Section I Hematology

Study of the Compo und Mic

roscop e
Learning Objectives
After completing this practical,
you will be able to (MUST KNO You may also be able to (DESIR
1. Iden tify different parts of the W): ABLE TO KNOW) :
co mpound microscope. 1. Describe the pri nciple of mic
2. List the uses of different part roscopy .
s of the microsco pe. 2. Explain the physical principles
3. Make microscopic adj ustm ents of construction of the compound
for view ing the object under low -pow microscope.
high-power, and oil-immersion obj er,
ectives. 3. Provide the solutions for com
4. List the precautions to be take mon problems encountered in mic
n while using the microscope. 4. Explain the basic principles of roscoh
5. Handle the microscope carefull working of other types of microsc
y. o pes.

INTRODUCTION ma gn ify an ob jec t to a po

int w h ere it ca n b e see n
the hu ma n eye. Be for e a '-' ith
Th e mi cro sco pe is usu all stu de nt lea rns mi cro sco
y use d in ph ysiology to stu sho uld un de rst an d a few py. he
the m orp ho log y of blo od dy ph ys ica l ter ms fun da m e1
cell s an d for ma king differ to mi cro sco py- th e res olu 1 ta\
cell co un ts . Th ere for e, ent tio n, the wo rki ng dis tan
a physiologis t sho uld an d the nu me ric al ap ert ure ce
co mp ete nt in mi cro sco py be .
an d a stu dent of physiolo
sh o uld lea rn the bas ic gy 1. Resolution Th e lim it of u sef
pri nci ples of mi cro sco py ul ma gn ifi ca tio n o a
ob ta in the be st pe rfo rm . To mi cro sco pe is set by its
an ce from a mi cro sco pe, res olv ing po we r , tha t is
on e ne ed s to un de rst an ability to rev eal clo sely its
d the op tic al pri nci ple s, ad jac en t str uc tur al detail
ba sics of the co nst ruc tio the sep ara te an d dis tin ct. Re , as
n of the mi cro sco pe, an d so lut ion , the ref ore , desc1
scientifi c ba sis of rou tin e the how small ind ivi du al ob jec Jes
car e an d ma int en an ce of ts ca n lie clo se to eac h o
in str um en t. the an d still be rec og nis abl e. ier
G en era lly, the hu m an eye
A mi cro sco pe ma gn ifie s sep ara te (or res olve) do ts l,an
the image of an ob jec t an tha t are 0 .25 mm apa rt·
in simpl e ter m s, it is a ma d li gh t mi cro sco pe can sep the
gnifying glass . Th e mo de ara te do ts tha t are 0.2 5
co mp o un d mi cro sco pe (li rn ap art ; an d the ele ctr on mi 1111
gh t mi cro sco pe) is on e of cro sco p e ca n sep ara te
mo st fr eq u entl y use d eq uip the tha t are 0 .5 nm ap art . 01s
me nt in me dic al lab ora tor
fo r stu de nts. Im pro pe r ies
use of the mi cro sco pe lea
to los s of cla rit y of the ds Note: 1 nanometre (nm)
im age, which res ult s in
los s
= 0.001 micrometre (~Lm)
of def ini tio n. Th ere for e,
co nd itio n , op tic all y an d me
it mu st be kept in excelle
nt = 0.000 001 mm
ch ani cally.
Mi cro sco pe s de sig ned by Th e_res olv ing po we r is
dif fer ent ma nu fac tur ers th e mt cro ex pre sse d qu an tita ti\'L 3
'
d iffe r gre atly in de tai ls of sco pe 's lim it of res olu
the ir co nst ruc tio n an d mo th e mi tio n (LR) , fr is.
of op era tio n . Th ere for e, de nim um di sta nc e be tw ee
the ma nu fac tur er' s ma nu n tw o visible boc 31
an d in str uc tio n s for op era al wh ich the y are see n as sep
tin g the mi cro sco pe mu st ara te an d no t in contac th
fo ll ow ed pro pe rly before be on e an oth er• Th e LR is i
using the in str um ent . de ter mi ne d acc ord ing
following for m ula: ihr
I Physical Terms LR = 0.61
Th e co mp ou nd micro sco
xW
pe con sis ts of two a ·r NA
. .
len ses : the obJec ·
t1ve an d the eye\Jie ce It 111cg111 ymg
· ·s
1 use d to wh ere W is th
' . ht
e wa ve 1en gth of the lig \ .
th e nu me rical a ra \ : .- . ,ll 1
f h
pe rtu re o t e ob_.1ec't1\e
-: . · ,
111 u~,
 Study of the Compound Microscope

