of histological preparations.

The banded appearance that show some preparations is typically caused by an incorrect angle of incidence of the blade.
Other sometimes occurs a banded to try to cut too fine a fabric that does not have the hardness and/or sufficient consistency.

Some preparations are small bubbles of air trapped in the midst of mounting.
This appliance is usually given when the medium ofmounting is very dense and solidifies quickly.

Sometimes, during the process of preparation of samples, preferably during the final Assembly, "slip" about cutting specks of dust, small hairs and other elements of the environment are set out in the preparation to use the mounting medium.
These inclusions can also be precipitated due to small dye crystals color.

The nicks are produced by small imperfections from the edge of the blade when cutting on the microtome.
These nicks cause drag and destruction of a straight band of tissue whose width corresponds to the width of the imperfection of the blade edge and occupying all of the tissue in the direction of the Court.

The folds are often occur during the cutter on the slide Assembly.
Sometimes it's a simple ripple of tissue, sometimes it a great fold that fold onto itself.

We are convinced that reading this topic will help the student to understand the histological processing that is required to get the cuts that will study the microscope and this redundARA in a better understanding of the tissue cells and structures observed during the study.



This interactive diagram shows, step by step, how to make paraffin blocks, to subsequently obtain sections using the microtome.



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