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WARNING: ALL THE MATERIALS (i.e.

, TEXT DOCUMENTS, DIGITAL


SLIDES, PHOTOMICROGRAPHS, PICTURES, VIDEOS, ETC.) THAT YOU
WILL USE IN THIS LABORATORY COURSE IN HISTOLOGY ARE
COPYRIGHTED. YOU ARE NOT ALLOWED TO DOWNLOAD, UPLOAD,
SHARE, COPY, OR EDIT ANY PART OF ANY MATERIAL, UNLESS
SPECIFICALLY GIVEN PERMISSION TO DO SO. YOU ARE HOWEVER
ALLOWED TO TAKE PHOTOGRAPHS OF THE MATERIALS AS THEY
APPEAR ON THE MONITOR, BUT ONLY FOR YOUR PERSONAL USE. YOU
CANNOT POST THE PHOTOGRAPHS IN ANY SITE ON THE INTERNET,
SEND THEM TO ANYONE BY E-MAIL OR ANY ELECTRONIC MEANS, OR
MASS PRODUCE THEM.
THIS LABORATORY IS MONITORED BY A CLASSROOM MANAGEMENT
PROGRAM THAT CAN DETECT VIOLATORS OF THE ABOVE RULE.
VIOLATORS WILL BE SUBJECTED TO DISCIPLINARY ACTION.

ORIENTATION INTO THE LABORATORY


COURSE IN HISTOLOGY
As you know by now, this course in MEDICAL HISTOLOGY that is being offered by the De
La Salle College of Medicine consists of a theoretical section, which refers to the
lectures that are delivered to the class in the lecture hall, and a practical section, which
refers to the laboratory sessionsthe subject of this orientation session. The two sections
of this histology course are not separate or independent of each other. The laboratory
sessions have been designed to complement your understanding of the concepts you
encountered in the lectures and your readings by giving you the opportunity to see and
identify microscopic structures.
Up to a few years ago, the laboratory sessions in Medical Histology in this medical school,
as in most other medical schools, consisted mainly of microscopy sessions. During these
laboratory sessions, the student is issued a light microscope (LM) and several laboratory
slides (glass slides) and, with the aid of a printed manual, textbook and atlases, he/she
looks for and identifies structures that are to be found in the histologic specimens (tissue
specimens; histologic sections; tissue sections) that are mounted on the glass slides.
When necessary, the student seeks the help of a faculty member who is ever present in the
lab to verify and confirm his/her findings. Seeing and verifying or confirming help the
student better understand and remember concepts that were expounded in the lectures.
Starting school year 2013-14 however, the laboratory sessions in histology have been held
in a digital or virtual setting. This simply means we (i.e., the Department of Anatomy)
have replaced the microscopes with computers. Instead of viewing histologic specimens
under the light microscope, you will view them on the computer monitor. Consequently, in
the laboratory sessions, you will no longer be required to search for structures with the use
of a light microscope (LM). Rather, you will recreate the experience of using a microscope
with the use of softwares, principally Aperio ImageScope, a freeware that is rather user-
friendly. In lieu of glass slides, you will study digital slides, which, like glass slides,
contain histologic specimens (tissue specimens; histologic sections; tissue sections).
Unlike glass slides, however, the digital slides that you will be using are not contained in a
slide box, but in a computer folder labeled HISTOLOGY SLIDES.

During the laboratory sessions, by using Aperio Imagescope, you will be able to move
around the histologic specimens on the computer screen and, under different
magnifications, identify microscopic structures. ImageScope offers a wider range of
magnifications than the light microscope where magnification is limited to only few
settings, i.e., 40x, 100x, 400x and1000x. When you study any of the digital slides, always
initially view the slide under low magnification.

In addition to the digital slides, you will likewise occasionally study photomicrographs
(i.e., photographs), which are static images (i.e., images that cannot be moved around) of
a single light microscopic field taken at a particular magnificationmostly 40x, 100x,
400x or 1000x.