Eyepi ece - - - - - ~ = : : : ) ,
L'11
For example, if green light (wavelength 0. 55 ~1111 )
and oil-imm ersion objective (N A 1. 3) are used, the LR
will be 0. 25 µm (0.61 x 0. 55 / I .3 = 0.25 µm ).
2, Working distance Wo rkin g di stance is the distan ce Body tube
between th e objective and the objecti ve slide. Coarse
adjustm ent
The workin g distance decreases with increas ing screw
Fixed and
magnific ation. It is 0. 15- 1.5 111111 in the case or the Fine
revolving
nose piece
oil-imm ersion objective, 0.5- 4 mm in th e case of adjustment
screw Objectives
high-power, and 5- 15 111111 in th e case of low-power
objective.
Handle Stage
3. Numerical aperture (NA) The numeri cal aperture of a
lens is th e ratio of the diameter of the lens to its focal
length . Any particular lens has a constant numeri cal
aperture and this value is dependent on the radius of
the lens and its fo cal length (the distance from the
object being viewed to the lens or the objecti ve).
NA of a lens is an index of the resolvin g power. As
the NA increases, the resolution (or di stance from
each oth er at which objects can be distin guished ) Fig. 5.1 Parts of the compound microscope.
decreases. That means the greater th e NA, the greater
is th e resolvin g power. The NA fo r low -power, high- 1. Base The base support s th e microscope and is
power and oil -immersion objectives are 0.3 0, 0.65 horseshoe-shap ed to provide maximum stab ility.
and 1.30 respecti vely. The NA is also described as an
2. Pillars Two upright pillars project upwa rds fro m th e
index of the light-gathering power of a lens, th at is, the
base, and th e handle of the microscope is hinged to th e
amount of light entering th e objective. The NA can be
pillars.
decrease d by decreasing the amount of light that passes
through a lens. Therefore, th e illumina tion has also to 3. Handle (arm) The arm support s the magnifying
be increase d in the same order when the objectives are and adjustin g systems. It is also th e handle by which
changed fro m low-power to high-po wer. the microscope can be carried with out damagin g th e
delicate parts. It is curved and th e microscope can be
tilted at the hinged joint when des ired.
PARTS OF THE COMPOUND MICROSCOPE
4. Body tube The body tube is the part th rough whi ch
There are two commo nly used compound microscopes: the light passes to the eyepiece. The length of the
monocular and bin ocular. Basicall y, th ey are the same tube is usually 160 111111 . This is the tube th at actuall y
except that th e monocular microsco pe has one eyepiece conducts the image.
(ocular) whereas the bin ocular mic roscope has two 5, Stage The fi xed stage is th e hori zo ntal platfo rm on
eyepieces. The structur e of th e compound microscope which th e object being observe d is placed. The centre
can be discusse d under four main systems: of the stage has an aperture centre through whi ch the
I. The support system (th e fram ework) converging cone of light passes. Most microscopes
2. The illumina tion system have a mechani cal stage which makes it much easier to
3. The magnifi cation system manipul ate the objects being observe d. It is calib rated
4. The adju stment system and fitt ed on the fixed stage. There is a spri ng- mounted
clip to hold th e slide or the countin g chambe r in
I The Support System pos ition, and two screws for moving these transversely.
or forwa rds and backwa rds.
The support system is the framewo rk of the microscope
6. Nosepiece The fixed nosepiece is attach ed to the:
that holds its components. The fra mework consis ts of
lower end of the body tube and th e revolvin g nosepiecc
several units (Fig. 5.1) .
U "I Hematology

is m o unted under it. T h e revolvin g nosepiece carr ies The plane mirror is u sed _fo r th_e oil -immersion objectiv~
the objective len ses of di fferent m agn ifyi ng powers . whereas the concave m irror 1s used for th e low- and
high- power objectives.
I The Illumination System Condenser
A microscop e canno t fun ction optimally withou t
pro pe r illuminatio n. The illumination system provides The condenser foc uses the rays o f ligh t reflected
unifo rm a nd soft-brig ht illumination o f the entire fiel d fro m the mirror on to the object u nder examination
: iew e d under the microscope. The illumination system and also helps in resolving the image . It is mounted
is,_ therefore, an importa n t part of th e com pound li ght below the stage of the m icroscope w ith a rack and
mi c rosc o pe. pin ion mechan ism fo r adjust ing its foc u s. M icroscopes
There are six types of illu m inatio n systems based o n generally use a substage Abbe-type con denser. This
w hich th e microscopes work : Abbe condenser is composed of two lenses uncorrected
fo r spherical and ch rom a tic aberration. T herefore.
1._ Bright-field or light microscope This u ses white ligh t,
for better microscopic examination, a good q uality
e ither ext ernal sunlig ht o r internal tun gsten filament
achromatic condenser should be used . The condenser
la mp , as the source o f illumina tion. When viewed
can be raised and lowered beneath th e stage by m eans
under the microscope, objects look d ark or coloured,
o f an adjustment knob . l t m u st be correctly positioned
contrasted against a lighted background.
to focus the light p roperly on the object being viewed.
2. Dark-field microscope A special da rk -fi eld condenser is because, being a lens, it has a fixed n u mer ical aperture.
u sed that lights up the object, li ke stars against a dark T he numerical aper tu re of the condenser should ie
s ky . equal to or sligh tly less than the n u mer ical aperture of
3. Fluorescent microscope T hi s uses a special ultraviolet the objective being u sed. Cha ng ing the pos itio n of the
la mp as th e source of illumination . A flu orescent dye is condenser can vary its numerical aperture. T herefo e,
a ttach ed to the object th roug h laboratory procedures; the position of the condenser m u st always be adjusted
thi s glows whe n exposed to ultravio let radiati on. with each objective used, to get the maximu m focus
4. Polarising microscope o f_ light and the accurate resolving power of 1 1e
microscope.
5. Phase-contrast microscope
. Wh:~ a low -power objective is u sed , the conden ~r
6. Interference-contrast microscope ts _pos_1tion~d _at th_e lowest level; w ith a high -pm ~r
The illumina ti o n sys tem of a compound microscope obiect1ve, It is raised optimally; and with an l !-
co n s ists of a lig ht sou rce, cond enser and iris d iaph ragm . immersion ob· t· ·
. Jee ive, It is raised fu lly. Most mod ·n
microscopes do not n d d
d .
I Light Source ee a con enser adjustment: 1e
~~n ~ns~r ,s_ placed at the highest position and 1e
i u m111at1on is adjusted . ·1
Th e illuminatio n sys tem begins wi th a source of light. th · .· d' pnman Y by openino· or elm 1g
e 11 is iaphragm . o
T his m ay b e in ternal or extern al.
Internal source ln m ost m o d ern com poun d microscopes, I Iris Diaphr!!rim
th e re is a b uilt-in light source w ith an electric lamp,
The iris d iaph rao-m
w hi c h provides b etter con trol of illumination . Th e lamp passes th rough ~h regu 1ates the amount of light 31
hou s in g has a frosted tu ngsten lamp, wh ich is placed e material d . ·,
1
located at the bott f
0 111 o the
u n er observat ion. 1 •
directly under the stage. aperture, which can be d al
con enser. It has acer
External source ln the students' compou nd microscope for less li o-ht ace d ' opened fo r more light or ck ·d
t h ere is no in -b u ilt light sou rce . Th ese m icroscopes use o , or
1ever provided w·th mg to necessity, . .i
by m eans '
an external source of ligh t. T l: is can _b e fro m an electric .
1 i a shutte Th .
i egu ates the amou t f r. e size of the ar er
11 0
d in a la m p box w ith a w 1t1dow, or fro m the und er b J
lamp h ou Se . . . . o servation. Reo-. light . th at passes to the
of ligh t are refl ected by a mm 01 towa1 ds means affects the o_ulation of the lio-h t b, , ,.
s un . Th e ray S B d .
. t The m irror is located at the base of the numen ca! c- .
the o b JeC . . d _Y re uc1ng the field . aperture of the conJL'
. It has two surfaces, plane an concave . diaphragm. th _s ize With the h ,] l1I tli,'
m icroscope . e numerical c r
aperture or till' L·, '' " l,
is dec reas ed. Thu s , pro per illun11·nat·ion pro ce d ure
. . . 1 ·
incl udes a com bm at1o n of light inte ns·t1 y regu at1on ,
. . .
pos itio n, and field size
light so_urce pos1t1on , con den ser
regulation .

