Assignment Topic

“Microscope”
Microbiology
Submitted To:
Dr.Farkhanda Jabeen
Submitted By:
Faiza Kanwal
Roll No. M17-01
M.Sc 1st Semester

Department of Botany
University of the Punjab
Lahore-Pakistan

Contents:
  Definition
 Rise of modern light microscope
 Electron microscope
 Fluorescence microscope
 Types
 Optical microscope
 Modern transmission electron
microscope
 First atomic force microscope
 Principle of microscope
 Parts of microscope
 Functions of microscope
 Conclusions
 References

Definition:
 An optical instrument used for viewing very small
objects, such as mineral samples or animal or plant
cells, typically magnified several hundred times.

A microscope (from the Ancient Greek: μικρός,

mikrós, "small" and σκοπεῖν, skopeîn, "to look" or "see")
is an instrument used to see objects that are too small
to be seen by the naked eye. Microscopy is the science
of investigating small objects and structures using such
an instrument. Microscopic means invisible to the eye
unless aided by a microscope.

There are many types of microscopes, and they

may be grouped in different ways. One way is to
describe the way the instruments interact with a sample
to create images, either by sending a beam of light or
electrons to a sample in its optical path, or by scanning
across, and a short distance from, the surface of a
sample using a probe. The most common microscope
(and the first to be invented) is the optical microscope,
which uses light to pass through a sample to produce
an image. Other major types of microscopes are the
fluorescence microscope, the electron microscope
(both, the transmission electron microscope and the
scanning electron microscope) and the various types of
scanning probe microscopes.

Rise of modern light microscopes :

Carl Zeiss binocular compound microscope, 1914

The first detailed account of the microscopic
anatomy of organic tissue based on the use of a
microscope did not appear until 1644, in Giambattista
Odierna's L'occhio della mosca, or The Fly's Eye.[15]

The microscope was still largely a novelty until the

1660s and 1670s when naturalists in Italy, the
Netherlands and England began using them to study
biology, both organisms and their ultrastructure. Italian
scientist Marcello Malpighi, called the father of histology
by some historians of biology, began his analysis of
biological structures with the lungs. Robert Hooke's
Micrographia had a huge impact, largely because of its
impressive illustrations. A significant contribution came
from Antonie van Leeuwenhoek who achieved up to
300 times magnification using a simple single lens
microscope. He sandwiched a very small glass ball lens
between the holes in two metal plates riveted together,
and with an adjustable-by-screws needle attached to
mount the specimen. Then, Van Leeuwenhoek re-
discovered red blood cells (after Jan Swammerdam)
and spermatozoa, and helped popularise the use of
microscopes to view biological ultrastructure. On 9
October 1676, van Leeuwenhoek reported the
discovery of micro-organisms.

The performance of a light microscope depends

on the quality and correct use of the condensor lens
system to focus light on the specimen and the objective
lens to capture the light from the specimen and form an
image. Early instruments were limited until this principle
was fully appreciated and developed from the late 19th
to very early 20th century and until electric lamps were
available as light sources. In 1893 August Köhler
developed a key principle of sample illumination, Köhler
illumination, which is central to achieving the theoretical
limits of resolution for the light microscope. This method
of sample illumination produces even lighting and
overcomes the limited contrast and resolution imposed
by early techniques of sample illumination. Further
developments in sample illumination came from the
discovery of phase contrast by Frits Zernike in 1953,
and differential interference contrast illumination by
Georges Nomarski in 1955; both of which allow imaging
of unstained, transparent samples.
Electron microscopes:
In the early 20th century a significant alternative to
the light microscope was developed, an instrument that
uses a beam of electrons rather than light to generate
an image. The German physicist, Ernst Ruska, working
with electrical engineer Max Knoll, developed the first
prototype electron microscope in 1931, a transmission
electron microscope (TEM). The transmission electron
microscope works on similar principles to an optical
microscope but uses electrons in the place of light and
electromagnets in the place of glass lenses.

Development of the transmission electron

microscope was quickly followed in 1935 by the
development of the scanning electron microscope by
Max Knoll.[17] Although TEMs were being used for
research before WWII, and became popular afterwards,
the SEM was not commercially available until 1965.