The digital slides that you will study have been purchased. The photomicrographs, on the
other hand, have been acquired through a variety of means. Some have been taken by the
staff of the Department of Anatomy under the light microscope using the glass slides that
have been issued in previous years to the medical students of this institution. Some have
been purchased, but quite a number have been downloaded or will be directly accessed via
the INTERNET from websites all over the world. Whenever a downloaded material from a
website is used, it will be properly acknowledged and cited.

Aside from digital slides and photomicrographs, electron micrographs (i.e., images of
specimens taken with the electron microscope), videos and other audio visual
materials will be utilized in the laboratory sessions. As with the photomicrographs,
whenever a downloaded learning material from a website is used, it will be properly
acknowledged and cited.

By going virtual, we have effectively shortened the time it will take you, the students, to
look for, identify and study histologic structures because scanning glass slides is decidedly
more tedious and time-consuming than panning (i.e., scanning) digital slides. Doing away
with the microscope also stresses the fact that this laboratory section of the histology
course is not meant to teach you how to use a microscopea skill which we presume you
already acquired during your undergraduate years. We hope that the time we have
redeemed for you by going virtual will be devoted to further reading and more in-depth
study of the particular topic that is under consideration.

Incidentally, we also assume that you are well-versed with regard to surfing the WEB and
that you have considerable experience in using common computer softwares (e.g.,
microsoft word, microsoft power point, etc.)

VENUE AND CONDUCT OF HISTOLOGY LABORATORY SESSIONS

All the histology laboratory sessions and examinations will be held in this Health
Informatics Laboratory, which is located on the 2nd floor of the College of Medicine.
Inasmuch as you will be logging in to a computer that has access to the INTERNET every
time you attend a laboratory session in Histology, you can utilize the WEB in answering
some of your queries or in clarifying certain matters that may be unclear to you regarding
the subject matter under study. Access to the INTERNET notwithstanding, you will, many
times, find it necessary to consult your textbook and an atlas. Hence, always bring your
textbook and an atlas to the histology lab.

The Laboratory course consists of 19 modules. These modules are intended for
independent work among the students. Thus, during the laboratory sessions, your
interactions with your classmates should be minimal. If you have any queries direct
them to a preceptor (faculty member). In fact, you are encouraged to interact with the
faculty members on items that will help you understand subject matters that you may find
unclear. But the faculty members expect your inquiries to be sensible and valid, hence,
make sure you have read enough about a particular topic before you ask for help on it.

APERIO IMAGESCOPE

All the modules in this laboratory course will require you to view and study digital slides
using Aperio ImageScope, a user friendly, high-performance software created by
ScanScope. This software has numerous features, including the following:

Instantly pan and zoom to any region and magnification of a multi-gigabyte


digital slide.
View multiple digital slides concurrently in synchronized windows.
Apply image enhancements, in real-time, for contrast, brightness and gamma.
Integrate with Aperio software for remote viewing, authoring annotations, digital
slide conferencing, and image analysis.
Use the Image Quality (IQ) feature to optimize the appearance of your digital slides
based on their staining.
Use the Annotations summary view window to perform quick and easy analysis of
IHC digital slides.
Record and replay tracks showing your movement through a digital slide.
In so far as this histology course is concerned, you need to learn just the first two features
listed above. Accordingly, the tutorial below is designed to let you take advantage of these
two features only. But as you go through the course, you might find it advantageous to
learn the other features of the software. You can do so by tinkering with the software on
your own or with the help of the user manual for Imagescope, which you can access via
the website below. You do not have time this session to visit the website. Simply copy the
websites address and access it when you have the time.

http://media.rrc.uic.edu/RRC/RHTIC/WikiAperioTraining/Scanning/MAN-0001-Rev-J.pdf

APERIO IMAGESCOPE TUTORIAL

You will now go through a tutorial to acquaint you with the features of Aperio
ImageScope that you need to learn. To be able to follow this tutorial, you need to split your
monitor into two so that you can use ImageScope while reading this tutorial at the same
time. If you do not know how to split the monitor, the steps below will enable you, not only
to split the monitor, but to start ImageScope as well.

