MicroscoPy

only in the twentieth

microscope emerged
century 73
electron microscoPy about
midway RP for
greatly
of
extended out
powers of low
Adve he century
ion. Today a variety of microscopes power objective S50nm
w u g h

resolution.
biologist. Basic principles, designs 2x0-25
v i s u a l

vailatble
to =
1100 nm =
1-1 pm
es
microscopes and applications are discussed
an
RP for
a
high power
jecive S50 nm
are
b

f e
these
l o w i n d e t a i l .
objective= 2x0-65
Light Microscopy 423-07 nm =
0.42 um
RP for an
instruments including the eye, pend oil immersion objective 550 nm
All optical instrumn

al parameter known as resolution. x1:25

220 nm =022
Depending
o n a p h y s i c a

6
the unaided
human eye is about 0.1
of are of
upon the lens
system, microScopes
Microscopy R e s

mm (1004).
o l u t i o n

In other words, two

minimum
her appear
to be
objects within 100
in contact. The (11)
compound
two
types (i) simple
consists of a microscope.
microscope
A simple
ana

of
each
which they
are resolvable as
separate single lens system while a microscope
compound
at
by Abbe's relationship. microscope consists of two or more lens
ities is explained
dsias Depending upon the systems.
source of illuminatüon, the
6

microscope can be classified as

do n sin o. (1) Light microscope
G) Bright field microscope
i) Dark field microscope
whered =minimum distance (ii) Ultraviolet microscope
A=wavelength of fadiation (iv) Phase contrast microscope
Microscopy is defined as a lens or combination of lenses used to index between the specimen and (v) Fluorescent microscope
n=refractive
magnify and observe near objects so that details invisible to the the first lens (2) Electron microscope
naked eye can be revealed. At the
beginning of the A comparison of the major features of various
Enlightenment, magnifying property of an optical lens was Age
of = numerical aperture
n sin it clear that high kinds of microscopes is given in the table 1.
known and such lenses were used already Abbe's relationship
makes
by various scientists in Europe microscope can be achieved by
resulting in the invention of the telescope and of the resolution in a
Bright-field Compound Microscope
2-stage
microscope. These developments initiated a period of discoveries manipulating these variables i.e. wavelength of
the microscopic field in radiation, the refractive index and the The bright-field compound microscope (mieroscope
mainly by Anthony van Leeuwenhoek (1632- iluminating
1723), Robert Hooke (1635-1703), Marcello the limit of aperture of a good that uses a direct light source for illumination) is
Malpighi aperture. 85° is
and Jan Swammerdam (1628-1694) index is easy to alter, a type of microscope, which is most commonly used
(1937-1680). These followed an interval of optical microscope. Refractive
almost 150 years during which but only within narrow limits, n for air is 1,
whereas for general laboratory observations. It consists of a
owing to the lack of further
development of the instruments, hardly any scientific discoveries were transparent oils have an n upto 15, accounting
for series of two lens system between the eye and the
made but source (sun and mirror or
a
"microscopic delectation of the mind and the
eye", became
the popularity of the oil immersion lenses of modern object and a direct ight
known.
microscopes. lamp) where light shines directly on the specimen,
On the realization of is observable.
important optical principles and the Thus, a good light microscope with a numerical and dark objects in a bright field
progressive mastery of the design of instruments for A typical bright-field microscope
(Fig.1) consists
astronomy and
navigation, the barriers of difference in the traditional theoretical perture of 1.4 and light at the short and of using
and the Visible spectrum (0-4 u or 400 nm) will resolve of four basic parts
practical thinking were broken. The demands for instruments for firm support and stability
The base to provide
research in astronomy and
microscopy grew with the blossoming of WO points at about 0.174 separation. This is ()
to the microscope;
natural sciences. Increasing interest,
following French Revolution, in mensely better than the unaided eye. The resolving The which is
stage,
a flat platform that holds
medical sciences during first half of the 19" PWCr of a lens can be expressed as follows (2)
century, led to the the slide in position;
realization that microscope is for carryingg
capable of revealing more than which is movable, used
entertaining spectacles and curiosities. Although the first functional 6A (3) The arm
comfortable
be set in a
RP= and can
microscope was produced by a Dutchman Zacharias Janssen in the n sin a
the microscope
and
the worker;
first decade of the sixteenth
century, the present Thus TCsolving position for the magnifie-
design of light power of a low power, hign for transmitting
POwer and oil-immersion objectives can be calculated (4) The body tube

follows- image
741
ocular lens: further
magnifies
the image produced by the
objective lens. usualy 10
times
Microse Microscoyy

