BBY 1103 CELL BIOLOGY AND GENETICS – Lecture two

2.0 MICROSCOPY

Microscopes are devices that provide magnified images of minute objects. Microscopy is
the practice of using a microscope to observe small structures that the naked eye cannot
view. The physical principles applied in microscopy are magnification, resolution, and
contrast.
 Resolution is the ability to discern fine details. It describes the minimum
viewable distance between two distinct points of a specimen.

 Contrast is the difference in light intensity between the image and the adjacent
background relative to the overall background intensity.

 Magnification is the ability of a microscope to produce an image of an object at

a scale larger (or even smaller) than its actual size.

2.1 BASIC COMPONENTS OF A MICROSCOPE

The main components of a microscope are the objective lenses, eyepiece, condenser, iris
diaphragm, stage and light source.
 Eyepiece (Ocular lens) functions to deliver the image to the eye or camera. They
also magnify the image, but it is an empty magnification. Meaning, the eyepiece
enlarges the image but does not increase the resolution. The eyepiece has a lower
magnification than the objective lens.

 The objective lens functions to magnify the object. It is composed of a number of

individual lenses combined within the barrel of the objective. A microscope may also
include a nosepiece onto which a number of objective lenses are mounted.

 The condenser functions to focus the light or electrons on the specimen. The
condenser also eliminates stray light/electrons and provides a uniform illumination.
The sub stage condenser occurs between the light/electron source and the stage.
 Iris diaphragm controls the amount of light or electrons reaching the specimen. In
some microscopes adjusting, the voltage applied controls the light/electron intensity.

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Evans Omwango
Department of Natural Sciences, Mount Kenya University
 The stage provides specimen support. A stage that can move on x and y-axes is
sufficient. For applications requiring angular measurements, a rotating stage is used.
Clips secure the specimen slide on the stage.

 Microscopes use tungsten lamps as the light source or electron gun as the
electron source. The illumination set up uses a collector lens to project the image
of the lamp and a diaphragm to control the size of the illuminated field.

2.4 TYPES OF MICROSCOPES

Classification of microscopes is based on their system of illuminating the sample under

observation. These include;
o Light microscopes
o Fluorescent microscopes
o Electron microscopes

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Evans Omwango
Department of Natural Sciences, Mount Kenya University
2.4.1 LIGHT MICROSCOPES

Compound light microscopes use visible light to produce a magnified image of an

object (or specimen). The word compound refers to the fact that two lenses, the
objective lens and the eyepiece, work together to produce the final magnification. Light
microscopes have limited resolution and therefore limited capability in regards to the
size of a particle that they can examine.

Variations to light microscopy

Many modifications of light microscopes have specialized its applications. These include
bright field, dark field, phase contrast, polarized light, differential interference contrast
and Hoffman modulation contrast microscopy.

 Bright field microscopy

This is the basic microscope set up e.g. the compound light microscope. It is used for
basic observation and image recording

 Dark field microscopy

Dark field microscopy ideally works on unstained samples causing them to appear
brightly lit against a dark background. The microscope uses a dark field stop (annular
aperture) placed between the light source and the condenser. This produces a hollow

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Evans Omwango
Department of Natural Sciences, Mount Kenya University
cone of light that illuminates the specimen from the side. The light diffracted or
scattered by the specimen then enters the objective resulting in an image against a dark
background. The dark-field microscope images have low resolution and poor details. It
is useful for visualization of small particles such as bacteria.

 Phase contrast microscopy

Some specimens do not produce good contrast, and simply cause a phase shift in the
light rays passing through them. Phase contrast microscopy converts the phase
differences to intensity differences with special lenses. In a phase contrast microscope, a
condenser annulus replaces the condenser diaphragm so that the sample is illuminated
only with light that passes through the annulus.

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Evans Omwango
Department of Natural Sciences, Mount Kenya University
 Polarized light microscopy
Polarized light refers to light that has been filtered to remove all its planes of vibration
except one. A polarizing filter does not change the constituent colors of the light, the
wavelengths, frequencies, or speed of propagation. However, the intensity of the light
decreases since some of the energy filters out. The field of view is slightly less bright
because less total light reaches the eyepiece.

The primary difference between light and polarizing light microscopes is the presence of
two polarizing filters. The first, called the polarizer, is in the light path between the
lamp and the sample. The second, called the analyzer, occurs after the sample and
before the eyepiece. Light emerging from the lamp passes through the first filter before
following the usual path to the sample. After passing through the sample, the emergent
light passes through the second filter, which is oriented perpendicular to the polarizer.
Polarized light microscopy is most useful as an analytical technique.