Before you proceed, close all windows and programs that are open except for this one.

SPLITTING THE MONITOR AND STARTING IMAGESCOPE

Press and hold down the key on the keyboard with the windows icon, then press the
right arrow key. You will note that this window now occupies just the right half of the
monitor. Release both keys (i.e., windows icon and right arrow) if you havent done so yet.

Now, you will run Imagescope and make its window occupy the left half of the monitor.
This way, you will be able to study digital slides using ImageScope while utilizing this manual as a
guide.

Start or run ImageScope by double clicking the ImageScope icon on your desktop. After
ImageScope has loaded, press and hold down the key on the keyboard with the
windows icon, then press the left arrow key.

You have successfully split the monitor and started Imagescope. The left half of the monitor
should now be occupied by the Imagescope window and the right half by the window for
this manual.

OPENING DIGITAL SLIDES

For this tutorial, you will open or load two (2) digital slides using ImageScope. The steps
below will guide you in doing this:

1. On the ImageScope Menu (i.e., File, Edit, Image, View, Tools, Windows and Help)
that is located on the top portion of the ImageScope main window, click File, then
on the File menu click Open Image and the Open image window appears. On the
box to the right of the File name box, choose All Image Files.
2. Navigate to the location of the folder labeled HISTOLOGY SLIDES (the folder is on
your desktop) and double click the folder. Once the content of the folder are
shown, double click the folder labeled Module 0. Orientation slides. The folder
contains two (2) digital slides labeled Kidney and Skin, sole of foot.
3. Double click the digital slide labeled Kidney and the slide loads or opens. If an
aperio ad pops-up, simply close the ad.
4. Repeat step 1 above. Then, double click the digital slide labeled Skin, sole of foot
and the slide opens or loads.

VIEWING DIGITAL SLIDES

Your Imagescope window should look similar to the picture above. In particular, take note of
the following items in the picture above: Filmstrip, Zoom slider, Thumbnail window, and
Magnifier window. Now, check the ImageScope you have run, do you see the four items?

If Zoom slider, Thumbnail window or Magnifier window is/are missing in your


ImageScope main window, find the icon of the missing item/s on the Toolbar (see above
picture for location of toolbar). By placing the pointer over an icon that is displayed on the
toolbar, a text box that tells you what the icon is for will be displayed. When you find the
correct icon, click it and the tool appears on the main window.
You can show/hide any of the three items (i.e., Zoom slider, Thumbnail window and
Magnifier window) by clicking the icon of the item on the Toolbar. Practice hiding and
showing the three items.

If the Filmstrip is missing in your ImageScope main window, click View in the
ImageScope menu. Then, click Filmstrip (the 7th entry in the View Menu) and the item
will appear in the main window. You can hide the filmstrip by clicking Filmstrip again.

Incidentally, many of the ImageScope commands and features are available on the
Toolbar, but you can also access these features through the ImageScope menus. Verify
this by clicking View in the Imagescope menus. You will note that a number of
features/items that you can turn on and off (by clicking the item) have been displayed.

Below is a brief description of what the four tools mentioned in the paragraphs above can
do:

Filmstrip The filmstrip shows what slides are open. You can move between digital
slides by clicking the slides image in the filmstrip. Try moving from one slide to another
(i.e., kidney and skin) by clicking the two images that are displayed in the filmstrip, one
after the other.
Thumbnail window The histologic specimen (tissue specimen; histologic
section; tissue section) that is contained in digital slides is large and often you see
only a portion in the ImageScope main window. The thumbnail is a view of the complete
histologic specimen.
Zoom slider By dragging the slider up and down, you can magnify or shrink the
current view. Drag the slider to the highest magnification possible. Now look at the
thumbnail window, you will see a small rectangle /square which shows you the area of
the slide that is currently magnified. You can pan or scan the slide by dragging the slide
with the hand pointer in the main window, or by dragging the rectangle/square in the
thumbnail window. Practice panning or scanning the slide.
Magnifier window Move this tool by dragging it to the area you are interested to see in
magnified view; or, you can position the hand pointer on the area of the slide you want to
magnify and a magnified view of the area appears on the magnifier window. Practice
magnifying different parts of the specimen.