I The Magnification System

extremely imp orta nt
The 1~agnification sys tem plays an
bec aus e it macrnifies
role m the use of a mic ros cop e
view. The com ;ou nd
the ima ge of the obj ect und er
nifying lenses , the
rnic r?sc ope con sist s of two mag
total magnification
eyepiece and the objective. The
cop e is the pro duc t
pro vid ed by a com pou nd mic ros
d by the objective and
of the mag nifi cati on con trib ute
piece forms a virtual
tha t by the eye piec e. The eye
nifi ed ima ge form ed
mag nifi ed ima ge of the real mag
by the obj ecti ve.

I Eyepiece Fig . 5.3 The binocular research micr

oscope.

lens tha t magnifies the pro duc ed by the

The eye pie ce or the ocu lar is a multiplied by the mag nifi cati on
ima ge for me d by the objective.
It fits into the top of cati on of the object
objective gives the total mag nifi
es are pro vid ed wit h
the bod y tub e. Mo st mic ros cop bein g viewed.
h mag nify ing pow ers
two eye pie ces , 5 x and 10 x , wit
r, 2x, 8x and 20x
of 5 and 10 resp ecti vely . How eve
st mic ros cop es have
I Objectives
eye pie ces are also available. Mo orta nt par t of the
and the se are called Objectives are the mo st imp
a pro visi on to fit one eye pie ce thre e objectives are
5.2 ) , whe rea s som e mag nifi cati on system. Usually ,
mo noc ula r mic ros cop es (Fig. ece in a com pou nd
fitting two eyepieces scre wed into the revolving nos epi
mic ros cop es hav e a pro visi on for pivot tha t ens ure s a
bin ocu lar mic ros cop es mic rosc ope . The nos epi ece is a
at a tim e and the se are call ed thre e objectives are
duc ed by the eyepiece qui ck cha nge of objectives. The
(Fig. 5.3 ). The mag nifi cati on pro 40x or 45X : high-
(a) 10x : low -po wer objective, (b)
100 x: oil- imm ersi on
pow er objective, and (c) 90 x or
objective (Fig . 5.4 ) .
ve is usually
Low-power objective The low -po wer objecti
10 times. Thi s
10 x , whi ch mag nifi es the ima ge
g and obs ervatio n .
• obj ecti ve is use d for initial foc usin
low -po wer obj ectives
Som e mic ros cop es also have very
ves. Th ese are use d in
(3 x or 4 x) , the sca nni ng objecti
tion s.
initial sca nni ng of hist olo gic sec
of the low -po wer
The num eric al ape rtur e (NA)
t of the con den ser in
obj ecti ve is always less tha n tha
to achieve foc us, the
mo st mic ros cop es . The refo re,
mo re closely ma tch ed
num eric al ape rtur es mu st be
cim en. Thi s ~an be
by red uci ng th e light to the spe
ser and by clo smg thL'
ach ieved by low erin g the con den
iri s dia phr agm (iri s slightly ope
ned ) .. NA ?f
~b_iect ih'~
acit y ol tn11..:rL' :s(,' t '~'
CE
is rela ted to th e ma gnifica ti o n cap
(Table 5.1 ) .
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Section I Hematology

un de r th e oi l- im
m er si on ob j ec tiv e
ap er tu re of th e hi . . . Th . e nu rn Cr1.c:~
gh -p ow er ob1 ect1
to (o r sligh tly le ss ve 1s al mo~ t cl
th an ) th at of m o st
co nd en se rs . T h er c orn1 Tlon\ y u~:~
efo~e.' th e c~ nd
sli ghtly ra ise d an d en se r sh ou ld bt
th e m s b e pa rti all y
o pe n ed to achi e, l
m ax im um focu s.
Oil-immersion ob jec tiv e T h e oil -im m er si on
ge ne rally a 90 x or o bj ec tive i~
10 0 x le n s , w hi ch
im age 90 or 10 0 m ag n ifies the
tim es. T h e ob je ct
rest s on th e slides iv e le n s alm o~t
w he n in u se (Fig .
a sp ec ia l type of 5 . 5) . It re qu ire1
oil ca lle d im m er si
pl aced be tw ee n th e on oil , w hi ch ii
obj ec tive an d th e
co m m on ly used im sl id e. T h e most
F~g . 5.4 Objectives m er sio n oi l is ce da
of a mi crosc ope. Lo w- used to in crea se th r w oo d o il. Oil is
high-pow er ob 1ect1ve power objective (10 x) e nu m er ic al ap er tu
(40 x ) and oil -imm ers , re so lving po w er of re an d th us the
ion ob jective (100 x ). th e obj ec ti ve . Li gh
air at a gr ea te r sp t tr av el s th ro ugh
Table 5.1 Relation of NA of ob ee d th a n th ro u gh
jectives to the magnific th e imm er sio n oil , glas s ; th ro ugh
Objective ation . lig ht tra ve ls at th
NA th ro ugh glass. Th er e sa m e sp eed ~as
Magnification efor e, th e o il is u
Objective Eyepiec th e sp ee d at wh ic h se d to d ec re a~e
Lo w-power e Total lig ht tra ve ls to in cr
0.30 nu m eric al ap er tu re ea se th e effecti e
10 10 of th e obj ec tiv e . It
High-powe r 100 also d ec reas,',
0.6 5 45 th e defraction of lig
10 450 ht ra ys .
Oil -immersi on ~in~ e t~ e nu m eric al -
1.30 100 10 1000 ap er tu re of th e oi
obie ctJve is alw ays l-i m m er si c 1
High-p ower ob jectiv e It gr ea te r th an th at of
is us ua lly a 40 th e . ~o nd en se r sh th e co nd en se .
m ag nific at io n le ns x or 45 x ould be plac ed
. It m ag pos1t1o n an d th e iri at th e hi crh e t
tim es. Th i s obje ct ive nifies th e im ag e 40 s di ap hr ag m sh o ul
is used fo r m ore de or 45 (Tab \~ . 5 .2_) . d b e full y ~ pc 1
th e to ta l m ag ni fic at ta ile d stu dy, as m ag rn f ica t1on Th
of
e oil- im m er si on
le n s ·v
4 00 or 45 0 ti m es .
io n (w ith a 10 x ey
epiece) is us ua lly eyep iece Th f l 00 0 ti 90 g1 es a
It is used fo r a br
oa d view of bl oo d m or ph ol·og ic er
.
e or e it is _m es or O tim es w ith a to IO
t,
,
fi lm s or hi st olog ic exam· ' . cr \\
se cti on s pr io r to th slides.
0
en er a Y u se d fo r
eir examin ati on m at io n of bl oo d fi d et ai \e
lm s or hi sto l og i

