MICROSCOPY

Dr. Eric Agboli

 Outline

- Purpose of microscopy
- Various designations of microscope (Microscopy techniques)
- Other modifications to a microscope
- Care for a microscope
 What is a microscope?
❑The microscope is an instrument for producing enlarged images of
objects that are too small to be seen unaided.
❑Microbiology owes its existence to Antony van Leeuwenhoek.
❑In 1673, with the aid of a crude microscope consisting of a biconcave
lens enclosed in two metal plates, Leeuwenhoek introduced the world to
the existence of microbial forms of life.
❑Over the years, microscopes have evolved from the simple, single-lens
instrument of Leeuwenhoek, with a magnification of 300, to the present-
day electron microscopes capable of magnifications greater than
250,000.
❑Microscopic observations generally provide information only about the
morphology of microorganisms and not their physiological features.
 General working principle of microscope
❑Microscope will emit light onto or through objects (Sample placed on the
specimen stage) and magnify the transmitted or reflected light with the
objective and ocular lenses.
 Few terms in microscopy

❑Magnification = Ability of a microscope to produce an image of an object at a scale

larger (or even smaller) than its actual size via an objective lens.

❑Resolution = Ability to identify two light spots separately and is expressed as the
shortest distance between two points that can still be distinguished as distinct entities.

❑Numerical aperture (NA) = It measures the ability to gather light and resolve
fine specimen detail while working at a fixed object (or specimen) distance.

❑Aberration = Distortion or blurring of an image caused by imperfections in the lens’

shape
 Designation (Types?) of microscope
-Microscopes are designated as either Light Microscope or Electron Microscope.

❖Light Microscope
- Bright-field microscope
- Dark-field microscope
- Fluorescence microscope
- Phase contrast microscope
- Interference microscope

❖Electron Microscope
- Transmission electron microscope
- Scanning electron microscope
 Other modifications to a microscope
❑Compound Microscope
-A microscope that uses multiple lenses to enlarge the image of a
sample.
-Typically, a compound microscope is used for viewing samples at
high magnification (40 - 1000x), which is achieved by the
combined effect of two sets of lenses: the ocular lens (in the
eyepiece) and the objective lenses (close to the sample).
-Light is passed through the sample (called transmitted light
illumination).
1.Ocular (eyepiece) lens
2.Objective turret or Revolver (to hold multiple objective lenses)
3.Objective
4.Focus wheel to move the stage
5.Frame
6.Light source, a light or mirror
7.Diaphragm or condenser lens
8.Stage (to hold the sample)
9.Base
10.Phototube (for attaching a camera)
 Other modifications to a microscope
❑Stereomicroscope
-The stereo- or dissecting microscope is an optical
microscope variant designed for observation with
low magnification (2 - 100x).
- It uses incident light illumination (light reflected off
the surface of the sample is observed by the user).
-It uses two separate optical paths with two
objectives and two eyepieces to provide slightly
different viewing angles to the left and right eyes.
-In this way it allows a three-dimensional 1. Focus wheel
visualization of the sample. 2.
3.
Light source
Base
4. Ocular (eyepiece) lenses
 Other modifications to a microscope
❑Inverted microscope
-An inverted microscope is a microscope with its light source
and condenser on the top, above the stage pointing down,
while the objectives and turret are below the stage pointing
up.
-Inverted microscopes are useful for observing living cells or
organisms at the bottom of a large container (e.g., a tissue
culture flask – tissue culture examination) under more natural
conditions than on a glass slide, as is the case with a
conventional microscope.
-These microscopes may also be fitted with accessories for
fitting still and video cameras, fluorescence illumination,
confocal scanning and many other applications.
 Other modifications to a microscope
❑Inverted microscope

Inverted microscope for Fluorescence microscopy

Components of a compound microscope
 Bright-field Microscope

▪This instrument contains two lens systems for magnifying specimens: the
ocular lens in the eyepiece and the objective lens located in the nose-
piece.
▪The specimen is directly illuminated by a beam of tungsten light focused
on it by a sub-stage lens called a condenser, and the result is that the
specimen appears dark against a bright background.
▪A major limitation of this system is the absence of contrast between the
specimen and the surrounding medium, which makes it difficult to
observe living cells.
▪Therefore, most brightfield observations are performed on nonviable,
stained preparations.
Bright-field Microscope
 Dark-field Microscope