Table.omparison
ison o
offi . types ot microscopes.
five
fixed draw tube 75
coarse-adjustment »
body tube contains mirrors and Maximum uselul Appearance
knob
prisms that transmit the image from M i c r o s c o p e
magnification of specimnen
nmon uses Advantages
the objective to the
ocular lens
nose
1000
Specimen stained or Observing Disadvantages
fine-adjustment piece 1.Bright-field

knob
unstained; bacterium specimen,stained
Easy to use;
generally stained microbes. sections readily Lack of contrast;
objectives primary lenses
that magnify the and appear in stain.
available; allows to resolve very
inaby
small
arm
specimen colour of staining reactions to bacteria and
be interpreted.
vinuses
introduction of artifacts
stage: platform which 1000 x dunng
holds the slide Dark field Generally unstained; Viewing staining
stage clips on
position appears bright or cells unstained Allow living procedures.
not Staining reactions cannot
hold the slide
"lighted" observable microbes
-condenser in an with bright field
to be be sued for
examining
otherwise dark field viewed e.g.
iris diaphragm; controls Bright and coloured' microscope spirochetes.
stained specimens.
the amount of 1000 x
substage adjustment light entering Colour of the Detecting
the condenser
Fluorescence
specific
fluorescent dye infectious agents in ofRapid identification
knob
iníectious
Specimens that naturaly
tissue, detecting fluoresce or which are
mirror focuses immunological microorganism stained with a fluorescent
base the light rays reactions dye are only observed.
1000x Structures varying
A B
4. Phase-contrast

degrees darkness
Observing living
unstained cell;
Enhances subcellular Inability to evaluate
Fig. 1. The anatomy; allows staining reactions.
monocular compound revealing observation of
A
diagrammatic representation bright-field (or light) microscope. intracellular structure motulity,
of numerical (A) The principle parts
apearture. and their
The
function (B) phagocytosis and
optical biological activities.
lenses, the objectivesystem consists of two 1,00,000 x Viewed on Examination of
lens and ocular lens series of
5. Electron
Allows viewing of Very
of the expensive; only
The upper end of (eye piece). mirror is used fluorescent sereen Viruses and
objects too small to killed specimens are
lens (10x15x)
the
body tube holds the ocular (sunlight) and the flat for natural day
side for the light ultrastructure of cells; be observed by observed; introduction of
through which the image is viewed.
The lower end of (e.g. lamp). More
recently artificial light diagnosis certain light microscopy. artifacts in the specimen

light source which designed microscope has

virus diseases and
body tube which is a built-in during harsh preparation
to the
object being examined lodged close simplifies cancers; detecting
(larger) and fine light focusing
is fitted with The procedures.
nose coarse
piece holding generally a
rotating certain giant
which provide three
objective lenses, adjustment knobsare used for (smaller) foc molecules
and 100X. The magnification powers of l0X, 40X
of the
specimen. The coarse focusing the image
most
standard
greatest magnification achievable by or lowers the base adjustment which raises
tube, is used
10 X 100 =
bright-field
1000X. microscope is for low-power objective and the fine for focusing the Procedure for Microscope Operation (4) If the microscope does not have a built-in-
The resolution
microscope is 0-00027 mm (0:27 of such focusing high-power adjustment, is USeu
a light source place the microscope in front of

thousandth the thickness of um), about one- objectives. The area seen
and oil-immersion () Remove the microscope from the cabinet by a microscope lamp.
The stage human hair.a known as the
field of through a microscop grasping the microscope arm fimly with the
(5) Place the slide on the microscope stage and
may have two A vision.
stage, which moves by means clips or mechanical called microscope
a nght hand and the base with the left hand stage clips or other
means