45 x
10 0x

O bje cti v es

Sli d es

M icr os co p
( IC fie ld
ma gn ific ation ) s

I Distance (working dis

F g 5 5
obtained unde r the thr . . tance ) between th e objec tive len
ee ob1ect1ve s. se s and the objec t in
the three type Ot .
.
s ob 1ect1v
es . Note •1,1: en~
•Q IW .
 Study of the Compound Microscope

Table 5.2 Adjustments in the microscope for using differe

nt on the stage of the micro scope. Parts of the specimen
ll il
object ives. that are opti cally dense , have a high refractive index or
Objective Mirror Condenser State of iris are coloured with a stai n, cast a potential image like
position diaphragm a shadow which is magn ified in different stage s as it
Low-p ower Concave Lowes t Partially closed passes up th e microscope to the eye.
High-power Conca ve Slightly raised Partially
opened I Requirements
Oil-immersion Plane Fully raised Fully opened
I . Microscope
2. Light source
I The Adjusting System 3. Blood film
The adjus ting syste m consists of two adjustment
systems: th e coarse adjus tment sys tem and the fin
e I Procedure
t system is
adjus tment sys tem. Th e coa rse adjus tmen The microscope shoul d be handl ed carefully. Befor
e
e
used to obta in an ap prox imate foc us whereas th e fin using th e microscope, examin e it thoro ughl y. The
of
adj ustm ent system is used to obtai n the exact foc us student should fo llow the steps given below while using
the objec t aft er prior coa rse adjustment.
a comp ound microscope .
I. Place the microscope on the wo rking table in the
I Coarse Adjustment System upri ght pos ition and adj ust the height and positio n
Two coars e adjustment screw s are used for makin
g of your chair so that you are comfo rtable and
coarse adj ustm ents . Th ese screws are moun ted on prepared for prolo nged viewing. The eyepi eces of
the top of the handl e by a doubl e-sid ed micro meter the microscope should level with and be close to
mechani sm, one on each side. If one screw is rotate
d, your eyes whil e you are sittin g upright (Fig. 5.6).
its member on the opposite side also rotate s at th e Keep your fo rearm s on the table so that you can
sa me tim e. Therefore, there is no need to operate
the eas ily handl e the adjus tment screws . Yo u need not
adj ustm ent screws from both sides simultaneo usly. re move your glasses if you use them constantly.
t
If the left hand is used for handling the adju stmen
th e
screw , th e right hand ca n be used for manipulating
body tube or
body tube or th e mechanica l stage. The
rse
stage can be raised or lowered quick ly with the coa
adjus tm ent screw .

I Fine Adjustment System

fin e
Two fine adjus tmen t screws are used fo r makin g
th e
adjustmen ts. Usua lly these screws are mounted on
s by doubl e-
handl e below the coa rse adjus tment screw
sided micro mete r mechanisms, one on each side.
and
These screws are operated fo r fin e adjus tment
exact foc using of th e objec t.

METHODS

Method of Use of the Microscope ..

