CONDE, Ryan Carlo P.

BS-BIOLOGY III

January 19, 2016

MICROLEC
BRIGHT FIELD

'Brightfield' microscopes are the classical microscopes in which the sample is illuminated from the back
and the image is formed due to the absorbing properties of the imaged objects. The name "brightfield"
is derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing
field.
Simple light microscopes are sometimes
referred to as brightfield microscopes. To some
extent, brightfield microscopy is used in most
disciplines requiring microscopic investigation.
Because it is a simple method, this is the first
type of microscopy students learn in schools.
The life sciences, particularly microbiology and
bacteriology, have always relied on the
brightfield technique. This technique can be
used to view fixed specimens or live cells. Since
many organic specimens are transparent or
opaque, staining is required to cause the
contrast that allows them to be visible under the
microscope. Brightfield illumination has been
one of the most widely used observation modes
in optical microscopy for the past 300 years.
The technique is best suited for utilization with
fixed, stained specimens or other kinds of
samples that naturally absorb significant
amounts of visible light. Images produced with
brightfield illumination appear dark and/or highly
colored against a bright, often light gray or
white, background.

DARK FIELD ILLUMINATION

Is

simple and popular method for making unstained transparent specimens clearly visible. Such objects

CONDE, Ryan Carlo P.

BS-BIOLOGY III

January 19, 2016

MICROLEC

often have refractive indices very close in value to that of their surroundings and are difficult to image in
conventional brightfield microscopy.
Darkfield microscopy is an excellent tool for both biological and medical investigations. It can be
effectively used at high magnifications to photograph living bacteria, or at low magnifications to view
and photograph cells, tissues, and whole mounts. Marine biologists continue to use darkfield
illumination at low powers to observe and record data about fresh and salt water organisms such as
algae and plankton.

Other types of specimens, including many that are stained, also respond well to illumination under
darkfield conditions. Figure 7 illustrates darkfield photomicrographs of three types of specimen, all of
which produce good contrast in both brightfield and darkfield illumination. Details in the body of the
deer tick (Ixodes demmini) shown in Figure 7(a) can be washed out in brightfield, unless the
condenser aperture is stopped down to maximize contrast. However, in darkfield, most of the specimen
detail in the tick becomes visible and can be easily captured on film. The heavily stained helminth
trematode (Echinostoma revolutum, Figure 7(b)) also reveals considerably more detail when
illuminated under darkfield conditions, as does the silkworm trachea and spiracle illustrated in Figure
7(c). In addition to the examples presented above, a number of other specimens can also be viewed
and photographed under both brightfield and darkfield illumination to achieve the desired effects.

PHASE CONTRAST

Blue-Green (Spirulina)
Algae

Human Erythrocytes

CONDE, Ryan Carlo P.

BS-BIOLOGY III

January 19, 2016

MICROLEC

Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike, is a contrastenhancing optical technique that can be utilized to produce high-contrast images of transparent
specimens, such as living cells (usually in culture), microorganisms, thin tissue slices, lithographic
patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other
organelles).
In effect, the phase contrast technique employs an optical mechanism to translate minute variations in
phase into corresponding changes in amplitude, which can be visualized as differences in image
contrast. One of the major advantages of phase contrast microscopy is that living cells can be
examined in their natural state without previously being killed, fixed, and stained. As a result, the
dynamics of ongoing biological
processes can be observed and
recorded in high contrast with sharp
clarity of minute specimen detail.

FLUORESCENCE
The absorption and subsequent reradiation of light by organic and
inorganic specimens is typically the
result of well-established physical
phenomena described as being
either fluorescence orphosphores

CONDE, Ryan Carlo P.

BS-BIOLOGY III

January 19, 2016

MICROLEC

cence. The emission of light through the fluorescence process is nearly simultaneous with the
absorption of the excitation light due to a relatively short time delay between photon absorption and
emission, ranging usually less than a microsecond in duration. When emission persists longer after the
excitation light has been extinguished, the phenomenon is referred to as phosphorescence.
The technique of fluorescence microscopy has become an essential tool in biology and the biomedical
sciences, as well as in materials science due to attributes that are not readily available in other contrast
modes with traditional optical microscopy. The application of an array of fluorochromes has made it
possible to identify cells and sub-microscopic cellular components with a high degree of specificity
amid non-fluorescing material. In fact, the fluorescence microscope is capable of revealing the
presence of a single molecule. Through the use of multiple fluorescence labeling, different probes can
simultaneously identify several target molecules simultaneously. Although the fluorescence microscope
cannot provide spatial resolution below the diffraction limit of specific specimen features, the detection
of fluorescing molecules below such limits is readily achieved.

DIFFERENTIAL INTERFACE CONTRAST MICROSCOPY

The DIC microscope employs a mode of dual beam optics that transforms local gradients in optical
path length in an object into regions of contrast in the object image. It is an excellent mechanism for
rendering contrast in transparent specimens, differential interference contrast (DIC) microscopy is a
beam-shearing interference system in which the reference beam is sheared by a minuscule amount,
generally somewhat less than the diameter of an Airy disk. The technique produces a monochromatic
shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial
frequencies present in the specimen. Those regions of the specimen where the optical paths increase
along a reference direction appear brighter (or darker), while regions where the path differences
decrease appear in reverse contrast. As the gradient of optical path difference grows steeper, image
contrast is dramatically increased.
ELECTRON MICROSCOPE
The electron microscope is a type of microscope that uses a beam of electrons to create an image of
the specimen. It is capable of much higher magnifications and has a greater resolving power than a
light microscope, allowing it to see much smaller objects in finer detail. They are large, expensive
pieces of equipment, generally standing alone in a small, specially designed room and requiring trained
personnel to operate them.

CONDE, Ryan Carlo P.

BS-BIOLOGY III

January 19, 2016

MICROLEC

CONDE, Ryan Carlo P.

BS-BIOLOGY III

January 19, 2016

MICROLEC

Types of Electron Microscopes

Transmission Electron Microscope (TEM)

The original form of electron microscopy, Transmission electron microscopy (TEM) involves a high
voltage electron beam emitted by a cathode and formed by magnetic lenses. The electron beam that
has been partially transmitted through the very thin (and so semitransparent for electrons) specimen
carries information about the structure of the specimen
Scanning Electron Microscope (SEM)
Unlike the TEM, where the electrons in the
primary beam are transmitted through the
sample, the Scanning Electron Microscope
(SEM) produces images by detecting
secondary electrons which are emitted from
the surface due to excitation by the primary
electron beam. In the SEM, the electron beam
is scanned across the surface of the sample in
a raster pattern, with detectors building up an
image by mapping the detected signals with
beam position.