Transmission electron microscopes became

popular following the Second World War. Ernst Ruska,
working at Siemens, developed the first commercial
transmission electron microscope and, in the 1950s,
major scientific conferences on electron microscopy
started being held. In 1965, the first commercial
scanning electron microscope was developed by
Professor Sir Charles Oatley and his postgraduate
student Gary Stewart, and marketed by the Cambridge
Instrument Company as the "Stereoscan".

One of the latest discoveries made about using an

electron microscope, is the ability to identify a virus.[18]
Since this microscope reflects a visible, clear image of
small organelles, then in an electron microscope there
will no need for reagents to see the virus or harmful
cells, resulting with more efficient way for pathogen
detection.
Electron microscopes:
In the early 20th century a significant alternative to
the light microscope was developed, an instrument that
uses a beam of electrons rather than light to generate
an image. The German physicist, Ernst Ruska, working
with electrical engineer Max Knoll, developed the first
prototype electron microscope in 1931, a transmission
electron microscope (TEM). The transmission electron
microscope works on similar principles to an optical
microscope but uses electrons in the place of light and
electromagnets in the place of glass lenses. Use of
electrons, instead of light, allows for much higher
resolution.

Development of the transmission electron

microscope was quickly followed in 1935 by the
development of the scanning electron microscope by
Max Knoll.[17] Although TEMs were being used for
research before WWII, and became popular afterwards,
the SEM was not commercially available until 1965.

Transmission electron microscopes became

popular following the Second World War. Ernst Ruska,
working at Siemens, developed the first commercial
transmission electron microscope and, in the 1950s,
major scientific conferences on electron microscopy
started being held. In 1965, the first commercial
scanning electron microscope was developed by
Professor Sir Charles Oatley and his postgraduate
student Gary Stewart, and marketed by the Cambridge
Instrument Company as the "Stereoscan".

One of the latest discoveries made about using an

electron microscope, is the ability to identify a virus.[18]
Since this microscope reflects a visible, clear image of
small organelles, then in an electron microscope there
will no need for reagents to see the virus or harmful
cells, resulting with more efficient way for pathogen
detection.
Fluorescence microscopes:
Fluorescence
microscope with the filter cube turret above the
objective lenses, coupled with a camera.

The most recent developments in light microscope

largely centre on the rise of fluorescence microscopy in
biology.[citation needed] During the last decades of the
20th century, particularly in the post-genomic era, many
techniques for fluorescent staining of cellular structures
were developed.[citation needed] The main groups of
techniques involve targeted chemical staining of
particular cell structures, for example, the chemical
compound DAPI to label DNA, use of antibodies
conjugated to fluorescent reporters, see
immunofluorescence, and fluorescent proteins, such as
green fluorescent protein. These techniques use these
different fluorophores for analysis of cell structure at a
molecular level in both live and fixed samples.

The rise of fluorescence microscopy drove the

development of a major modern microscope design, the
confocal microscope. The principle was patented in
1957 by Marvin Minsky, although laser technology
limited practical application of the technique. It was not
until 1978 when Thomas and Christoph Cremer
developed the first practical confocal laser scanning
microscope and the technique rapidly gained popularity
through the 1980s.
Fluorescence microscopes:
Fluorescence
microscope with the filter cube turret above the
objective lenses, coupled with a camera.

The most recent developments in light microscope

largely centre on the rise of fluorescence microscopy in
biology.[citation needed] During the last decades of the
20th century, particularly in the post-genomic era, many
techniques for fluorescent staining of cellular structures
were developed.[citation needed] The main groups of
techniques involve targeted chemical staining of
particular cell structures, for example, the chemical
compound DAPI to label DNA, use of antibodies
conjugated to fluorescent reporters, see
immunofluorescence, and fluorescent proteins, such as
green fluorescent protein. These techniques use these
different fluorophores for analysis of cell structure at a
molecular level in both live and fixed samples.

The rise of fluorescence microscopy drove the

development of a major modern microscope design, the
confocal microscope. The principle was patented in
1957 by Marvin Minsky, although laser technology
limited practical application of the technique. It was not
until 1978 when Thomas and Christoph Cremer
developed the first practical confocal laser scanning
microscope and the technique rapidly gained popularity
through the 1980s.
Types:
Microscopes can be separated into several
different classes. One grouping is based on what
interacts with the sample to generate the image, i.e.,
light or photons (optical microscopes), electrons
(electron microscopes) or a probe (scanning probe
microscopes). Alternatively, microscopes can be
classed on whether they analyze the sample via a
scanning point (confocal optical microscopes, scanning
electron microscopes and scanning probe microscopes)
or analyze the sample all at once (wide field optical
microscope and transmission electron microscopes).