Fig. 5.6 Wo rking with the microscope. Note that the eye
at the level of the eyepiece in the upright s1tt1ng
position should be
I Principle position ; an examiner using spectacles should wear them while
ment.
. f cused beam of li ":crht viewing through the eyepiece. (Courtesy. Ph ysiolog y Depart
In the comp ound microscope, a o Hemat ology Labora tory, JIPME R, Puducherry, India.)
. b. t laced on a bcrlass sli de
~ca ns the specimen or o Jee P
.---~_
H_e matolo gy - - - - - ~ - - ~ - - - - - - - ~ - - ~
--~~-~~---~------
a nd th e o th er on th e screw to move th e s ta ge_
Note: Observers who wear glasses may be abl e to dispense with
• Brin g th e o bj ec t o f in teres t to the centre.
them when _using a microscope: if not, they must take care to
prevent their spectacle s to uching and scratchi ng the lenses of 7. A ft e r pre limin ary sc reeni n g u nder lo w- riowt
the eyepieces. o bj ec ti ve, procee d to exa m in e th e film und e r hi gh
powe r o bj ect ive . Th e s teps arc:
2. Ch ec k th a t th e eye pieces a nd obj ec ti ves a re free
• Brin g th e hi g h -p owe r objec tive (40x or 43 x
fro m du s t a nd o il. Use a fres h len s tiss u e fo r thi s
into pos iti o n b y ro ta tin g th e n osep iece ; 117ak
pur pose .
sure th a t th e obj ective c li c ks int o pl ace .
N_ote : Only benzol or xylol should be used to re move hardene d • Use th e co ncave mirro r , rai se th e co nd en~c
OIi. sli g htl y, a nd c hec k that th e iri s di a phragm i
pa rti a ll y o pe n so that th e illumin at io n is p roricrl
3. Provid e a d e q u a t e I·11 um111
· a t1·o n. 1{· yo u h ave to use ce ntred . Increa se th e illumi nation a s n eed ed. ·
the ex~e rna l la mp , pl ace it ab o ut 20 cm away fro m
• Repeat th e proces s o f fo c u s in g as describ cc
t~ e micro scop e , switch o n th e la mp a nd all ow th e
ea rli e r by u sin g th e co arse a dju stmen t kn ob anc
l~ght to fa ll o n th e mir ro r . lf you have to u se natu ra l
th e fin e a dju s tment kn o b in sequ e nce.
light , pl a c e th e mi c ro sco pe near th e w ind ow for
m aximu m illumin ati o n.
Note: As the high-po wer objective does not normally touch the
4 . S e lect a nd a dju s t th e mirror for o ptim al illumin slide (check carefully the distance between the slide and th e hioh-
ati o n
a c cordin g to the objecti ve to be u sed. Di rect the power objective at its lowest positi on) , the use of th e coarse 2 id
p ~th o f li~ht to pass th ro u gh th e ho le o f the stage fine adjustment knobs may not be so critical and they can 1e
w ith maxim um inte n sity w hil e settin g th e mirror switched freely.
(l o ok fro m th e side to ch eck illumin a ti o n ).
8. After scree ning und er th e low - powe r , nd
5. Place th e slid e with th e o bj ect o n th e stage, such
examin ing und er th e hi g h- power , u se t he ii-
th a t it is h e ld b y th e s ta ge cli ps a nd p ressed at bo th
e nd s so as to b e in close co ntact w ith th e surface o f immer sion obj ective to obta in grea te r d e tail s o f ,he
obj ect. The ste ps a re :
th e stage.
6 . M a ke vari o u s mi c ro sco pic adju s tm ent s to view • ~win g away ~h e hi g h-po w e r o b jecti ve a nd p ta
th e o bj ect unde r low-po we r obj ec ti ve (th e co rrect tm y drop o f imm e r sio n- o il o n th e s lid e over he
proce dure is to fir s t vi ew th e o bj ec t in low -powe r). path o f li ght.
Th e s te p s are: • Chan ge th e mirro r to th e plan e si d e
• Ra ise th e co nd
• Bring the low-po we r o bj ecti ve ( I Ox) into e n se i_ to m ax imum . · (to p ,ce
p os iti o n b y revolv ing th e no sepi ece (th e obj ec ti ve b e Iow th e s ta ge ) and
. . o pe n th e m . .s full y to ob .
11n
mu s t click into place ). max imum illumin ati o n
• Adju s t th e illumin ation to improv e co nt ras t. Use • Turn . th . e . nose pi ec e a n.d set th e o il- imm e1 on
th e c on cave mirror , place th e co nd ense r a t th e b
oh Ject1ve 111 pos it"
. . io n ; m a 1<e s ure th a t th e obj e \e
lowes t pos ition , a nd sli ghtl y o pe n th e iri s. as c 1icked mto pl ace.
• Use th e fin e ad·
Note: For tow-power, the illumination is cut down to the minimum . f JU St m e nt kn ob to act t he ol ~Cl
117 oc u s. If thi s f ·1 1
31 s, oo k fro m th
by reduc ing the aperture size . th e e
ye 1eve] w ith th
b
e sid e. er
1·d
• U se th e coa rse focu sin g a dju s tm e nt to foc us th e
obj ective f e s I e a n d lower Iie
ca re ull y w it]1 11 1
kn o b until th . . t e coarse adju ~t ' n
s pe cim e n o n th e slide . e o tl-1m me 1. .
th e o il L s ion o b JCct1
. .
ve t OL c'
·
Note : Never use th e fine focusing adjustment until the specimen stop wh ower
e nt1
th
1 eo il -i
° e b " ·
Ject1ve fu rth er d own 1d
.
has been made visible and brought nearly into focus with the th e slid e ( . m m crs1on objecti ve to L "
ca uti o n ·
co arse adju stment. the p repa ra t · ) · avoi·d pressin g ha n ' n
10 17
th e coarse ad· · Firs t , focus th e o bjec t •h
• Use th e fin e adju s tm e nt o nl y to obt a in and
the gap b t JU St mcnt kn o b wh ile in crL 1~
main ta in exac t foc u s. Put o ne ha nd on the .
F1na l! y foeeween _ th , l"d
c s I c a nd the obll.
focusi n g k no b (coars e o r fin e , o ne at a tim e) ' u s t11 e ob · .
adju stm ent kn o b . JCct wi th th e h elp ul" th ·
 Study of the Compound Microscop~