Familiarize yourself with the four features of ImageScope described above by panning or
scanning the two digital slides, one-after the other, using different magnifications.

Note: Although the digital slides you have opened contain sections of the kidney and the skin,
respectively, you are not expected to identify any structure that is present in either organ during
this session. Simply utilize the slides to acquaint yourself with the features of Imagescope.

If you still have time, explore the other icons in the Toolbar. You may also want to briefly
explore the ImageScope menus by clicking the headings (i.e., File, Edit, Image, View, Tools,
Windows and Help).
INTRODUCTION INTO THE
LABORATORY COURSE IN HISTOLOGY

COMPETENCIES

At the end of this module on Introduction to the Laboratory Course in Histology, the
student can:

1. Discuss the steps involved in the preparation of tissue or histologic sections for
study under the light microscope.
2. Describe how the most common dye combination, i.e., hematoxylin and eosin (H&E),
stains various components of cells and tissues.
3. Distinguish between the light microscope (LM), scanning electron microscope
(SEM) and transmission electron microscope (TEM) as to their operating principle,
resolving power and usefulness in the study of histology.
4. Demonstrate proficiency in using AperioImagescope.

TOOLS OF HISTOLOGY

The field of histology encompasses the study of the microscopic structure of organs, tissues
and cells (cytology). A good grasp of the microscopic structure of the body is a prerequisite
to understanding how the different cells, organs and tissues function in health and in
disease.

For basic histology the most commonly used tool is the compound light microscope, or
simply, light microscope (LM). However, the electron microscope (EM) is often a useful
adjunct because knowledge of the ultrastructure of cells and tissues is oftentimes
necessary for a more thorough understanding of their architecture.

RESOLVING POWER OF LIGHT AND ELECTRON MICROSCOPES

The resolving power of a microscope is defined as the shortest distance at which two
points can be seen as separate. Magnifying the image that was taken by a microscope
beyond the microscopes resolving power results in empty magnification. The image
becomes bigger, but no additional details shall be revealed. A blur simply becomes a bigger
blur.

The light microscope (LM), has a resolving power of 0.2 m (i.e., 2 millionth of a meter), at
best; while the most common electron microscopes, the transmission electron
microscope (EM; TEM) and scanning electron microscope (SEM) can resolve objects
that are 0.2 nm (i.e., two billionth of a meter), and 10nm, apart, respectively.
LIGHT MICROSCOPE

The light microscope (LM) is otherwise called a compound light microscope because it uses
not only one, but several lenses that are specifically arranged to magnify an image. The
figure below shows a typical monocular LM with its parts labeled.

In light microscopy, a light beam is focused and made to pass through the specimen to be
studied that is placed on the stage. The light passing through the specimen is then made to
enter an objective lens. Present day microscopes usually have 3 or 4 objective lenses (the
microscope on the right has 3) that are on a movable turret located immediately above the
specimen to be studied. The objective lenses are labeled 4x, 10x, 40x and 100x. They
magnify the image 4, 10, 40 and 100 times, respectively. The 4x objective lens is often
referred to as scanning objective, the 10x low power objective (LPO), the 40x high power
objective (HPO) and the 100x objective oil immersion objective (OIO). The image from the
objective lens is further magnified by an ocular lens or eyepiece. The LM is designed to
have only one ocular lens at a time, usually a 10x lens (if the microscope is binocular, the
two ocular lenses have the same magnification power, e.g., both are 10x). If an ocular
lens/es with a different magnification has to be used, say 5x or 12.5x, the 10x lens/es
has/have to be pulled out from the body tube and replaced.

