Introduction to Cell Biology

Preparation of Cells and Tissues for Microscopic Examination

The most common procedure used in the study of tissues is the preparation
of histological sections that can be studied with the aid of the light
microscope. Under the light microscope, tissues are examined via a light
beam that is transmitted through the tissue. Because tissues and organs are
usually too thick for light to pass through them, they must be sectioned to
obtain thin, translucent sections. However, living cells, very thin layers of
tissues, or transparent membranes of living animals (eg, the mesentery, the
tail of a tadpole, the wall of a hamster's cheek pouch) can be observed
directly in the microscope without first being sectioned. It is then possible to
study these structures for long periods and under varying physiological or
experimental conditions. In most cases, however, tissues must be sliced into
thin sections and attached on glass slides before they can be examined.
These sections are precisely cut from tissues previously prepared for
sectioning using fine cutting instruments called microtomes.

The ideal microscope tissue preparation should be preserved so that the

tissue on the slide has the same structure and molecular composition as it
had in the body. This is sometimes possible but—as a practical matter—
seldom feasible, and artifacts, distortions, and loss of components due to the
preparation process are almost always present.To prepare tissue for
examination with the microscope it should be processed as follow :

Fixation

If a permanent section is desired, tissues must be fixed. To avoid tissue

digestion by enzymes present within the cells (autolysis) or by bacteria and
to preserve the structure and molecular composition, pieces of organs should
be treated as soon as possible after removal from the animal's body. This
treatment—fixation—can be done by chemical. In chemical fixation, the
tissues are usually immersed in solutions of stabilizing agents called
fixatives. Because the fixative needs some time to fully diffuse into the
tissues, the tissues are usually cut into small fragments before fixation to
facilitate the penetration of the fixative and to guarantee preservation of the
tissue. One of the best fixatives for routine light microscopy is a buffered
isotonic solution of 4% formaldehyde.

In view of the high resolution afforded by the electron microscope, greater

care in fixation is necessary to preserve ultrastructural detail. Toward that
end, a double fixation procedure, using a buffered glutaraldehyde solution
followed by a second fixation in buffered osmium tetroxide, has become a
standard procedure in preparations for ultrastructural studies. The effect of
osmium tetroxide is to preserve and stain lipids and proteins.

Embedding

Tissues are usually embedded in a solid medium to facilitate sectioning.

To obtain thin sections with the microtome, tissues must be infiltrated after
fixation with embedding substances that impart a rigid consistency to the
tissue. Embedding materials include paraffin and plastic resins. Paraffin is
used routinely for light microscopy; resins are used for both light and
electron microscopy.

The process of paraffin embedding, or tissue impregnation, is ordinarily

preceded by two main steps: dehydration and clearing. The water is first
extracted from the fragments to be embedded by bathing them successively
in a graded series of mixtures of ethanol and water (usually from 70% to
100% ethanol). The ethanol is then replaced with a solvent miscible with the
embedding medium. In paraffin embedding, the solvent used is usually
xylene. As the tissues are infiltrated with the solvent, they generally become
transparent (clearing). Once the tissue is impregnated with the solvent, it is
placed in melted paraffin in the oven, typically at 58–60°C. The heat causes
the solvent to evaporate, and the spaces within the tissues become filled with
paraffin. The tissue together with its impregnating paraffin hardens after
being taken out of the oven. Tissues to be embedded with plastic resin are
also dehydrated in ethanol and—depending on the kind of resin used—
subsequently infiltrated with plastic solvents. The ethanol or the solvents are
later replaced by plastic solutions that are hardened by means of cross-
linking polymerizers. Plastic embedding prevents the shrinking caused by
the high temperatures needed for paraffin embedding and gives much better
results.

Sectioning:
The hard blocks containing the tissues are then taken to a microtome (Figure
1–1) and are sectioned by the microtome's steel or glass blade to a thickness
of 1–10 µm. Remember that 1 micrometer (1 µm) = 0.001 mm = 10–6 m; 1
nanometer (1 nm) = 0.001µm = 10–6 mm = 10–9 m. The sections are floated
on water and transferred to glass slides to be stained.