I Precautions
1. The microscope should be placed on the working
table in a stable position.
I Common Difficulties in Microscopy
A number of difficulties may be encountered by
beginners. The following tips are given to overcome
-
2. The height of the observer's chair should be raised them.
to a position that allows for comfortable handling I. Inability to achieve focus or obtain a clear image.
of the microscope.
i) The failure to find focus may be due to the slide
3. Objectives and eyepieces should be free from dust not being brought close enough to the objective
and oil. Xylol or benzol should be used to remove to remain within its focal distance, or there
hardened oil. Lenses should never be touched with may be no visible material on that small area
the fingers. of the slide within the field of the objective.
4. If natural light is used, the microscope should First, move the slide so that the focus material
be kept near the window; if the lamp is used, it is brought into the field. This procedure will
should be kept about 20 cm from the microscope, ensure that there is visible material to focus
to prevent heating of the microscope. on . Then, with the eyes level with the stage,
5. The mirror , the position of the condenser and the use the coarse adjustment to raise the slide
until it again comes as close as possible to the
aperture of the iris should be checked in order to
objective without touching it. Finally, apply
get proper illumination.
the eyes to the eyepieces and use the coarse
6. While changing the objective, it should be noted
adjustment to move the objective away from
that the objective clicks into its proper position .
the slide until the specimen is seen in focus.
Otherwise the objective may not remain in position.
ii) Check that no dirt or dried oil has adhered to
7. Never bring down the objective with the coarse the objective lens. If so, clean it thoroughly.
adjustment while looking through the microscope.
iii) Check that the slide carrying the object has not
You should look from the side. been placed upside down on the stage. If so,
8. Examination of the specimen under low and high reverse it.
power should always precede examination under iv) Check that the immersion oil has not become
the oil-immersion objective. sticky. If so, wipe off the old oil and replace.
g. The stage of the microscope should always be v) Check whether the specimen is covered with a
brought down before bringing the oil-immersion layer of dried oil or dirt (left on it by a previous
objective into position. Otherwise it may damage viewer). lf so, clean it with a lens paper
the preparation. . . moistened with benzol or xylol.
10. The distance between the slide and the obiect1ve vi) Check whether the coverslip placed on the
should always be checked while using the coarse specimen is too thick or whether the mountant
adjustment screw, especially for high-power and is so thick that the objective cannot reach close
oil-immersion objectives. to the specimen to bring it within its focal
11. The stage should be always kept ~lea~ and not be length.
soiled with specimen material , stam , 011 or water . vii) If none of the above steps improve the
12. The microscope should be kept covered when not performance of the microscope, ~ou. should
consider the possibility that the obiect1ve may
in use.
be faulty. Exchange the objective with one
from another good microscope and if a sharp
image is obtained, discard the fau lty one.
DISCUSSION 2 _ A dark shadow in the fi eld resulting in loss of
. . · scopy . and the definition of the image. . .
The common difficulties 111 micro i) This is usually due to a dirty ~yep1~ce . If the
. are and mamtenance
solutions to these , tips for routme c . shadow moves when the eyepiece 1s rotated.
of microscopes are
of microscopes , and other ty pes rem ove the eyepiece and clean it.
discussed here .
 =========================-- . e Care and Maintenance of the
ct o,,,~r
i~ ;em; a;to;lo~g~y
...=~H
ii ) It may also be due tot e pl
h, ·esence [Link]'a1·1· bubble
ove the oil
I Routin
Microscope . ------
ly maintamed can be usect lo
;n the ;,n,ne,•s;on oH. It ;s better to'.""
from the sl;de and put fresh oil on n. A microscope ;r prope.r th and scratches (caused b,
3. Poor illumination. •t I Fungal gi ow . \
· ·opna eY many years. h ns damag e micro scope s in .
i) Check whethe r the condenser 15 appi dust particl es) on t e Ie '
positioned, that 1.s, rac I<e d fu lly upwards. . .
Sometimes it slips downwards in its rnount mg short time. ing pomts . be noted carefully for
The follow must
ring. It should be pushed up so that it c~n be . f the micro scope :
racked up to within 1 mm below the specnn en
i;out; ~:::v: r the micro scope is r_o be tran sported
slide, for the oil-imm ersion objective. anu
. carry 1t. bY hO!ding its arm with h b one hand
ii ) Check whether the iris is kept full y open whi le
using an oil-imm ersion objective. keeping th e other hand under t e ase . .
. use durincbr the day ' the micro scope
111. Check that the illumin ation is properly
centred. 2. When not m .

En sure that the concav e surface of the mirror should be covered, preferably with a plastic cover
faces th e light wh ile usin g the low- and At the end of the day, blow off the du st particle)
high-power object ives and the plane surface from the surface and store th e micro scope in a
face s the light while using the oil-imm ersion warm and dry place. Do not store the micro scope
objective, and that it is in the correc t position in its wooden box.
to reflect light centra lly into the conden ser. 3. Remove oil from the oil-im mersi on obj ecti,t
4. Presence of unclea r image under th e oil-i111mersion immed iately after use by wiping with a clean len1
objective. paper.
i) It sugges ts a problem in the slide or with the 4. Clean the eyepiece frequ entl y becau se it i1
objective. First check the slide to see whether vulnerable to dirt as it is placed at the top o1 the
it contains dirt. lf so, clean it with benzol. microscope and usuall y comes in conta ct wit!, the
ii ) Check the objecti ve. and if required, clean it observer's eye. An air syring e can be used fo1 thi1
proper ly. purpo se.
iii ) Check for ai r bubbles in the specim en by
5. Do not remov e the eyepiece from the microscope
holding the slide again st the light.
for a long duration ; otherwise dust will ente1 the
5. Object not comin g into focus even when the
body tube and will be depos ited on th e rear len\ of
objecti ve is in the lowermost position (and the fin e the objectives.
adjustment screw not bringing the objective any
closer to the slide). Thi s happens when the fine 6. While working with the oil-immersi on object ive.
adjustment screw reach es the end of th e thread do not pull out the specimen- slid e from th e ,·a~e
before the object is brought to focu s. To correct without lowering down the stage or swincrb in, 0: 1
th Ob' ' .
thi s, turn back th e fin e adjustm ent screw in the e Jective ; the slide may scratc h the obj e ire.
reverse direction for several turn s and then focu s '."so rei~ember that you shoul d not pu sh th oil·
nnm ersio 11 0 b. ·
the objec_t carefu lly with th e coarse adju stment Jective on th e sl ide · it may da -1ge
knob to fmd the focu s. Finally, sharpen the focu s bot~ the slide and the objective. '
7
by turning the fin e adjust ment knob. . Whil e handling th r· and coarse ad1·ust nt~
. e Ille
Note. It is a good practice to keep the fine adi·ustrn ent kno b.1nthe to _achieve focu s, if the screw s offer u1 ual
'ddl · · 1i
rn, e po_s,t,on . o check the position , turn the fine ad ·u resistance, do not use force to overcome it ' 1i1
knob to either extreme end, then turn back cou t' l stment may damage th
until the knob reaches the midpoint. ' n mg each tu rn · 6etter to c e screw an d the pinion mecl1, ' 11 ·
It is
8 8 ontac t the mech an ic . I
· efore storing th •
6. The /telc/ of view lookin g ova / Ch I . the Ienses. e ITIICroscope after work · clc I h
. the correct.
. .ve 1.s pIaced 111
obJect1 .. < if
ec
h h pos1t1on. · Not e 9. Cleani11g of fe11 se •,\
I at w enever the objective is ch O
click into position . an°ed , it mu st with ringers . Lens .
s Lense s sh ould nc\ er bl' tl
to avoi·d du st scr es are cl ea ned \\'ith , ,,c( 1,
t h ·
· ([Link] c · b. 1f the lcn~ i~ t,1h'11 ,
cleaning .
- or O Jectl\ c ). 1,._l'l'I' 1t ,,,.
 Study of the Compound Microscope