Wide field optical microscopes and transmission

electron microscopes both use the theory of lenses
(optics for light microscopes and electromagnet lenses
for electron microscopes) in order to magnify the image
generated by the passage of a wave transmitted
through the sample, or reflected by the sample. The
waves used are electromagnetic (in optical
microscopes) or electron beams (in electron
microscopes). Resolution in these microscopes is
limited by the wavelength of the radiation used to image
the sample, where shorter wavelengths allow for a
higher resolution.

Scanning optical and electron microscopes, like

the confocal microscope and scanning electron
microscope, use lenses to focus a spot of light or
electrons onto the sample then analyze the signals
generated by the beam interacting with the sample. The
point is then scanned over the sample to analyze a
rectangular region. Magnification of the image is
achieved by displaying the data from scanning a
physically small sample area on a relatively large
screen. These microscopes have the same resolution
limit as wide field optical, probe, and electron
microscopes.

Scanning probe microscopes also analyze a single

point in the sample and then scan the probe over a
rectangular sample region to build up an image. As
these microscopes do not use electromagnetic or
electron radiation for imaging they are not subject to the
same resolution limit as the optical and electron
microscopes described above.

Optical:
 The most common type of microscope (and the
first invented) is the optical microscope. This is an
optical instrument containing one or more lenses
producing an enlarged image of a sample placed in the
focal plane. Optical microscopes have refractive glass
and occasionally of plastic or quartz, to focus light into
the eye or another light detector. Mirror-based optical
microscopes operate in the same manner. Typical
magnification of a light microscope, assuming visible
range light, is up to 1250x with a theoretical resolution
limit of around 0.250 micrometres or 250 nanometres.
This limits the practical magnification limit to ~1500x.
Specialized techniques (e.g., scanning confocal
microscopy, Vertico SMI) may exceed this
magnification but the resolution is diffraction limited.
The use of shorter wavelengths of light, such as the
ultraviolet, is one way to improve the spatial resolution
of the optical microscope, as are devices such as the
near-field scanning optical microscope.
Sarfus, a recent optical technique increases the
sensitivity of standard optical microscope to a point it
becomes possible to directly visualize nanometric films
(down to 0.3 nanometre) and isolated nano-objects
(down to 2 nm-diameters). The technique is based on
the use of non-reflecting substrates for cross-polarized
reflected light microscopy.

Ultraviolet light enables the resolution of

microscopic features, as well as to image samples that
are transparent to the eye. Near infrared light can be
used to visualize circuitry embedded in bonded silicon
devices, since silicon is transparent in this region of
wavelengths.

In fluorescence microscopy, many wavelengths of

light, ranging from the ultraviolet to the visible can be
used to cause samples to fluoresce to allow viewing by
eye or with the use of specifically sensitive cameras.

Phase contrast microscopy is an optical

microscopy illumination technique in which small phase
shifts in the light passing through a transparent
specimen is converted into amplitude or contrast
changes in the image. The use of phase contrast does
not require staining to view the slide. This microscope
technique made it possible to study the cell cycle in live
cells.

The traditional optical microscope has more

recently evolved into the digital microscope. In addition
to, or instead of, directly viewing the object through the
eyepieces, a type of sensor similar to those used in a
digital camera is used to obtain an image, which is then
displayed on a computer monitor. These sensors may
use CMOS or charge-coupled device (CCD)
technology, depending on the application.
 Digital microscopy with very low light levels to
avoid damage to vulnerable biological samples is
available using sensitive photon-counting digital
cameras. It has been demonstrated that a light source
providing pairs of entangled photons may minimize the
risk of damage to the most light-sensitive samples. In
this application of ghost imaging to photon-sparse
microscopy, the sample is illuminated with infrared
photons, each of which is spatially correlated with an
entangled partner in the visible band for efficient
imaging by a photon-counting camera.

