Vilma Greene, Ph.D.

Bio263 Thurs 9:15 - 2:15pm SB D335

O: Thurs!a": 2:15-3:15 pm
#$reene%&'.'un".e!u
(mail is )he *es) +a" )o rea'h me
There is no la* manual
3 ,a* -epor)s . 1/0 ea'h . 3/0
1i!)erm . 2/0
2inal (3am . 2/0
,a* Presen)a)ion . 150
4o)e Boo5s . 1/0
Par)i'ipa)ion . 50
Course Assessment
2orma) o6 )he Papers
7
2ollo+ 6orma) o6 a 8ournal ar)i'le
.
9n)ro!u')ion
7
Ba'5$roun! in6orma)ion, o*8e')i#es o6 "our s)u!"
.
1a)erials an! 1e)ho!s
7
Or$anism, rea$en)s, e&uipmen), pro'e!ures, e)'.
.
-esul)s
7
Da)a an! o*ser#a)ions presen)e! in 6orm o6 )a*les,
6i$ures, $raphs, s)a)is)i's, e)'. an! a legend e3plainin$
ea'h.
.
Dis'ussion
7
9n)erpre) )he resul)s
.
-e6eren'es
4o)e Boo5s
7
: re'or! o6 "our e3perimen)al pro'e!ures an! o*ser#a)ions.
7
Purpose o6 )he la* plus )he pro)o'ol mus) *e en)ere! *e6ore la*
7
4ea)l" an! le$i*l" en)er "our ins)rumen) se) up, !a)a, 6i$ures,
e)'. in "our no)e*oo5.
7
,a* en)r" mus) *e 'omple)e! *e6ore lea#in$ 6or )he !a".
7
Su$$es)ion: Brin$ "our 'ameras )o )a5e pi')ures an! lap)ops )o
$raph !a)a ;a) leas) 1 per $roup<.
Basic Lab Math
Molarity
(moles/liter)
= mass (g) .
MW (g/mole) x Volume (l)

Dilutions

C
i
x V
i
= C
f
x V
f

Percent solution
- W/V ! "aCl
- V/V #$! %t&'
Microscopy
Microscope

Must accom(lis) t)ree tas*s.

Magnification

+esolution

Visi,ility
Compound Microscope components
=en)erin$ s're+s
Con-enser iris le.er
Lenses
/ocus lig)t rays at a s(ecific (lace
calle- t)e focal (oint
Distance ,et0een center of lens
an- focal (oint is t)e focal lengt)
1trengt) of lens relate- to focal
lengt)
s)ort focal lengt) more
magnification
2i$ure 2.2
Path of Light
Sub-stage Condenser
2at)ers lig)t from t)e lig)t
source an- concentrates it into
a cone of lig)t t)at illuminates
t)e s(ecimen 0it) uniform
intensity o.er t)e entire fiel- of
.ie0
%ac) time an o,3ecti.e is
c)ange-4 t)e su,-stage
con-enser is a-3uste- to
(ro.i-e t)e (ro(er lig)t cone
for t)e numerical a(erture of
t)e ne0 o,3ecti.e.
5s most im(ortant in securing
correct illumination4 contrast4
an- -e(t) of fiel-.
)tt(6//micro.magnet.fsu.e-u/(rimer/
Objective Lens

7)e o,3ecti.e )as se.eral ma3or functions6

Must gat)er t)e lig)t coming from eac) of t)e .arious (arts or (oints
of t)e s(ecimen.
Must ,e a,le to reconstitute t)e lig)t coming from t)e .arious (oints
of t)e s(ecimen into t)e .arious corres(on-ing (oints in t)e image.
1)oul- ,e correcte- for s()erical an- c)romatic a,errations -
8c)romatic
Color fringes aroun- t)e s(ecimen
1traig)t lines a((ear cur.e-
1)oul- ,e correcte- for cur.ature of fiel- - Pro-uce flat fiel- (lan
Dry o,3ecti.e lenses 1canning4 lo0 (o0er an- )ig) (o0er.
&il immersion lens.
)tt(6//micro.magnet.fsu.e-u/(rimer/
Ocular Lens
7)e eye(iece ser.es to furt)er magnify t)e real image (ro3ecte-
,y t)e o,3ecti.e.
7)e eye(iece can ,e fitte- 0it) scales4 mar*ers or (ointers
(often referre- to as reticle) in suc) a 0ay t)at t)e image of
t)ese inserts can ,e su(erim(ose- on t)e image of t)e
s(ecimen.
5nter(u(illry -istance a-3ustment -ial an- eye (iece -io(ter
8llo0s you to .ie0 0it) ,ot) eyes simultaneously
8llo0s you to focus image for eac) eye in-e(en-ently
Correct for near an- far sig)te-ness ,ut not astigmatism.
1)oul- ,e ac)romatic an- s)oul- ,e flat fiel-.
)tt(6//micro.magnet.fsu.e-u/(rimer/
Ocular Lens
)tt(6//000.microsco(e--e(ot.com/ret9c)oose.as(
7otal Magnification
Power o Objective Power o Ocular
)tt(6//000.cas.muo)io.e-u/m,i-0s/microsco(es/Magnification.)tml
Plate magnification = si:e of -ra0ing (mm) ; actual si:e (<m)
Microscopic !esolution