surface. The eyepiece is pulled out from the tube that allows only shorter wavelength light to pass
tn J
while the objective is unscrewed from the nosepiece. through the specimen. If the specimen contains an
First, blow off the dust particles from the surface of object that fluoresces, it absorbs the short wavelength
the lens with the help of an air syringe or a rubber light and emits ligh t of a longer wavel ength. A barrier
bulb or a paint brush, followed by gentle rubbing filter is placed in the microsc ope tube or eyepiece ,
with a lens paper. For cleaning the lens, breathe on which filters onl y the wavelength of emitted light for
it through the mouth but do not clean it by spitting the particular flu orescent system. The flu orescence
or blowing on it. The oil-immersion objective microscope is usually used in immun ology laborato ries
requires proper cleaning . Use clean tissue paper lo study fluorescent antibodies.
for removing oil by repeated gentle rubbing on the
surface. Move the cloth across and not circularly. I Polarising Microscope
Note: Do not use organic solvents like ethanol and xylene A polarising microscope differs from an ordinary
frequently because the solvent may dissolve the cement holding microscope in that it has two polarising devices, a
the lens in the socket. polariser and an analyser. The polarise r (the filter)
absorbs light waves radiating in all direction s and
OTifER TYPES OF-MiCROSCOPES ~ ~ allows light waves from a particular directio n to pass
through the filter . The polari ser is placed usually
between the light source and the specimen and the
There are other types of microscopes that are not
analyser is placed between th e objective and the
routinely used in laboratories but these microscopes
eyepiece. The polari ser and the analyser are rotated
are specially designed and have some adva ntages over
until the two are at right angles to each other. This
compound microscopes. They are based on different
causes disappearance of light through the microscope
illumination systems . the character of light delivered to
because light waves are cancelled when they are at right
the specimen in different systems varies.
angles to each other. However, some objects have the
property of birefringence, that is, the ability to rotate
I Dark-Field Microscope (polarise) light. These objects bend light and can be
A special condenser is used in this microscope, which seen in this microscope. they appear light under a dark
allows light waves to cross on the specimen rather than background.
pass through the specimen. Therefore, the field in view
looks dark, as light does not pass from the condenser I Phase-Contrast Microscope
to the objective. However, if an object is placed on the
An important property of light is its phase. If two light
stage, light is deflected as it hits the object and passes
waves are completely in phase, they show interfere nce,
through the objective which is seen by the viewer .
the resultant amplitud e is greater and brighter light
Thus, the object under study appea rs light against a
is seen. When an obi'ect is seen without stainin cr the
i:, ,

dark background. This microscope is usually used in

indirect waves passing through the object are retarded.
the microbiology laboratory to study spirochetes m
The principle of phase-contrast microscope lies in
exudate s from leptospiral or syphiliti c infection s.
further retardation of th ese indirect waves. This is
achieved by inserting a phase plate within the objective
I Fluorescence Microscope lens . Since some diffracted light passes through
Certain compounds when irradiated by short the grooves of the plate, a halo appears arou nd the
wavelengths, say ultraviol et light, absorb the radiation object. The advantage of this microscope is that the
and then re- emit light energy of longer wavelength , cells or the organisms in wet preparations (without
that is, visible light. This phenomenon is called prior dehydration or staining) can be observed. As the
fluorescence. In flu orescence microsc ope, the material name of the system indicates, the structur es observed
in the specimen that fluoresces becomes visible. show added contrast compared with the bright-field
The flu orescence microscope is a dark-fi eld microscope, microscope. The retarda tion of the speed of light makes
which has been modified by incorporat ing two special the sys tem sensitive to differenc es in refractive index.
fi lters . The condenser is preceded by an exciter filter Objects with differences in refractive index show added
 Her nat olo ']y

uiflcrcnccs in the int ens ity and thr ee- dim ens ion al im age . It
shade of light passing t he is very use ful for
th ro ugh th em. Therefore, one . d. . \I t
can observe un stained prepara tl·ons such as uri na ry se 1ments, showin g r·
we t pre parations with goo details wi tho ut the ne ed for . . . int
d resolution and detail. spe cia l sta mm g.
Th is mi crosco pe is used in
henrntology for counting
platelets, by usin g a dir ect me
thod. I Electron Microscope --------
I interference-Contrast Microscope Th e ele ctr on micro sco pe use
instead of light rays. Th e ma
s a be am of electron,
Thi s micro sco pe yields a thr gn ifi ed im age is visiblt
ee -dime nsional im age of on a fluoresce nt scr een an
the object. A special bea m- spl d ca n be rec ord ed on
itting prism is added to photographic film . T he ma gn 0
the co nd en ser . Th e two split ific ati on ob tained is ver)
beams are then polari sed ; high. It gives the im age on
on e passes th rou gh the spe the ph oto gra ph ic plate.
cim en , which alters the at a magnification of ab ou
am pli tud e of the light wave, an t 50 00 to 20 ,000 timei.
d the oth er (which serves Th e negative is the n enl arg ed
as a refere nce) do es no t pas to ab ou t 10 tim es thui
s th rou gh the specimen. enabling a total magnification
·1 he tw o dissimilar light of ab ou t 12 0,000 x or
bea ms then pass sep aratel y more. In thi s micro sco pe, ele
th rou gh the obj ective an d are ctr om ag ne tic field~ are
recombined by a second use d in place of len ses . Th is
pri sm. Th is rec om bin ati on is u su ally use d fo, the
of li ght waves pro vid es
study of finer details of org an
ism s, cells or tissue~