❑This is similar to the ordinary light microscope; however, the condenser

system is modified so that the specimen is not illuminated directly.
❑The condenser directs the light obliquely so that the light is deflected or
scattered from the specimen, which then appears bright against a dark
background.
❑Living specimens may be observed more readily with darkfield than with
brightfield microscopy.
Dark-field Microscope
 Fluorescence Microscope
▪This microscope is used most frequently to visualize specimens that are
chemically tagged with a fluorescent dye.
▪The source of illumination is an ultraviolet (UV) light obtained from a high-
pressure mercury lamp or hydrogen quartz lamp.
▪The ocular lens is fitted with a filter that permits the longer ultraviolet
wavelengths to pass, while the shorter wavelengths are blocked or
eliminated.
▪Ultraviolet radiations are absorbed by the fluorescent label and the
energy is re-emitted in the form of a different wavelength in the visible
light range.
 Fluorescence Microscope
▪The fluorescent dyes absorb at wavelengths between 230 and 350
nanometers (nm) and emit orange, yellow, or greenish light.
▪This microscope is used primarily for the detection of antigen-antibody
reactions.
▪Antibodies are conjugated with a fluorescent dye that becomes excited in
the presence of ultraviolet light, and the fluorescent portion of the dye
becomes visible against a black background.
Fluorescence Microscope
Fluorescence Microscopy limitation

❑Fluorophores gradually lose their ability to fluoresce as they are

illuminated in photobleaching.
❑Fluorescence microscopy has enabled the analysis of live cells, but
fluorescent molecules generate reactive chemical species under
illumination that enhances the phototoxic effect, to which live cells are
susceptible.
❑Fluorescence microscopy only allows observation of the specific
structures labeled for fluorescence.
Phase contrast Microscope

▪The phase-contrast microscope is a modified version of the bright-field

microscope that helps visualize living cells without affecting the cells’
viability.
▪It is termed phase-contrast because it consists of a unique phase
contrast condenser (annular ring) and a phase contrast objective (phase
plate). These parts amplify the phase difference of transparent
specimens.
Phase contrast Microscope

▪Observation of microorganisms in an unstained state is possible with this

microscope.
▪Its optics include special objectives and a condenser that make visible cellular
components that differ only slightly in their refractive indexes.
▪As light is transmitted through a specimen with a refractive index different from
that of the surrounding medium, a portion of the light is refracted (bent) due to
slight variations in density and thickness of the cellular components.
▪The special optics convert the difference between transmitted light and refracted
rays, resulting in a significant variation in the intensity of light and thereby
producing a discernible image of the structure under study.
▪The image appears dark against a light background.
Phase contrast Microscope
 Interference Microscope

❑There are several types such as Nomarski Interference Microscope, that

are now used in many microbiology labs.
❑Like the phase contrast microscope these interference microscopes
work on the principle that light from two light waves can be combined to
increase or to decrease brightness.
❑These microscopes are designed so that a beam of light can be split.
❑One part of the light passes through the specimen and another part
goes around the specimen.
❑The beams are then recombined in a specialized prism and viewed.
❑The results is a colored image that has a three-dimensional appearance.
Interference Microscope
Interference Microscope

Modern interference microscope with a Mirau-type objective

 Electron Microscope

▪This instrument provides a revolutionary method of microscopy, with

magnifications up to one million.
▪This permits visualization of submicroscopic cellular particles as well as
viral agents.
▪In the electron microscope, the specimen is illuminated by a beam of
electrons rather than light, and the focusing is carried out by
electromagnets instead of a set of optics.
▪These components are sealed in a tube in which a complete vacuum is
established.
 Electron Microscope

▪Transmission electron microscopes (TEM) require specimens that are

thinly prepared, fixed, and dehydrated for the electron beam to pass
freely through them.
▪As the electrons pass through the specimen, images are formed by
directing the electrons onto photographic film, thus making internal
cellular structures visible.
▪Scanning electron microscopes (SEM) are used for visualizing surface
characteristics rather than intracellular structures A narrow beam of
electrons scans back and forth, producing a three-dimensional image as
the electrons are reflected off the specimen's surface.
Electron Microscope
Electron Microscope
Electron Microscope
Electron Microscope The SEM uses an electron
beam with lower acceleration
voltage that scans the surface
of the sample to create a 3D
image of its microstructure.
Thus, in the SEM, the sample
thickness is limited only by the
chamber size. In contrast, in
the TEM the beam penetrated
the sample limiting its
thickness (ultrathin sections).
As a result, the ultrastructure
becomes visible. Detecting
transmitted electrons also
means that the beam
acceleration voltage, therefore
also the resolution, of the TEM
is usually higher compared to
the SEM.

BSE come from deeper regions

of the sample, while SE
originate from surface regions.
Comparison of various types of microscopes
 Care for the Microscope
❖Remove all unnecessary materials such as books, papers, purses, and
hats from the laboratory bench where mic is placed.
❖Clean all lens systems; the smallest bit of dust, oil, lint, or eyelash will
decrease the efficiency of the microscope. Use lens paper or Xylol.
❖Place the low-power objective in position and bring the stage and
objectives close together.
❖Center the mechanical stage.
❖Coil the electric wire around the body tube and the stage.
❖Carry the microscope to its position in its cabinet in the manner
previously described.
 THE END

THANK YOU
 Assignment 1

Discuss the application of microscopy in biology

(1 page max)