of
adjustment monocular possessing a single ocular
a
and carry close to the body and gently keep it
secure it with
to hold the
slide. Attached to knobs, two ocular
lenses is microscope and that lens provided.
substage consisting of condenser, anthe stage is the
a called
binocular possessing a
On the laboratory table in an upright position.
(6) Adjust the miTor so that the flat side reflects
and a mirror. The
lenses that
iris
condenser consists of diaphragm Requirements microscop 4 Place the microscope with its arm facing the light into the barrel
the of the microscope.
(10x) objectkve in
several (7) Bring the low power
diaphragm
concentrate light on the
controls the angle and slide. The iris Bright-field (light) microscope er approximately 15 cm from the edge of
the laboratory table. the nose piece.
amount Light source position by rotating and
used. The mirror reflects of the eye piece/eye pieces
illumination via the
light from the sourcelight
of Lens paper,
Immersion
Clean the lenses of the
microscope with lens (8) Look through there is a minimum until
condenser. The concave side Xylol oil
paper before using it. Remove oily substance raise the
condenser
the
illumination i.e. is seen as a circle in
light
Prepared slide of an
organism
glass parts by wiping with a lens paper
of the field
called "a bright full
OIStened with xylol. But it is to be removed center

moon".
dately with lens paper dipped in 95%
Raise the
condenser up to the stage
with the

the lens is later on wiped dry (9) adjustment knob.

iwith a nd help of the condenser
fre_h lens m
I77

lamp
Aicn Interference Microscopy
76 Arpng
both ejes
phase plate
vular,

thnugh
the focus by The nterterence mieroscope works on the
sharp
10
Lovk
ih
yimen
into
Simlar to that of prineiph
yem.
bnnE phase contrast microscope but
offers additional advantage of
usng fineadjutme

dhaphragm
te prxiuc
optimum
condenser data. t
detects smali and continuous changes
giving quantiative
ins lens objective lens
Adrus refractive index whereas phase microscope reveals
with the
lumanatk into
focus

imag* omes
only sharp boundaries. The variations phase
When
the 1s r o a t e d refracted ray 1in
ca
objerive.
the Roe pienr be transtormed in coloured images that a living cen
w-puer or 100 x)by unrefracted ray
40 x
may resemble stained
the next lens (1e. des1red into
a
preparation.
Nomarski interference. This technique is used
the objertive Specimen
sa2nging specimen
sumy tube. Only
without raising
the body support to give
the image a characteristic etfect that is better
piace. knob
of the fine-adjustment than the phase contrast image. Observations on cel
sight adjustment focus because
objective condenser lens

wil bring the objevt

into sharp ens division are generally made using this
technique
all objectives are parafocal
13When using the
oil-immersion objectve. swing Ultra-Microscopy
the hagh-pOwer objective partally
out
of way annular
is also called as dark field
oil on the
This technique
drop ot immersIon diaphragm the light can that
and place a
microscopy. It works on principle
arta ot the siide you obserning are and bnng be scattered at boundaries between regions having
the otl-immersion objective nto posiuon.
different refractive indexes. Certain modifications
are
ocular
piece and focus
14 Now iook through the eye
made in the ordinary microscope. An ordinary
knoo.
the object with fine-adjustment illuminates the
condenser is replaced by one that
Before putting the microscope to its cabinet.
15) condenser does
ner compliction ot the exercISe. move the nose object obliquely. Use of dark field
not allow the light to enter the objective direcly
low-powe objective into
piece to bring a
scattered light
Therefore. the object appears bright in
positon. ciean the oil from the immersion lens
using a special lens paper and also clean offi and the background remains dark.
the detection of
the slide with a tissue paper (Fig. 2). Dark field microscopy allows
Fig. 2. The light microscope. Note the aperture angle, a smaller than those seen with the ordinary light
objects such objects.
Phase Contrast which is related to the resolving power by equation. When
However, it can not resolve
using an oil immersion lens, the space between the microscope.
Interference Microscopy specimen and the objective lens (circled) is filled wih
These transparent oi. Polarization Microscopy
microscopical methods have been developed
tissues show
using special optical techniques. Their basic principle of cells and
ertain components
lies on the fact that although biological structures the examined under polar1zed
objective adjusted
are retard or advance the
to behaviour when
are highly transparent to visible light. they cause particular
wavelength of light by additional 4 wavelengh
prase changes or relardations in ransmitted (1/4 ) with respect to central
radiations. annular phase plate that
light and by
For exampie. ray of light can be adjusted to
introduces 4 wavelengu
variation in the back focal
pass through a material which does not absorb light This plane of the objecive
phenomenon is called as effect. contrast microcope. Rays
ihat
(A) and a material which absorbs
light (C). In A. phase effect results from the phase ig. 3. Diagram of the phase
region of changing
refractive index will be |
the wave of
light is thrown over a material which the direct interference betwee sthrough a