2.4.2 ELECTRON MICROSCOPES

The general principal of electron microscopy is similar to light microscopy except that
electrons are used instead of visible light. The use of electrons instead of light overcomes
the limitation of resolution seen in light microscopes. The illumination source is a
tungsten filament, which emits high velocity electrons. A condenser focuses the electron
beam lens onto the specimen. The condenser lens, however, is an electromagnet.
These electrons impede the various structures within the specimen differently. They are
scattered or absorbed by the atoms of the specimen. A series of magnetic objective
lens focus the electrons that pass through the specimen onto either a photographic
plate or a fluorescent screen. The electrons interact with the photographic plate or

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Department of Natural Sciences, Mount Kenya University
fluorescent screen and generate an image. The differential loss of electrons due to the
subtractive action of the sample will generate an image in much the same way as the
absorption of light creates an image in light microscopy.

The two main types of electron microscopes are transmission electron microscope
(TEM) and Scanning electron microscope (SEM).

Transmission Electron Microscope

The Transmission electron microscope (TEM) has four essential systems:

 Electron source comprising an electron gun, that produces the electron beam,
and condenser system, which focuses the beam onto the sample.

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Evans Omwango
Department of Natural Sciences, Mount Kenya University
  The Image-producing system, consisting of the objective lens, movable
specimen stage, objective, intermediate and projector lenses, which focus the
electrons to pass through the sample and form a magnified image

 The image-recording system, which converts the electron image into a form
perceptible to the human eye. The image-recording system consists of a
fluorescent screen for viewing and focusing the image and a digital camera for
permanent records.

 A vacuum system, which creates a vacuum in the column.

TEM produce two-dimensional, black and white images and can easily resolve structure
such as ribosome, microtubules, microfilaments and large molecules such as proteins.
TEMs can achieve a resolution of approximately 0.1 nm, which is thousand times better
resolution, than the light microscope. Electrons have poor penetrating capability and get
absorbed in thick specimen.

Scanning electron microscope (SEM)

Scanning electron microscopes are composed of:

 Electron gun that emits an electron beam.

 Electron column, where the electron beam travels under vacuum and focuses by
electromagnetic deflection to scan the surface of a specimen.

 Condenser Lenses that cause the electron beam to converge and pass through a
focal point. The condenser lenses are primarily responsible or determining the
intensity of the electron beam when it strikes the specimen.

 Apertures that to reduce and exclude extraneous electrons in the lenses. The final
lens aperture located below the scanning coils determines the diameter or spot size
of the beam at the specimen. The spot size on the specimen will in part determine
the resolution and depth of field. Decreasing the spot size will allow for an increase
in resolution and depth of field with a loss of brightness.

 Scanning System: Images are formed by rastering the electron beam across the
specimen using deflection coils inside the objective lens.

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Evans Omwango
Department of Natural Sciences, Mount Kenya University
 Specimen Chamber where specimens are mounted and secured onto the stage.

 Electron detectors that collect the signal from the interaction of the electron
beam with the specimen and converts the signal into digital images.

 Vacuum System produced by a pump.

The electron gun produces an electron beam, which travels in the vacuum column
through electromagnetic fields and lenses, which focus the beam down toward the
sample. When the incident beam touches the surface of the sample, it produces signals.
The emitted signals are trapped by electrical detectors, converted into digital images and
displayed on a screen. SEM use much lower accelerating voltages to prevent beam
penetration into the sample since the requirement is generation of the secondary
electrons from the true surface structure of a sample.

2.5 IMAGE PROCESSING TECHNOLOGIES

Image processing technologies such as cameras allow images to be digitized and

manipulated electronically. This can correct imperfections in optical systems and can
overcome limitations of human eye. The human eye is not very effective in dim light and
cannot distinguish small differences in intensity against a bright background. Image
enhancement can remedy both of these limitations. However, it cannot increase the
resolution. This is due partly to the limit of resolution determined by wavelength (λ).

2.6 MICROSCOPY SPECIMEN PREPARATION

In their natural state, most of the cells and microorganisms under the microscope lack
color and contrast. This makes it difficult to detect important cellular structures and
their distinguishing characteristics without artificially treating specimens.
 Preparation of specimens for Light microscopy
There are two basic types of preparation used to view specimens with a light
microscope: wet mounts and fixed specimens.