Electron Microscope:

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removed.
Modern transmission electron microscope:
The two major types of electron microscopes are
transmission electron microscopes (TEMs) and
scanning electron microscopes (SEMs). They both
have series of electromagnetic and electrostatic lenses
to focus a high energy beam of electrons on a sample.
In a TEM the electrons pass through the sample,
analogous to basic optical microscopy. This requires
careful sample preparation, since electrons are
scattered strongly by most materials. The SEM has
raster coils to scan the surface of bulk objects with a
fine electron beam.

First atomic force microscope:

The probe and the surface of the sample are
measured and mapped. A near The different types of
scanning probe microscopes arise from the many
different types of interactions that occur when a small
probe of some type is scanned over and interacts with a
specimen. These interactions or modes can be
recorded or mapped as function of location on the
surface to form a characterization map. The three most
common types of scanning probe microscopes are
atomic force microscopes (AFM), near-field scanning
optical microscopes (MSOM or SNOM, scanning near-
field optical microscopy), and scanning tunneling
microscopes (STM). An atomic force microscope has a
fine probe, usually of silicon or silicon nitride, attached
to a cantilever; the probe is scanned over the surface of
the sample, and the forces that cause an interaction
scanning optical microscope is similar to an AFM but its
probe consists of a light source in an optical fiber
covered with between -field a tip that has usually an
aperture for the light to pass through. The microscope
can capture either transmitted or reflected light to
measure much localized optical properties of the
surface, commonly of a biological specimen. Scanning
 tunneling microscopes have a metal tip with a single
apical atom; the tip is attached to a tube through which
a current flows. The tip is scanned over the surface of a
conductive sample until tunneling current flows; the
current is kept constant by computer movement of the
tip and an image is formed by the recorded movements
of the tip.
Principle of Microscopes:
A general biological microscope mainly consists of
an objective lens, ocular lens, lens tube, stage, and
reflector. An object placed on the stage is magnified
through the objective lens. When the target is focused,
a magnified image can be observed through the ocular
lens.
Parts and Specifications:
Historians credit the invention of the compound microscope
to the Dutch spectacle maker, Zacharias Janssen, around the
year 1590. The compound microscope uses lenses and light to
enlarge the image and is also called an optical or light microscope
(vs./ an electron microscope). The simplest optical microscope is
the magnifying glass and is good to about ten times (10X)
magnification. The compound microscope has two systems of
lenses for greater magnification, 1) the ocular or eyepiece lens
that one looks into and 2) the objective lens, or the lens closest to
the object. Before purchasing or using a microscope, it is
important to know the functions of each part
.
 Eyepiece Lens: the lens at the top that you look through.
They are usually 10X or 15X power.

Tube: Connects the eyepiece to the objective lenses

 Arm: Supports the tube and connects it to the base

Base: The bottom of the microscope, used for support

Illuminator: A steady light source (110 volts) used in place

of a mirror. If your microscope has a mirror, it is used to reflect
light from an external light source up through the bottom of the
stage.

Stage: The flat platform where you place your slides. Stage
clips hold the slides in place. If your microscope has a
mechanical stage, you will be able to move the slide around by
turning two knobs. One moves it left and right, the other moves it
up and down.

Revolving Nosepiece or Turret: This is the part that holds

two or more objective lenses and can be rotated to easily change
power.

Objective Lenses: Usually you will find 3 or 4 objective

lenses on a microscope. They almost always consist of 4X, 10X,
40X and 100X powers. When coupled with a 10X (most common)
eyepiece lens, we get total magnifications of 40X (4X times 10X),
100X, 400X and 1000X. To have good resolution at 1000X, you
will need a relatively sophisticated microscope with an Abbe
condenser. The shortest lens is the lowest power; the longest
one is the lens with the greatest power. Lenses are color coded
and if built to DIN standards are interchangeable between
microscopes. The high power objective lenses are retractable
(i.e. 40XR).

This means that if they hit a slide, the end of the lens will
push in (spring loaded) thereby protecting the lens and the slide.
All quality microscopes have achromatic, par centered, par focal
lenses.