The magnification (linear magnification) attained with the LM is the product of the
magnification of the eyepiece and the objective that is in use. For example, the LPO, which
has a magnification of 10x, when used with a 10x eyepiece, gives a linear magnification of
100x. With the same eyepiece, the HPO which magnifies the specimen 40x and OIO which
magnifies the specimen 100x shall give magnifications of 400x and 1000x, respectively.

In the LM, the lower the power of the objective used, the wider is the view one gets of the
specimen. The low power objective (LPO) gives a slightly more than 2 mm diameter view
of the specimen, while the high power objective (HPO) 0.4 mm diameter and the oil
immersion objective (OIO), 0.2 mm diameter. The HPO and OIO are generally used only
when studying very minute parts of a specimen like individual cells or parts of cells.

The specimens or sections that are studied under the LM, and much more so with the
TEM microscope, are very thin slices of the cell, tissue or organ under consideration. They
are thin enough to allow the passage of light or a beam of electrons. Because of their
thinness, these specimens are virtually two-dimensional structures. Hence, the student
must develop the ability to make three dimensional interpretations of what he/she sees
in LM glass or digital slide or electron micrographs. This may be difficult at first, but it must
be learned.

The figure below should help you orient yourself on how sections of a three-dimensional
structure such as an irregularly-shaped tube (on the left) and an egg (on the right) would
look like in two-dimensional slides.
PREPARATION OF TISSUE SPECIMEN FOR LIGHT MICROSCOPY

The preparation of a histologic specimen (tissue specimen; histologic section; tissue


section) for light microscopy entails the processing of a small tissue block of an anatomical
part of the body so that it becomes firm enough to allow very thin (usually less than 10m)
slices to be made. These thin slices are then stained. Staining is necessary to provide the
contrast needed to distinguish the different parts of a specimen because biological
specimens are inherently of low contrast; and, to allow easier identification of parts due to
their respective staining properties.

The most commonly used staining procedure in histology involves the use of hematoxylin
and eosin (H & E), both of which are water-soluble dyes. Hematoxylin is a blue basic dye
whereas eosin is a red acid dye. Hematoxylin generally binds with acidic structures and
eosin generally binds with basic structures in a specimen. Parts of the specimen that take
up hematoxylin are colored blue and are referred to as basophilic. The parts that take up
eosin are colored various shades of red and are referred to as acidophilic or eosinophilic.
Examples of basophilic material in a cell are ribosomes, which contain ribonucleic acid
(RNA) and nuclei, which contain both deoxyribonucleic acid (DNA) and RNA. Some cell
organelles like mitochondria, and granules in the cytoplasm of some cells such as those
found in the Paneth cells of the intestines, acidophils of the adenohypophysis and the
eosinophils of blood take up eosin avidly.

The image below shows a high power view (400x) of a section of the liver stained with
H&E, note the contrast that the stains impart on the different parts of the tissue. The blue
structures are structures that have taken up hematoxylin while the pinkish ones have taken
up eosin.

Liver, H & E, 40x


Many other types of stains are used to distinguish the various parts of cells and tissues. The
images below, showing sections of the liver, illustrate three of these stains and their
specific purposes:
STEPS IN THE PREPARATION OF HISTOLOGIC SPECIMENS USING H&E STAINS

1. Obtaining the specimen - With a sharp knife, the specimen is obtained as close as
possible to the time of death or the time of removal of the organ from the body. It
must be small enough to ensure that the fixative shall be able to adequately
penetrate the interior. This means that the actual tissue will only be a few
millimeters long in any one direction. The orientation of the specimen in relation to
the entire organ is also considered. It must contain parts that will enable the
microscopist to orient himself/herself properly and make an intelligent
interpretation of the architecture of the organ.

2. Fixation - This process makes use of protein coagulants or cross-linking agents to


allow preservation, as close to life as possible, of the structures in the specimen.
However, it must be kept in mind that tissue shrinkage may occur as a result of
fixation. One of the most commonly used fixatives is 10% formaldehyde. The fixative
not only arrests chemical process, it also has antimicrobial properties.