8,ility of a lens to se(arate or -istinguis) small o,3ects t)at

are close toget)er

=imit of resolution = $.#> / ".8. . 8,,e>s e?uation

1maller t)e limit of resolution4 t)e ,etter resol.e- t)e image

NA = n sine
n ? re6ra')i#e in!e3 o6 )he me!ium *e)+een
)he o*8e')i#e lens an! )he 'o#er slip ;air, +a)er, oil<
@ ? hal6 )he 'one an$el o6 li$h) en)erin$ )he o*8e')i#e
'on)rolle! *" 'on!enser iris
Microscopic !esolution
Lenses and Light !eraction

=ig)t is refracte- (,ent) 0)en (assing from one

me-ium to anot)er

+efracti.e in-ex

a measure of )o0 greatly a su,stance slo0s t)e .elocity

of lig)t

Direction an- magnitu-e of ,en-ing is -etermine-

,y t)e refracti.e in-exes of t)e t0o me-ia forming
t)e interface
Oil Immersion
-e6ra')ion a) )he air- 'o#erslip in)er6a'e 'auses some ra"s )o
miss )he o*8e')i#eA Those ra"s 'an rea'h )he o*8e')i#e i6 oil is use!.
&il -
8ir
Co.er sli(
1(ecimen
1li-e
)tt(6//en.0i*i(e-ia.org/0i*i/&il9immersion
7
4earl" )ransparen) o*8e')s 'an *e !i66i'ul) )o see
7
=on)ras) - !i66eren'e in appearan'e *e)+een par)s o6 an
o*8e') an! )he *a'5$roun!
7
=on)rol )he in)ensi)" o6 )he li$h) sour'e
7
=lose )he 'on!enser 9ris . re!u'es )he 'one an$le . poor l.r.
7
S)ain !i66eren) par)s o6 )he 'ell
7
D"es - 2eul$en s)ain 6or D4:, D:P9 ;2luores'en) !"e 6or
D4:<, =oomasie *lue s)ains pro)eins, 6luorophores.
7
Various 'ell s)ru')ures )a5e up !i66eren) !"es . :*sor* li$h)
!i66eren)l"
7
Vi)al s)ains . 8anus $reen
Visibility
2i$ure 2.15'
Escherichia coli
Visibility
"he Light Microscope

@ses lig)t to illuminate a s(ecimen

Pro-uces a -ar* image against a ,rig)ter ,ac*groun-

8re com(oun- microsco(es

image forme- ,y action of A lenses

'as se.eral o,3ecti.e lenses

Parfocal an- (arcentral

Many ty(es
,rig)t-fiel- microsco(e
-ar*-fiel- microsco(e
()ase-contrast microsco(e
7)e P)ase-Contrast Microsco(e
7 Light microscopy suited for
transparent specimens, unstained
tissue
7 Enhances the contrast between
intracellular structures haing slight
differences in refractie
inde!
7 "ifferences in refractie inde! conerted
to differences in intensity#
7 E!cellent way to obsere
liing cells
Contrast Due To
Interference
Constructi.e interference Destructi.e interference
Bright ield
Phase contrast
/luorescence Microsco(y
@ses /luoro()ores.
8llo0 examining fixe- tissue
as 0ell as li.e cells.
8llo0s for locali:ation stu-ies
Multicolor images are se.eral
single color images
com,ine-
+e- 8ctin (P)alloi-in)
2reen Microtu,ules (/57C
con3ugate- 8,)
Blue D8P5 la,ele- "uclei
Com(uteri:e- image
-etection
De(t) of fiel-

