Eyepiece Lens: the lens at the top that you look through.

. They are usually 10X or 15X power.

%:-e Connects the eyepiece to the objective lenses
72 Supports the tube and connects it to the base
,se The bottom of the microscope, used for support
:2in,947: A steady light source (110 volts) used in place of a mirror. f your microscope has a mirror,
it is used to reflect light from an external light source up through the bottom of the stage.
$9,e: The flat platform where you place your slides. Stage clips hold the slides in place. f 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.
#ev4vin N4sepiece 47 %:77e9 This is the part that holds two or more objective lenses and can be
rotated to easily change power.
-ec9ive 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 DN 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, parcentered, parfocal
lenses.
#,ck $94p This is an adjustment that determines how close the objective lens can get to the slide. t 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 f 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)
4ndense7 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). f 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. f you go to 1000X
then you should have a focusable condenser lens with an N.A. of 1.25 or greater. Most 1000X
qlqa
microscopes use 1.25 Abbe condenser lens systems. The Abbe condenser lens can be moved up and
down. t is set very close to the slide at 1000X and moved further away at the lower powers.
i,ph7,2 47 7is: 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.


Liqht microscopy
Lhe moL common mlcrocope ln ue Loday
llmlLed Lo abouL 02 um
@he LoLal magnlflcaLlon l Lhe producL of Lhe ob[ecLlve len magnlfy Lhe eyeplece
f Lhe ob[ecLlve len magnlfle 100fold
and Lhe eyeplece magnlfle 10x Lhe
flnal magnlflcaLlon recorded by human eye
or on fllm wlll be 1000x
1be tesolotloo oo stooJotJ llqbt mlctoscopy ls llmlteJ to oboot 02m

@he reoluLlon of a mlcrocope len l
numerlcally equlvalenL Lo u Lhe mlnlmum
dlLance beLween Lwo dlLlngulhable
ob[ecL
@he horLer Lhe wavelengLh of llghL Lhe value of u wlll be maller and Lhe reoluLlon wlll be
beLLer
W depend on 3 parameLer all of whlch muL be conldered ln order Lo achleve Lhe beL
polble reoluLlon
1 Lhe bolfooqle %angular aperLure) or of Lhe cone of llghL enLerlng Lhe len from Lhe
peclmen
2 Lhe teftoctlve loJex N of Lhe alr or fluld medlum beLween Lhe peclmen and Lhe
ob[ecLlve len
3 Lhe oveleoqtb of lncldenL llghL


-
2
83

uecrealng Lhe value of or lncrealng elLher N or wlll decreae Lhe value of and Lhu
lmprove Lhe reoluLlon
@he half angle %angular aperLure) depend on Lhe wldLh of Lhe ob[ecLlve len and lL dlLance
from Lhe peclmenMovlng Lhe ob[ecLlve len cloer Lo Lhe peclmen wlll lncreae ln and
reduce and Lhe beLLer Lhe reoluLlon

@he refracLlve lndex%-) meaure Lhe degree
Lo whlch a medlum bend a llghL ray LhaL pae Lhrough lL Lhe refracLlve lndex of alr
l deflned a -10
Cne ueful way Lo decreae u and Lhereby lmprove Lhe reoluLlon l Lo ue an lmmerlon oll a
Lhe medlum beLween Lhe peclmen and Lhe ob[ecLlve len
:lnce Lhe refracLlve lndex of uch oll l 13
Lhe reoluLlon wlll be lmproved 13fold
:llnally Lhe horLer Lhe wavelengLh of
lncldenL llghL Lhe lower wlll be Lhe value of
u and Lhe beLLer Lhe reoluLlon
9tepotloq somples fot llqbt mlctoscope
:peclmen for llghL mlcrocopy are commonly flxed wlLh a oluLlon conLalnlng alcohol or
formaldehyde compound LhaL denaLure moL proLeln and nuclelc acld
:uually Lhe ample l Lhen embedded ln paraffln or plaLlc and cuL lnLo Lhln ecLlon of one or a few
mlcromeLer Lhlck
:lnce Lhe reoluLlon of Lhe llghL mlcrocope l02 m and mlLochondrla and chloroplaL are 1 m
long %abouL Lhe lze of eubacLerla) LheoreLlcally one hould be able Lo ee Lhee organelle
:ow ever moL cellular conLlLuenL are noL colored and aborb abouL Lhe ame degree of vllble
llghL o LhaL Lhey are hard Lo dlLlngulh under a llghL mlcrocope
:@he flnal Lep ln preparlng a peclmen for obervaLlon Lherefore l Lo Laln lL ln order Lo vluallze Lhe
maln LrucLural feaLure of a cell or Llue
mmunof/uorescence Microscope
f|uorescence m|croscope l an opLlcal mlcrocope ued Lo Ludy properLle of organlc or
lnorganlc ubLance ulng Lhe phenomena of fluorecence and phophorecence lnLead of or
ln addlLlon Lo reflecLlon and aborpLlon
lor locallzlng proLeln wlLhln cell and ued anLlbodle peclflc for Lhe delred proLeln
chemlcal l ald Lo be fluorecenL lf lL aborb llghL aL a peclflc wavelengLh and emlL llghL aL a
peclflc and longer wavelengLh wlLhln a vllble pecLrum
uye for mlcrocopy are rhodamlne Cy3 whlch emlL red llghL and fluoreceln whlch emlL green
llghL
lluorecenL mlcrocopy can alo be applled Lo llve cell and organell wlLhln Lhe cell
lluorecence mlcrocopy can alo meaure Lhe local concenLraLlon of Ca
2+
lon and Lhe
lnLracellular p

@he confoco/ sconninq microscope
@he confocal cannlng mlcrocope avold Lhe problem of Lhe
lmmunofluorecence mlcrocopy
@he lmmunofluorecence mlcrocopy ha lL llmlLaLlon
@he confocal cannlng mlcrocope creaLlng a vaLly harp lmage
L any lnLanL ln Lhl confocal lmaglng only a lngle mall parL of a ample l
lllumlnaLed wlLh exclLlng llghL from a focued laer beam whlch rapldly move
Lo dlfferenL poL ln Lhe ample focal plane
9fsecontrfst m|croscope
eneraLe an lmage ln whlch Lhe degree of darkne and brlghLne of a reglon of Lhe ample
depend on Lhe refracLlve lndex of LhaL reglon
9CM l a vallable Lool for examlnlng unflxedllvlng cell
@he 9CM l epeclally ueful ln examlne Lhe LrucLure and movemenL of Lhe larger organelle uch a
Lhe nucleu and mlLochondrla ln llve culLured cell
B@hl Lechnlque l ulLable for obervlng only
lngle cell or Lhln cell layer
/ectron microscope


@he fundamenLal prlnclple of elecLron mlcrocopy are lmllar Lo Lhoe of llghL mlcrocopy Lhe
ma[or dlfference l LhaL elecLromagneLlc lene noL opLlcal lene focu a hlghveloclLy elecLron
beam lnLead of vllble llghL
@hu Lhe llmlL of reoluLlon for Lhe elecLron mlcrocope l LheoreLlcally 0003 nm %le Lhan Lhe
dlameLer of lngle aLom) or 40000 Llme beLLer Lhan Lhe reoluLlon of Lhe llghL mlcrocope and 2
mllllon Llme beLLer Lhan LhaL of Lhe unalded human eye