3. Dehydration - Removal of the water content of the specimen is accomplished by


running the tissue sample through a series of increasing concentrations of an
organic solvent like ethanol. Removal of the water is needed in preparation for the
impregnation of the tissue sample by paraffin, a material that is not miscible with
water.

4. Clearing - Xylene is generally used for this purpose. It is miscible with alcohol which
it replaces in the specimen. After passing through xylene, the tissue is usually
translucent. Xylene is miscible with paraffin, the embedding medium.

5. Embedding - The tissue sample is placed in melted paraffin for a period of time to
allow impregnation of the interior by this substance. Then, it is placed in a small
plastic, metal, or cardboard box where melted paraffin is then poured.

6. Sectioning - The paraffin is allowed to set, then is trimmed and placed on the
microtome for sectioning. The sectioning of the specimen must be oriented
according to the needs of the histologist i.e., cross-section, longitudinal section,
oblique section, etc. The thin sections include both a part of the specimen and a part
of the paraffin. These are floated on a water bath set at just below the melting point
of the paraffin being used. At this temperature, the paraffin (and the slice of tissue)
should flatten out and have no wrinkles.

7. Mounting - Egg albumin is wiped on the surface of the glass slide on which the
specimen is going to be mounted. It is then air-dried. This glass slide is placed below
the slice of paraffin with the tissue and then slowly raised. At the same time, the
section is positioned properly on the glass slide with the aid of a dissecting needle.
To facilitate drying and to make sure that the specimen remains flat on the slide, it is
placed on a slide warmer.
8. Deparaffinization and Staining - Hematoxylin and eosin are water-soluble dyes,
therefore the tissue section impregnated with paraffin will not, as is, take up these
stains. The paraffin has to be removed by passing it through xylol. Since xylol is not
miscible with water, it is removed by passing it through decreasing concentrations
of alcohol. This will also hydrate the specimen. Only after this can the specimen be
stained with H & E and then covered with a cover slip.

At this point, reinforce your understanding of the process of tissue preparation


described above by watching the video from the link below. Note that the steps in the
preparation of histologic specimens that are described in the video differ slightly from the
steps enumerated above. Ctrl + click the link to launch the video. (After watching the
video, close its window and return to this document.)

http://www.youtube.com/watch?v=TLm37BbR1mo

ELECTRON MICROSCOPE

In electron microscopes, in contrast to light microscopes where visible light is focused on


a specimen, electromagnets focus a beam of electrons on a specimen. An electron
microscope has greater resolving power than a light optical microscope because electrons
have much shorter wavelengths (about 100,000 times shorter) than visible light. Present
day electron microscopes can magnify biological structures by as much as 150,000 times
good enough to visualize macromolecules.

The two common types of electron microscopes are the transmission electron
microscope (TEM) and scanning electron microscope (SEM).

In TEM, preparation of the specimen involves the same basic steps as in light microscopy,
but the tissue blocks are much thinner. Unlike the LM, the image produced by the TEM is
not viewed directly with the eye. Instead, the image is captured by an electron sensitive
film producing a negative from which a black and white photograph (positive) can be
printed. The black and white photograph is otherwise called electron micrograph.

SEM, on the other hand, is used to give a highly magnified view of the surface of a solid
specimen. In contrast to LM and TEM which shows a 2-dimensional view of an object, SEM
affords a 3-dimensional view of a structure or cell that is under study. In SEM, a beam of
electrons scans the surface of an object that has been previously coated with a heavy metal
such as gold or palladium. The electrons that are reflected and ejected from the heavy
metal coat are captured by electron detectors that collate and interpret the behavior of the
electrons to create a 3-dimensional image of the object being studied that is then displayed
on a monitor that can be photographed or stored digitally.
Below are diagrams and photographs of a Transmission Electron Microscope ( upper two )
and a Scanning Electron Microscope (lower two).

fei.com

binoc
ulas.
net
The images below show how a lymphocyte, a type of white blood cell, looks like under
the LM (upper left, at tip of arrow), TEM (upper right) and SEM (bottom).

End of Module

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