has different refractive index than the geometric image given by the central
afracted and after passing through the phase plate, wil
medium in of the
objective and lateral pa with unrefracted rays. This produces a
passing through this object, the amplitude of the retarded. or advanced to a
image, which has been einterference
Ellect. increasing contrast and making it easier to
wave is not
changed but the velocity is alered total 4
negative contrast,wavelength.
In bright or Cerhe unstained, living cells.
When the refractive index of the material is of rays are added and the two ses
than the medium. it causes higher the object
the transmittance of
delay or retardation in than the
surroundings. n darkappears brighter he
light (Fig. 3 contrast the two seis ot or
positive ckness and the difference between
In phase contrast rays
are used
microscope, special objectives making the image or the object are
subtracted rECve indices of the object and the medium.
to
intensify small phase differences. The surroundings (ig 3). A darker than the ed
Pplications. Phase contrast microscopy is u
lateral plane of the objective and central
plane of appears in various uransparent object thus
shades n e to observe living cells and tissuc mother pollen dividing
gray, virginica
depending on
and has been found
useful Fig. 4.
Tradescantia

2000 x.
parasites
obserui" g cells interterence
(contrast)
cultured in vitro during mitosiS.
78

propagated through
a
polarized 1ght
is
ight When
materal with the same velocity. 9
component
of the inpringing direction, they are
iicroscopy usung an
tuorescent materialepi-illiuminator.
independent o1 The basie t y
Such structures have the same
called as isotropic. that can be studied in ussucs
are listed in
all directions. However, certain the tollowing table.
index of relraction in

structures affect the veloc1ty of light. Such objects Table 2

Strongly autoluorwent substances which may Ctur

anisotropic or birefringent (two different

n tissues. (all
are called proteins are to oine extent
autofluorescent).
indexes of refraction).
For exanple, connective tussue fibres are 10
Substance Florescence Colour
birefringentif the index of refraction is greater along
the length of fibre than in the perpendicular plane Collagen ibres blue-Erec
9 Elastic ibres
and non birefringent in the opposite case. bluc green

Birefringence (B) lorms the principle of a wavelength Protein bound NADH;

Vitamn A
blue
8
polarzation microscopy. may be expressed It
Fig. 5. Excitation (lel) Lipotuscm ofange
and
quanutat1vely as the difference between the two emission Porphyrs
luorophore. spectrumofof a type td

