MICROSCOPY TECHNIQUES

Prepared By:
SMS
Microscopes
 Microscopes are instruments
designed to produce magnified
visual or photographic images of
small objects. The microscope
must accomplish three tasks:
produce a magnified image of the
specimen, separate the details in
the image, and render the details
visible to the human eye or
camera. This group of instruments
includes not only multiple-lens
designs with objectives and
condensers, but also very simple
single lens devices that are often
hand-held, such as a magnifying
glass. Microscopes are an
important tool of biologists, and
are used to study cells, tissues,
and microorganisms.
LIGHT MICROSCOPY
 Components of Light Microscopes:

 All modern optical microscopes designed for

viewing samples by transmitted light share the
same basic components of the light path. In
addition, the vast majority of microscopes have
the same 'structural' components(numbered
below according to the image on the right):
 Eyepiece (ocular lens) (1)
 Objective turret, revolver, or revolving nose piece
(to hold multiple objective lenses) (2)
 Objective lenses(3)
 Focus knobs (to move the stage)
◦ Coarse adjustment (4)
◦ Fine adjustment (5)
 Stage (to hold the specimen) (6)
 Light source (a light or a mirror (7)
 Diaphragm and condenser(8)
 Mechanical stage (9)
LIGHT MICROSCOPY
 The optical microscope, often referred to as the "light microscope",
is a type of microscope which uses visible light and a system of
lenses to magnify images of small samples. Optical microscopes are
the oldest design of microscope and were possibly invented in their
present compound form in the 17th century. Basic optical
microscopes can be very simple, although there are many complex
designs which aim to improve resolution and sample contrast.
 There are two basic configurations of the conventional optical

microscope:
The simple microscope and
The compound microscope.
 A magnifying glass is, in essence, a single lens simple microscope.

In general, microscope optics are static; to focus at different focal

depths the lens to sample distance is adjusted, and to get a wider or
narrower field of view a different magnification objective lens must
be used..
LIGHT MICROSCOPY

 Simple microscope:
A simple microscope is a
microscope that uses a lens or
set of lenses to enlarge an object
through angular magnification
alone, giving the viewer an erect
enlarged virtual image. Simple
microscopes are not capable of
high magnification. The use of a
single convex lens or groups of
lenses are still found in simple
magnification devices such as
the magnifying glass, loupes,
and eyepieces for telescopes and
microscopes.
LIGHT MICROSCOPY

 Compound microscope
A compound microscope is a microscope
which uses a lens close to the object being
viewed to collect light (called the objective
lens) which focuses a real image of the object
inside the microscope (image 1). That image
is then magnified by a second lens or group
of lenses (called the eyepiece) that gives the
viewer an enlarged inverted virtual image of
the object (image 2).The use of a compound
objective/eyepiece combination allows for
much higher magnification, reduced
chromatic aberration and exchangeable
objective lenses to adjust the magnification. A
compound microscope also makes more
advanced illumination setups, such as phase
contrast
Type of Light Microscopy
 Bright-field microscopy is the simplest of all the optical
microscopy illumination techniques. Sample illumination is
transmitted (i.e., illuminated from below and observed from
above) white light and contrast in the sample is caused by
absorbance of some of the transmitted light in dense areas of the
sample. Bright-field microscopy is the simplest of a range of
techniques used for illumination of samples in light microscopes
and its simplicity makes it a popular technique. The typical
appearance of a bright-field microscopy image is a dark sample
on a bright background.
 Bright-field microscopy typically has low contrast with most
biological samples as few absorb light to a great extent. Staining
is often required to increase contrast, which prevents use on live
cells in many situations. Bright field illumination is useful for
samples which have an intrinsic colour, for example chloroplasts
in plant cells.
LIGHT MICROSCOPY
 Application:
Optical microscopy is used extensively in microelectronics,
nanophysics, biotechnology, pharmaceutic research, mineralogy,
microbiology, medical diagnosis, histopathology when dealing
with tissues, or in smear tests on free cells or tissue fragments .
 Advantages:

Easy to use, Inexpensive (relative to electron microscopes),Can

look at live samples, Can magnify up to 2000 times

 Disadvantages:

At very high magnifications with transmitted light, point objects

are seen as fuzzy discs surrounded by diffraction rings
 PHASE CONTRAST MICROSCOPY

first described in 1934 by Dutch physicist Frits Zernike is an

optical microscopy technique that converts phase shifts in
light passing through a transparent specimen to brightness
changes in the image. Phase shifts themselves are invisible,
but become visible when shown as brightness variations
• Principle of Phase Contrast Microscopy
The ring shaped illuminating light (green) that passes the
condenser annulus is focused on the specimen by the
condenser. Some of the illuminating light is scattered by the
specimen (yellow). The remaining light is unaffected by the
specimen and forms the background light (red). When observing
unstained biological specimen, the scattered light is weak and
typically phase shifted by -90° — relative to the background
light. This leads to that the foreground (blue vector) and the
background (red vector) nearly have the same intensity,
resulting in a low image contrast(a). The background light is
phase shifted -90° by passing it through a phase shifting ring.
This eliminates the phase difference between the background
and the scattered light, leading to an increased intensity
difference between foreground and background (b). To further
increase contrast, the background is dimmed by a gray filter ring
(c). Some of the scattered light will be phase shifted and
dimmed by the rings. However, the background light is affected
to a much greater extent, which creates the phase contrast
effect.
PHASE CONTRAST MICROSCOPY
 Application:
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).
 Advantages:
Superior to bright-field optics. Fine details which are invisible
under bright-field optics show up in high contrast
 Disadvantages:
Not ideal for thick samples that may appear distorted
Halo effects or 'phase artifacts' may be present distorting details
around the perimeter of the sample
CONFOCAL MICROSCOPY
 Confocal microscopy is an optical imaging
technique used to increase optical resolution and
contrast of a micrograph by using point
illumination and a spatial pinhole to eliminate out-
of-focus light in specimens that are thicker than
the focal plane. It enables the reconstruction of
three-dimensional structures from the obtained
images.
Principles of Confocal Microscopy
The confocal principle in epi-fluorescence laser scanning microscopy
is when Coherent light emitted by the laser system (excitation source)
passes through a pinhole aperture that is situated in a conjugate plane
(confocal) with a scanning point on the specimen and a second pinhole
aperture positioned in front of the detector (a photomultiplier tube). As
the laser is reflected by a dichromatic mirror and scanned across the
specimen in a defined focal plane, secondary fluorescence emitted
from points on the specimen (in the same focal plane) pass back
through the dichromatic mirror and are focused as a confocal point at
the detector pinhole aperture.
CONFOCAL MICROSCOPY
 Application:
The broad range of applications available to laser scanning confocal
microscopy includes a wide variety of studies in neuroanatomy and
neurophysiology, as well as morphological studies of a wide spectrum of cells
and tissues. Other applications include resonance energy transfer, stem cell
research, photobleaching studies, lifetime imaging, multiphoton microscopy,
total internal reflection, DNA hybridization, membrane and ion probes,
bioluminescent proteins, and epitope tagging
 Advantages:

The non-­invasive confocal optical sectioning technique enables the

examination of both living and fixed specimens under a variety of conditions
with enhanced clarity.
 Disadvantages:

The harmful nature of high-­intensity laser irradiation to living cells and tissues.
the high cost of purchasing and operating multi-­user confocal microscope
systems, which can range up to an order of magnitude higher than comparable
widefield microscopes, often limits their implementation in smaller laboratories
FLUORESCENCE MICROSCOPY
 fluorescence microscope is an optical microscope that uses
fluorescence and phosphorescence instead of, or in addition
to, reflection and absorption to study properties of organic or
inorganic substances.The "fluorescence microscope" refers to
any microscope that uses fluorescence to generate an image,
whether it is a more simple set up like an epifluorescence
microscope, or a more complicated design such as a confocal
microscope, which uses optical sectioning to get better
resolution of the fluorescent image.
FLUORESCENCE MICROSCOPY
 Principle of Fluorescence Microscopy:

The specimen is illuminated with light of a specific wavelength (or wavelengths) which
is absorbed by the fluorophores, causing them to emit light of longer wavelengths
(i.e., of a different color than the absorbed light). The illumination light is separated
from the much weaker emitted fluorescence through the use of a spectral emission
filter. Typical components of a fluorescence microscope are a light source (xenon arc
lamp or mercury-vapor lamp are common; more advanced forms are high-power
LEDs and lasers), the excitation filter, the dichroic mirror (or dichroic beamsplitter),
and the emission filter (see figure below). The filters and the dichroic are chosen to
match the spectral excitation and emission characteristics of the fluorophore used to
label the specimen.In this manner, the distribution of a single fluorophore (color) is
imaged at a time. Multi-color images of several types of fluorophores must be
composed by combining several single-color images.
Most fluorescence microscopes in use are epifluorescence microscopes, where
excitation of the fluorophore and detection of the fluorescence are done through the
same light path (i.e. through the objective). These microscopes are widely used in
biology and are the basis for more advanced microscope designs, such as the
confocal microscope and the total internal reflection fluorescence microscope (TIRF).
FLUORESCENCE MICROSCOPY

 Application
Fluorescence microscopy is used in medicine, environmental studies, food
sanitation, biological research, education and industry. One of the main
applications is for rapid medical diagnosis.
 Advantages:

Highly sensitive and selective (because one can label specific structures with
appropriate probes) ,Versatile (antibodies to label proteins, probes for
nucleic acids, probes for a variety of cellular components, fluorescent
proteins for live studies),Potentially provide superb contrast because objects
are self-luminous against a dark background.An extremely small number of
fluorescent molecules (as few as 50 molecules per cubic micrometer) can be
detected.
 Disadvantages:

Fluorescence detection sensitivity is compromised by background signals,

either from endogenous sample constituents (autofluorescence) or from
unbound or non-specific reagents
POLARIZATION MICROSCOPY
 Polarized light microscopy can mean any of a number of
optical microscopy techniques involving polarized light.
Simple techniques include illumination of the sample with
polarized light. Directly transmitted light can, optionally, be
blocked with a polariser orientated at 90 degrees to the
illumination. These illumination techniques are most
commonly used on birefringent samples where the polarized
light interacts strongly with the sample and so generating
contrast with the background.
POLARIZATION MICROSCOPY
 Principle:
The polarized light microscope is designed to observe and photograph
specimens that are visible primarily due to their optically anisotropic character.
In order to accomplish this task, the microscope must be equipped with both a
polarizer, positioned in the light path somewhere before the specimen, and an
analyzer (a second polarizer), placed in the optical pathway between the
objective rear aperture and the observation tubes or camera port. Image
contrast arises from the interaction of plane-polarized light with a birefringent
(or doubly-refracting) specimen to produce two individual wave components
that are each polarized in mutually perpendicular planes. The velocities of
these components are different and vary with the propagation direction
through the specimen. After exiting the specimen, the light components
become out of phase, but are recombined with constructive and destructive
interference when they pass through the analyzer. Polarized light is a contrast-
enhancing technique that improves the quality of the image obtained with
birefringent materials when compared to other techniques such as darkfield
and brightfield illumination, differential interference contrast, phase contrast,
Hoffman modulation contrast, and fluorescence.
POLARIZATION MICROSCOPY
 Application:
Polarized light microscopy is used extensively in optical mineralogy. Polarized light
microscopy is capable of providing information on absorption color and optical path
boundaries between minerals of differing refractive indices, in a manner similar to
brightfield illumination, but the technique can also distinguish between isotropic
and anisotropic substances.
 Advantages:

Basic identification of the materials was first performed by light microscopy and
gross analysis. This provides a large base of published information against which to
check analysis and analytical technique.The analysis is specific to fibers. The
minerals present can exist in asbestiform, fibrous, prismatic, or massive varieties all
at the same time. Therefore, bulk methods of analysis such as X-ray diffraction, IR
analysis, DTA, etc. are inappropriate where the material is not known to be fibrous.
 Disadvantages:

Even using phase-polar illumination, not all the fibers present may be seen. This is
a problem for very low asbestos concentrations where agglomerations or large
bundles of fibers may not be present to allow identification by inference.The
method requires a great degree of sophistication on the part of the microscopist.
An analyst is only as useful as his mental catalog of images. The mineralogical
training of the analyst is very important. It is the basis on which subjective
decisions are made.
Electron Microscopy
 An electron microscope (EM) is a type of microscope that uses
an electron beam to illuminate a specimen and produce a
magnified image. The electron microscope uses electrostatic
and electromagnetic lenses to control the electron beam and
focus it to form an image. These electron optical lenses are
analogous to the glass lenses of a light optical microscope.
Electron Microscopy
 Principle
The conventional electron microscope requires that the electron beam be in
a vacuum, because electrons cannot ordinarily travel an appreciable
distance in air at atmospheric pressure. The column of the electron
microscope is evacuated by pumps, and the specimens and any other
necessary apparatus are introduced into the vacuum by means of air locks.
Unlike the optical microscope, in which the lenses are of fixed focus and the
distance between specimen and objective lens is varied, the electron
microscope has variable-focus lenses,and the distance between specimen
and objective lens and the separation of the lenses remain constant. The
magnification is determined mainly by the value of the current (for magnetic
lenses) through the intermediate and projector lens coils. The image is
focused by changing the current through the objective lens coil. Another
difference is that the optical microscope is usually operated so that the
image is a virtual one, while in the electron microscope the final image is
invariably real and is visualized on a fluorescent screen or recorded for
study on a photographic plate in traditional instruments.
Electron Microscopy
 Application:
Electron microscopes are used to investigate the ultrastructure of a wide
range of biological and inorganic specimens including microorganisms,
cells, large molecules, biopsy samples, metals, and crystals. Industrially,
the electron microscope is often used for quality control and failure
analysis. Modern electron microscopes produce electron micrographs,
using specialized digital cameras or frame grabbers to capture the image.
 Advantages:

An EM has a higher resolving power than a light microscopeand can

reveal the structure of smaller objects because electrons have
wavelengths about 100,000 times shorter than visible light photons
 Disadvantages:

Electron microscopes are expensive to build and maintain.The samples

largely have to be viewed in vacuum, as the molecules that make up air
would scatter the electrons.
THE END