i•Mwl
I. Make mic r osc opi c adj ust me
nts for foc usi ng a film und
er a low -po we r obj ect ive
Ste ps .
1. Cha nge to co nca ve mir ror.
2. Sli ght ly ope n t he iris d 5. Bring the cond ens er to t he
ia phr agm . low est pos iti on .
3 . Bri ng the low -po we r obj 6. Pl ace the slide on the st age
ect ive into pos itio n . 7 M k of t h .
[Link] t h e fine adj ust me nt . a e coarse adj ust me n ts to e m icro sco pe.
scr ew for fin al foc usin g. f h .
o cus t e ima ge.
II. M ake mic ros cop ic
adj ust me nts for foc usi ng
a fil m und er a h igh -po we
r obJ·ect ·
Ste ps 1ve.
1. Ch ang e to con cav e
mir ror.
2. Par tial ly ope n the iri 5. Slig htly raise the con
s d ia_phr ag m. d
3. Bri ng the hig h- pow er _ _ 6. Pla ce th e slide on the enser.
obJect1ve into pos 1t1on . sta e .
4. Use t h e fin e adj ust me . 7. Ma ke coa rse adJ· ust
.
nt scr ew for f ina l foc u sing g of th e mi c ros cop e .
. rne nts to f
No te: To get t he foc u s Ocu s the i ma ge.
easi ly und er a hig h - pow
er obj ect ive , it is bet ter to
Ill. Make mic ros co p ic . . foc us th f
adJust m ent s for foc usi ng . . e ii rn firs t in a Io
a film und er an oil- imm ers . .
ion obj ect ive .
Ste ps W- pow er obJ ect 1ve.
1. Cha ng e to pla n e mir
ror. 6. Brin g
2 . Op en the iri s dia phr agm the con den ser t
full y. 7 Pl
3. Place a dro p of oi l at . ace t h e sli·de on t h o t he hig h
th e cen tre of the sme ar. 8. Brin g the oil- irnr ne
est posit io
4 . Mo ve th e slid e on t e sta ge of t he rnic:r n.
he sta ge of the mic roscop 9. Use t he fine . rs1on ob · .
brin g the cen tre of t he slid e to adju strn osc o pe .
e u nde r vie w so t hat the Jec:t1ve into . .
ent screw f pos1t1on.
obj ect iv e tou che s t he o il.
_ or fina l foc usi ng.
5. M ake co arse adj u stm ent
s to foc us the_1mage . .
. . ..
No te: To ge t t h e fo cus easi ly und er an oil - 1mm ers1on obJ
. . ect1ve, 1t 1s bet ter to exa
_ :)Ow
m·1
r _ :er
_~ obJ~. ect ive__
.:,_ s. __ _ _ _
_______
ne the f 1
irn sr1

- __ _ __ _ _
_ _ __ _ _ rst in I0 v-.- ard
1,1c 1•
 Study of the Compouod ~
I
· ~
VIVA )

1. Who invented the compound microscope?

2. What is the principle of microscopy?
3. What do you mean by resolution?
4· What is th~ use of the terms numerical aperture (NA) and working distance in microscopy?
5· How are different adjustments made in the microscope while using different types of objectives?

An s: Objective Mirro r Condenser position State of iris diaphragm

Low-power Concave Lowest Partially closed
High - power Concave Slightly raised Partially opened
Oil-immersion Plane Fully raised Fully opened

6. What are the functions of the condenser and iris diaphragm?

7. How is the NA of objectives related to magnification?

Ans : Objective NA Magnifica ti on

Objective Eyepi ece Total
Low - power 0.30 10 10 100
High-power 0.65 45 10 450
Oil-immersion 1.30 100 10 1000

8. Why is a special grade of oil (immersion oil) used in the oil-immersion objective?
An s: Immersion oi l (cedar wood oil or liquid paraffin) is used to increase the NA and thus the resolving power of the
objective. Light travels through air at a greater speed tha n through glass. Thus, to increase the effective NA of the objective,
oil is used to slow down the speed at which light travels (speed of light is measured in terms of the refractive index).
increasing the gathering power of the lens and decreasing the diffraction of light rays. The refractive indices of air, glass and
immersion oil are 1.00, 1.515 and 1.515, respectively.
9. Why should the eyepiece, objective and condenser lenses never be cleaned with paper tissue, gauze or ordinary cloth?
Ans: These optical lenses are softer than ordinary glass. Therefore, cleaning with paper tissue, gauze or ordinary cloth will
scratch the lens. To clean the lenses of the microscope, a lens paper is used . Before polishing with a lens paper, care must
be taken to see that nothing is present on the surface that will scratch the optical glass in the polishing process. Potentially
abrasive dust or dirt is blown away using an air syringe before polishing.
70. Why should the oil be removed from the oil-immersion objective immediately after use?
Ans: Oil is removed from the oil- immersion objective by wiping with a clean lens paper immediately afte r use.
If it is not removed, it may dry on the outside surface of the objective or may seep inside the lens and damage it.
77. What microscopic adjustments are made when the field of view is not clear?
72. What is the cause of dark shadows in the field of view, and how can this be prevented?
73. What microscopic adjustments are made if the image is not clear in the oil-immersion objective?
74. What is the cause of oval field of view, and how do you correct it?
75. In microscopy, what does the term parfocal mean?
Ans : It means that if one objective is in focus and a switch is made to another obj ective, the foc us wil l not be lost. Thus
the microscope can be focused under low-power and then switched to the high-power or oil-immersion objective and it will
still be in focus except for fine adjustment. Usually, after focusing in low-power if you switch to hi gh-pow er, on e comp lete
rotatio n of the fi ne adjustment screw (which adjusts about one micron) brings the object into cl ear focus.