indexes of refraction (N-No). Thus = Ne-No

In practice. these differences
result into wavelength is known as Stokes shift. On Electron Microscopy
retardation of polarized light.
This retardation of light, the ahson
depends upon the thickness () of the fluorophore molecules sopo Electron microscopy developed along with light
electronic state isbecome e
specimen. that is to say their
Therefore. microscopy or optical microscopy. Optical
excited state. When this excited changed to microscopy could function under certain limits ot
A
ground state, the
molecule retums
B =
surplus energy is dissipated resolution. In the light microscope, magnification is
A
polarizing
emitted as
fluorescenceor utilized asinte largely determined by the objective and a maximum
additional devices microscope
is
i.e. the
cquipped with two photochemical reaction. The spectral magnification of 100 or 120X can be achieved. Since
polarizer and the of
typical fluorophore are shown in characteristig
prepared from a analyzer
both of which can be a
4 the ocular lens can increase this image only 5 to 15
film or from Nicol Polaroid a
pure substance, the Figure 5. F a total useful magnification of 500 to 1500X
prisms of times,
1s
placed below the substage calcite. The polarizer emission spectrum
independent of the wavelength can be achieved.
analyzer is placed above condenser whereas the The fact that the used for excitatio using |
the objective lens. emitted light ol a Tuorescence microscope
In practice there were no lenses that could bee
is of Fig. 6. Optical diagram
long 1 lamp. 2
wavelength than that used for
a
dia-illumination
or dark-ground
illumination.
used to focus very short wavelengths.
To overcome
FluorescenceMicroscopy basis of excitation forms th exeitation filter, 5 field lens and
luorescence microscopy. collector lens. 3
mirror, 6 condenser, 7 object this problem Dr. Ruska (University of Berlin))
Fluorescence Design diagphragm. substage
discovered electron optical system that led
to this
when Kohler microscopy dates from about 1904
filter, 10 ocular, 11
of 9 barrier
a
Fluorescence (tuorescent). 8 objective,
(EM)
tissues under
reported observing Nuorescence
ultraviolet
radiation in a of This
microscope differs from the
Microscope observerS eye or camera. well
n
established science of electron microscopy
1931.
Coons (1941)
introduced the fluorescentmicroseope. microscope. Two filters convenuo i m m u n o f l u o r e s c e n c e and for other In EM, charged particles such as electrons
technique with antibody A primary (excitation) are used in this microscop it great value in also
magnetic fields. Since particles
a

immunology. consequent revolution in between the filter is requiring the demonstration of small respond to
Development
staining methods, of
instrumentation, light source andPlaced
the
placed somewhere
somewn purposes exhibit wave properties, focusing
them with magnetic
and of
during past few years. applications has been
of
allows light only over a
ot specimen. 1nefiler amounts of substances. of a produces an image. If a
metal filament is

fluorescence The basic rapid

technique of correspondingcomparatively wavelength
narrow Du
of the
heapparent colour and contrast
uorescent specimen depends not only upon the filter
lenses

placed in vaccum
electrons
tube and heated, it emits
Price and microscopy have
Schwartz (1956).
been described
by
fluorophore to the
being observed. excitation p
The of the that can be accelerated by
an electrical potential
Holborow (1970). Haitinger (1959) secondary filter to
system but the characteristics
also upon path with properties
prevent barrier filter is and tend to follow a straight
or

reacting the light observer's eye (or photographic film).

of the
Basic Principles observer's excitation intendeu those of light.
stream
Like light, he of
object and eye
(Fig. 6).
eye. It is wavelength from
placed Applications
similar to

corpuscular and vibratory

has
character

Essentially. fluorescence is The usual between electrons a

can be expressed by
the equation
in which
light is absorbed optical phenomenon
an
microscope optucal
is such arrangenment of
uorescence techniques can be applied to all kinds
but the wavelength
12 3
fluorescentfluorescen
by that the a
as
fluorophore, and is substance. known microscopy
a
is
capable of biological major advantage of
material. A AEA
light of a instantaneously
larger wavelength. This re-emitted very small
quantity of asrevealinga
the specinmen OFescence microscopy is its very high sensitivily which the
change in out
against the dark tluorophore whichpresence o ICles below the resolution of light microscope Where E is the voltage through
S0,000 volts
background. It is the will stand
sensitivity of the fluorescence viewed by fluorescent microscope. electron s
accelerated.
Electrons at

technique whichextreme be Opaqu of 0-05 A°. This value

does not
s or relatively opaque objects such as vey have a wavelength numerical
gives offer improvement in resolution because
uons can also be examined by fluorescence (BC43)
80
The Scanning Electron Aic MMCoscOpy
aperture of
electron microscope
1s much
Therefore,
smaller than

magnetic coils Microscope (SEM) CroNe e l e c t r o n s e m i l t e d f r o n

rom
primary electron bear
of the specimen.
that
(1) Aheate
urface ated 81
that of light microscopes.
(cathode) tungsten filament that emits
the
are
condenser. Electrons with
are used which
act as a Although, scanning instruments
nts were avalabe
Microscope
teractea
Transm (iT
ssE M
io n )E
dleec
c ttr
roo