Light Microscopy

Conventional light, phase-contrast and fluorescence microscopy are all

based on the interaction of light and tissue components. With the light
microscope, stained preparations are usually examined by means of light that
passes through the specimen. The microscope is composed of mechanical
and optical parts . The optical components consist of three systems of lenses:
condenser, objective, and eyepiece. The condenser collects and focuses
light, producing a cone of light that illuminates the object to be observed.
The objective lenses enlarge and project the illuminated image of the object
in the direction of the eyepiece. The eyepiece further magnifies this image
and projects it onto the viewer's retina, a photographic plate, or (to obtain a
digital image) a detector such as a charged coupled device camera. The total
magnification is obtained by multiplying the magnifying power of the
objective and eyepiece.
Phase-Contrast Microscopy

Phase-contrast microscopy is based on the principle that light changes speed

when passing through cellular and extracellular structures with different
refractive indices. These changes are used by the phase-contrast system to
cause the structures to appear lighter or darker relative to each other, which
makes this kind of microscopy a powerful tool with which to observe living
cells. It also enable the researcher to observe unstained cells or tissue
sections and produces an apparently three-dimensional image .
Paramecium seen under phase contrast microscope.

Fluorescence Microscopy

When certain substances are irradiated by light of a certain wavelength, they

emit light with a longer wavelength. This phenomenon is called
fluorescence. In fluorescence microscopy, tissue sections are irradiated with
either ultraviolet (UV) light or laser, and the emission is in the visible
portion of the spectrum. The fluorescent substances appear brilliant or
colored on a dark background.

Fluorescent compounds that have an affinity for cell macromolecules may

be used as fluorescent stains. DAPI stain can combine with DNA in the
nucleus and give red colour . When observed in the fluorescence
microscope, the DNA– DAPI stain emits a red light. It is thus possible to
identify and localize nucleic acids in the cells. Another important application
of fluorescence microscopy is achieved by coupling fluorescent substances
(such as fluorescein isothiocyanate, FITC) to molecules that will specifically
bind to components of the tissues and will thus allow the identification of
these components under the microscope.
Electron Microscopy

Transmission and scanning electron microscopes are based on the

interaction between electrons and tissue components.

Transmission Electron Microscopy

The transmission electron microscope is an imaging system that theoretically

permits very high resolution (0.1 nm) (Figure 1–8). In practice, however, the
resolution obtained by most good instruments is around 3 nm. This high
resolution allows magnifications of up to 1000,000 times to be viewed with
detail. Very thin tissue sections ( about 60-70nm)can be observed with detail
at these higher magnifications .

The configuration of the electron microscope is very similar to that of the

optical microscope, although the optics of the electron microscope are
usually placed upside down . The first lens is a condenser that focuses the
beam of electrons on the section. Some electrons interact with atoms of the
section and continue their course, whereas others simply cross the specimen
without interacting. Most electrons reach the objective lens, which forms a
magnified image that is then projected through other magnifying lenses.
Because the human eye is not sensitive to electrons, the image is finally
projected on a fluorescent screen or is registered by photographic plates or a
charged coupled device camera. Because most of the image in the
transmission electron microscope is produced by the balance between the
electrons the resulting image is always in black and white. Dark areas of an
electron micrograph are usually called electron dense, whereas light areas
are called electron lucent

.
Electron micrograph of section of hepatocyte as seen by transmission
electron microscope.

Scanning Electron Microscopy

Scanning electron microscopy permits pseudo-three-dimensional views of

the surfaces of cells, tissues, and organs. This electron microscope produces
a very narrow electron beam that is moved sequentially (scanned) from point
to point across the specimen. Unlike the electrons in the transmission
electron microscope, those in the scanning electron microscope do not pass
through the specimen . The electron beam interacts with a very thin metal
coating previously applied to the specimen and produces reflected or emitted
electrons. These electrons are captured by a detector that transmits them to
amplifiers and other devices so that in the end the signal is projected into a
cathode ray tube (a monitor), resulting in a black-and-white image. The
resulting photographs are easily understood, since they present a view that
appears to be illuminated from above, just as our ordinary macroscopic
world is filled with highlights and shadows caused by illumination from
above. The scanning electron microscope shows only surface views.

Autoradiography of Tissue Sections

Autoradiography is the study of biological events in tissue sections

using radioactivity. Autoradiography permits the localization of radioactive
substances in tissues by means of emitted radiation effects on photographic
emulsions. Silver bromide crystals present in the emulsion act as
microdetectors of radioactivity in the same way that they respond to light in
common photography. The first step of autoradiography is to deliver a
radioactive compound to the cells. A variety of molecules, including
radioactive amino acids, radioactive nucleotides, and radioactive sugars, can
be used, depending on the purpose of the study. The tissue sections are
prepared and are covered with photographic emulsion. The slides are kept in
light-proof boxes; after an adequate exposure time they are developed
photographically and examined. When the silver bromide crystals present in
the photographic emulsion are hit by radiation they are transformed into
small black granules of metallic silver, thus revealing the existence of
radioactivity in the tissue. The structures that contain radioactive molecules
become covered by these granules. This procedure can be used in both light
and electron microscopy .
Appearance of radiolabeled materials under light microscope

Appearance of radiolabeled materials under EM