Rack Stop: This is an adjustment that determines how

close the objective lens can get to the slide. It is set at the factory
and keeps students from cranking the high power objective lens
down into the slide and breaking things. You would only need to
adjust this if you were using very thin slides and you weren't able
to focus on the specimen at high power. (Tip: If you are using thin
slides and can't focus, rather than adjust the rack stop, place a
clear glass slide under the original slide to raise it a bit higher)

Condenser Lens: The purpose of the condenser lens is to

focus the light onto the specimen. Condenser lenses are most
useful at the highest powers (400X and above). Microscopes with
in stage condenser lenses render a sharper image than those
with no lens (at 400X). If your microscope has a maximum power
of 400X, you will get the maximum benefit by using a condenser
lenses rated at 0.65 NA or greater. 0.65 NA condenser lenses
may be mounted in the stage and work quite well. A big
advantage to a stage mounted lens is that there is one less
focusing item to deal with. If you go to 1000X then you should
have a focusable condenser lens with an N.A. of 1.25 or greater.
Most 1000X microscopes use 1.25 Abbe condenser lens systems.
The Abbe condenser lens can be moved up and down. It is set
very close to the slide at 1000X and moved further away at the
lower powers.

Diaphragm or Iris: Many microscopes have a rotating disk

under the stage. This diaphragm has different sized holes and is
used to vary the intensity and size of the cone of light that is
projected upward into the slide. There is no set rule regarding
which setting to use for a particular power. Rather, the setting is
a function of the transparency of the specimen, the degree of
contrast you desire and the particular objective lens in use.
 Functions:
The microscope gets its name from the Greek words micro,
meaning small, and skopion, meaning to see or look, and it
literally is a machine for looking at small things. A microscope
may be used to look at the anatomy of small organisms such as
insects, the fine structure of rocks and crystals, or individual cells.
Depending on the type of microscope, the magnified image may
be two-dimensional or three-dimensional.
The mental image you probably have of an ordinary
microscope is that of an optical microscope. These microscopes
use lenses and visual light. You look through the eyepiece of the
microscope at a sample in real time. In contrast, imaging
microscopes use a beam of radiation or particles. This beam
bounces off or passes through the sample and is measured and
interpreted by a computer that creates and saves an image of the
sample for later viewing.

Function of Compound Microscope: The compound

microscope is the most familiar form of optical microscope. A
compound microscope utilizes multiple lenses to provide
magnification. A typical compound microscope will include a
viewing lens that magnifies an object 10 times, and four
secondary lenses that magnify object 10, 40, or 100 times. Light
is placed below the sample and travels through one of the
secondary lenses and the viewing lens, and is thus magnified
twice. For instance, if you use the 40 magnification lens with the
10 magnification viewing lens, the object you're viewing will be
magnified 10 times 40, or 400 times. While a compound
microscope can provide large amounts of magnification, the
image produced by visual light are usually of a lower resolution
than those produced by other microscopes.

Function of Dissection Microscope:

Another form of optical microscope is the dissection or
stereo microscope. This microscope uses two different viewing
lenses and produces three-dimensional images of the sample.
But it has a much smaller maximum magnification than a
compound microscope, and usually cannot magnify more than
100 times.

Imaging Microscopes:
Imaging microscopes are significantly higher in resolution
and magnification than optical microscopes, but are also much
more expensive. Different types of imaging microscopes utilize
beams of different types of radiation or particles to provide an
image of a sample. Confocal microscopes use laser light,
scanning acoustic microscopes use sound waves, and X-ray
microscopes, predictably, use X-rays. Electron microscopes use
electrons and can magnify a sample by up to 2 million times. The
transmission electron microscope creates a two-dimensional
image, while the scanning electron microscope creates a three-
dimensional image.
 A scanning probe microscope can create a computerized
image of individual atoms. This type of microscope measures the
surface texture of an object on a very small scale, and will note
where individual atoms protrude from that structure.
Conclusions:
It is device used to see the microbial
organisms which are not seen by naked eyes. There are various
types of microscopes used from seventeenth century. The highly
modified form of microscope is the stereo microscope. Stereo
microscope is used to see the micro life having only single celled.
These are found in every biological laboratory.
References:
1. Characterization and Analysis of Polymers, Hoboken,
N.J.: Wiley-Interscience. 2008.
2. Bardell, David (May 2004). "The Invention of the
Microscope",
3. Liz Logan (27 April 2016). "Early Microscopes Revealed a
New World of Tiny Living Things"
4. Pennycook, S.J.; Varela, M.; Hetherington, C.J.D.;
Kirkland, A.I. (2011), "Materials Advances through
Aberration-Corrected Electron Microscopy,
5. Www. Wikipedia.com