)
of the object and then are
the mid 1940s, biologists 1here is an electrons
focused on the plane
coil which acts as an found
this technique by 1960s. In
1) are attracted anode toward which
deflected by another magnetic Way (3) Apotential the
electrons
objective lens and gives
a magnified image

by a third magnetic
of the
lens
the beam of electrons are
and forth across the specimen. As to
Scanning microses
allowed
The
EM are, in principle, very simpler
microscope, except that the the
anode difference between the
object. This is received
move optics light imparts an
between 20,000 cathode and
which acts as an ocular or projection lens and again
the specimen, they may be elect Se O
of
of
electrons rather than a beam

electrons and accelerating voltage ot

scatteredns 200,000 volts
b e a m

of this
magnifies the image from the objective.
The final
electrons may be knocked from the U s e s
a

nciple
components
microscope (4) The beamcreating the beam to the
image can be observed on a fluorescent screen or
electrons, in either case, are Secong
the sample. The TBM
The
summarized as
ilows
foll
thus
(the cathode andemerging from the electron
recorded on a photographic plate. collecte
photomultiplier tube. Other types of anbe
into a anode gun
assembly) is condensed
In the EM the resolving power is so high that
also be induced in the specimen
nearly
the condenser parallel beam at the
a wide range of magnifications can be attained by and lenses, and specimen by
introducing one or more intermediate lenses. Direct
Creviceswill produce fewer detectable cathode
the
specimen, the beam after is
passing through
whereas projections will be highlighted.
electron (electron gun) magnified image of the projected
as the
magnifications as high as 1,000,000X may thus be
variations are used to modulate the beam anode (5) object.
Specimen onto the fluorescent
obtained and the micrographs further enlarged ( a c c e l e r a t i n g

photographically to 10,000,000X or more depending

across the picture tube, thus
producing an i objective and projector lenses. screen by the
image
voltage)

With the scanning EM, surface 6) The

upon the resolution achieved. view specimen for TEM are cut into ultrathin
specimen
H
is obtained by using the sections
Condenser using ultramicrotome.
Secondan lens the sections are not
thin, the
In case
electrons would
Simply be stopped and
visual display formed.
no
image would be
(long persistence When electrons
electron gun CRI) of the section
impinge on the section, portions
specimen through which electrons have passed
electron beam-
appear bright in the fluorescent screen while
deflection coils of the portions
specimen that have absorbed or scattered
objective lectrons because of their
inherent density or because
lens
photographic of heavy metals added
during specimen preparation
tirst display appear dark.
condenser lens-
(short persistence
CRT)
Scanning Transmission Electron
electron beam
In CRT
Microscopy (STEM)
second
Recently, a combination of the scanning and the
Condenser lens- Scan transmission EM has been developed in the so called
generator
STEM. The STEM uses a thin electron beam (a
deflection coils few A° in diameter) to scan the specimen. The
objective lens- projector scattered collected and
electrons that are are
video jens
obtained
amplifier analyzed by special detector and the image
is displayed on a television screen.
The STEM, is particularly useful for observing
the high contrast
target
viewing the biological specimens. Due to
Screen to study unstained
(specimen) photomultiplier thus obtained, it is possible
or camera reliable
molecules and to obtain
a more
proteins metals.
image than those stained by heavy
Fig. 7. The Scanning Electron
Microscope. The beam is swept back and forth across the
specimen in synchrony with a beam moving across the face Confocal Microscopy
of a television
Radiation (scattered
or
secondary electrons, picture tube. T Ohe Electron Microscope. Compare with Fig.
observation beam,
from etc.) the sample is used to modulate the The entire column, from
producing variations in brightness. filament to screen microscopy, now a well
established too
BC-43) ite very high vacuum. Note the small Confocal
electron microscopy. Du
both light and
ngle, greatly enlarged in this drawing complements
(BC4