In Wet mount, the specimen is placed on the slide in a drop of liquid. Specimens that
are already in a liquid form are deposited on the slide using a dropper. Solid specimens

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Department of Natural Sciences, Mount Kenya University
are placed on the slide before adding a drop of liquid to prepare the wet mount. The
liquid used can be simply water, but often stains are used to enhance contrast. Once the
liquid has been added to the slide, a coverslip is placed on top and the specimen is ready
for examination under the microscope.

Fixation
Fixing of a sample refers to the process of attaching cells to a slide. Fixation is achieved
by either heating (heat fixing) or chemically treating the specimen. In addition to
attaching the specimen to the slide, fixation also kills microorganisms in the specimen,
stopping their movement and metabolism while preserving the integrity of their cellular
components.

To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a
smear), and the slide is then briefly heated over a heat source.

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Department of Natural Sciences, Mount Kenya University
Chemical fixatives are often preferable to heat for tissue specimens. Chemical agents
such as acetic acid, ethanol, methanol, formaldehyde, and glutaraldehyde denature
proteins, stop biochemical reactions, and stabilize cell structures in tissue samples.
Chemical fixation kills microorganisms in the specimen, stopping degradation of the
tissues and preserving their structure so that they can be examined later.

Following fixation, the samples may be embedded into a supporting medium. Paraffin
and plastic resins are common embedding mediums for light microscopy. Once
embedded, sectioning is done with a microtome, which cuts the specimen into thin
slices, or sections, of a specified thickness.

In addition to fixation, staining is usually applied to color certain features of a

specimen before examining it under a light microscope. Biological samples do not
greatly impede light. Therefore, it is often necessary to stain them with dyes to provide
more contrast. Stains, or dyes, contain salts made up of a positive ion and a negative ion.
Depending on the type of dye, the positive or the negative ion may be the
chromophore (the colored ion); the other, uncolored ion is called the counterion. If
the chromophore is the positively charged ion, the stain is classified as a basic dye; if
the negative ion is the chromophore, the stain is considered an acidic dye.

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Department of Natural Sciences, Mount Kenya University
Dyes are selected for staining based on the chemical properties of the dye and the
specimen being observed, which determine how the dye will interact with the specimen.
In most cases, it is preferable to use a positive stain, a dye that will be absorbed by the
cells or organisms being observed, adding color to objects of interest to make them
stand out against the background. However, there are scenarios in which it is
advantageous to use a negative stain, which is absorbed by the background but not by
the cells or organisms in the specimen. Negative staining produces an outline or
silhouette of the organisms against a colorful background.

Because cells typically have negatively charged cell walls, the positive chromophores in
basic dyes tend to stick to the cell walls, making them positive stains. On the other hand,
the negatively charged chromophores in acidic dyes are repelled by negatively charged
cell walls, making them negative stains.

Some staining techniques involve the application of only one dye to the sample; others
require more than one dye. In simple staining, a single dye is used to emphasize
particular structures in the specimen. A simple stain will generally make all of the
organisms in a sample appear to be the same color, even if the sample contains more
than one type of organism. In contrast, differential staining distinguishes organisms
based on their interactions with multiple stains. Differential staining techniques
commonly used in clinical settings include Gram staining, acid-fast staining, endospore
staining, flagella staining, and capsule staining.

 Preparation of specimens for Electron microscopy

Samples to be analyzed using a TEM must have very thin sections. Cells are too soft to
cut thinly. Therefore, samples are usually fixed, embedded in plastic resin and then
dehydrated through a series of soaks in ethanol solutions. The ethanol replaces the
water in the cells, and the resin dissolves in ethanol and enters the cell, where it
solidifies. Thin sections are then cut using an ultramicrotome. Finally, samples are
fixed to fine copper wire or carbon-fiber grids for support. To obtain more contrast,
samples are stained with salts of heavy metals, such as osmium, uranium and lead.

When samples are prepared for viewing using an SEM, they must also be dehydrated
using an ethanol series. However, they must be even drier than is necessary for a TEM.

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Department of Natural Sciences, Mount Kenya University
Critical point drying with inert liquid carbon dioxide under pressure is used to displace
the water from the specimen. After drying, the specimens are sputter-coated with metal
to prevent specimens from becoming charged by the SEM’s electron beam.

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Evans Omwango
Department of Natural Sciences, Mount Kenya University