'ig)er at lo0er magnifications

7)e entire s(ecimen a((ears focuse-

=o0er at 'ig)er magnifications

1(ecimen can only ,e focuse- in one focal (lane 0)ile t)e
focal (lanes a,o.e an- ,elo0 are out of focus
Confocal Microsco(y
5n or-inary lig)t microsco(y4 an o,ser.er .ie0s t)e s(ecimen at -ifferent
-e(t)s ,y c)anging t)e (osition of t)e o,3ecti.e lens ,y rotating t)e
focusing *no,.
Different (arts of t)e s(ecimen go in an- out of focus
1(ecimen )as -ifferent le.els of focus -oesnCt allo0 for a cris( image
Confocal microsco(e (ro-uces an image of a t)in (lane situate- 0it)in
a t)ic*er s(ecimen.
8c?uires in focus images &(tical sectioning
'ig) resolution images 0it) limite- -e(t) of focus
7)ose images are su(er im(ose- to (ro-uce a t)ree -imensional
image.
5mage ?uality is en)ance- ,ecause image information from multi(le
-e(t)s is not su(erim(ose-.
+e- fluorescent anti,o-y )as staine- D"8 in t)e nuclues
2reen fluorescent anti,o-y is ,oun- to a telomerase ,in-ing (rotein.
&(tical sections of Deast nucleus $.Emm t)ic*
%lectron Microsco(y

'ig) magnifications #4$$$4$$$

'ig) limit of resolution

l.r. = $.#> / ".8. nm

1%M an- 7%M

5mage from electrons t)at )a.e ,ounce- off or

transmitte- t)roug) t)e o,3ect.
Light microscope
S#M
"#M
Pi(etting

F ------------ +eagents ------------ G

7u,e 2reen +e- Blue 7otal

8 #$ <l #H <l H <l E$ <l

B H <l #H <l #$ <l E$ <l

C $ <l #$ <l A$ <l E$ <l

Io)ler 5llumination

/ocusing of fiel- iris at t)e (lane of o,3ect.

%.enly 5lluminates fiel- of .ie0.

Bring u( su,stage con-enser all t)e 0ay
&(en fiel- iris an- con-enser 5ris all t)e 0ay
@sing #$J o,3ecti.e focus t)e s(ecimen
/ocus eac) ocular for -ifferences in .ision of t)e t0o eyes
Close fiel- 5ris almost all t)e 0ay
=o0er t)e su,stage con-enser until a s)ar( images of
lea.es of fiel- iris a((ears
Center image of fiel- iris using centering scre0s
8fter itCs centere-4 o(en fiel- iris until lig)t fills t)e fiel- of
.ie0
+emo.e an eye(iece4 close t)e iris until A/E t)e -iameter of
,ac* focal (lane of o,3ecti.e is fille- 0it) lig)t.
Cali,ration of t)e ocular ruler
7o-ay
Cali,rate t)e ocular ruler
/or KJ4 #$J4 A$J an- K$J
2ra() o,3ecti.e .s. <m in eac) ocular unit . W)atCs t)e relations)i( ,et0een t)emL 'o0
many <m e?ual an ocular unit un-er oil immersion (-eri.e from t)e (lot)L
&nion %(i-ermis
&,ser.e 0it) an- 0it)out t)e io-ine stain ,rig)t fiel- microsco(y
Measure t)e lengt) of #$ -ifferent cells.
Measure #$ -ifferent nuclei
Calculate mean an- stan-ar- -e.iation using M1 %JC%=
Dra0/()otogra() a ty(ical onion e(i-ermal cell (=a,el4 (late magnification)
%lo-ea leaf "& 1785"5"2
@((er surface facing you.
Determine t)e t)ic*ness of elo-ea leaf EJ
"ote t)e line -i.ision on t)e fine focusing *no, as t)e u((er surface 3ust comes into focus an- again
0)en t)e lo0er surface 3ust goes out of focus.
&,ser.e cyto(lasmic streaming an- -etermine rate of cyclosis
7ime re?uire- to tra.el lengt) of cell ; lengt) of t)e cell (-o it for as many cells as (ossi,le)
Dra04 la,el an- inclu-e (late magnification
P)ase contrast microsco(y "& 1785"5"2
Ma*e sure t)e annular -ia()ragm matc)es t)e ()ase o,3ecti.e.
=oo* at onion e(i-ermal cells an- elo-ea leaf. W)atCs more clear t)an ,efore
C)ee* Cells Dra04 la,el4 inclu-e (late magnification.
Resolution --eyes ~0.1mm --light microscope
~0.2 m
Microscopic !esolution
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