2. Conventional bright-field microscopy, as well as fluorescence, phase-
contrast, confocal, and polarizing microscopy are all based on the
interaction of light and tissue components and can be used to reveal and
study tissue features
Bright-Field Microscopy
With Bright-Field Microscopy stained preparations are examined by means
of ordinary light that passes through the specimen.
3. In fluorescence microscopy, tissue sections are usually irradiated
with ultraviolet (UV) light and the emission is in the visible
portion of the spectrum. The fluorescent substances appear
brilliant on a dark background.
Fluorescence microscopy is applied for the detection of specific
structures, molecules, or proteins within a cell.
Another important application of fluorescence microscopy is
achieved by coupling fluorescent compounds to molecules that
will specifically bind to certain cellular components and thus
allow the identification of these structures under the microscope
Antibodies labeled with fluorescent compounds are extremely
important in immunohistological staining.
4. Phase-contrast microscopy, however, uses a lens system that produces
visible images from transparent objects
Phase-contrast microscopy is based on the principle that light changes
its speed when passing through cellular and extracellular structures with
different refractive indices.
(a) Bright-field microscopy: without fixation and staining, only the
two pigment cells can be seen.
(b)Phase-contrast microscopy: cell boundaries, nuclei, and
cytoplasmic structures with different refractive indices affect in-phase
light differently and produce an image of these features in all the cells.
5. (c) Differential interference microscopy: produces an image with
a more apparent three-dimensional aspect than in routine phase-
contrast microscopy.
6. Confocal microscopy avoids stray light and achieves greater resolution by
using
(1) a small point of high-intensity light provided by a laser
(2) a plate with a pinhole aperture in front of the image detector.
The point light source, the focal point of the lens, and the detector's
pinpoint aperture are all optically conjugated or aligned to each other in
the focal plane (confocal) and unfocused light does not pass through the
pinhole. This greatly improves resolution of the object in focus and allows
the localization of specimen components with much greater precision than
with the bright-field microscope.
7. Polarizing microscopes are conventional microscopes with
additional features that permit observation under polarized light.
The light source of such an instrument is equipped with a
polarizing filter, the polarizer, so that the light it supplies is
linearly polarized.
Polarizing light microscopy produces an image only of material
having repetitive, periodic macromolecular structure; features
without such structure are not seen
8. Transmission and scanning electron microscopes are based on the
interaction of electrons and tissue components.
Transmission electron microscopes (TEM) are microscopes that use a
particle beam of electrons to visualize specimens and generate a highly-
magnified image. TEMs can magnify objects up to 2 million times.
A scanning electron microscope (SEM) is a type of electron microscope
that produces images of a sample by scanning the surface with a focused
beam of electrons.
9. Histochemistry is the science that combines the techniques of
biochemistry and histology in the study of chemical constitution of cell.
Enzyme histochemistry usually works in the following way
tissue sections are immersed in a solution that contains the substrate of
the enzyme to be localized;
the enzyme is allowed to act on its substrate;
at this stage or later, the section is put in contact with a marker
compound;
this compound reacts with a molecule produced by enzymatic action on
the substrate;
the final reaction product precipitates over the site that contains the
enzyme.
10. Examples of enzymes that can be detected histochemically include the
following:
Phosphatases
Dehydrogenases
