lab1 microscopy (1)

8/10/2019 Lab1 Microscopy (1)

http://slidepdf.com/reader/full/lab1-microscopy-1 1/39

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

i!)erm 2/0

inal (am 2/0

a* Presen)a)ion 150

4o)e Boos 1/0

Par)i'ipa)ion 50

Course Assessment



orma) o )he Papers

7 ollo+ orma) o a 8ournal ar)i'le n)ro!u')ion

7 Ba'$roun! inorma)ion, o*8e')i#es o "our s)u!"

a)erials an! e)ho!s

7 Or$anism, rea$en)s, e&uipmen), pro'e!ures, e)'.

esul)s7 Da)a an! o*ser#a)ions presen)e! in orm o )a*les,

i$ures, $raphs, s)a)is)i's, e)'. an! a legend eplainin$

ea'h. Dis'ussion

7 n)erpre) )he resul)s

eeren'es



4o)e Boos

7 re'or! o "our eperimen)al pro'e!ures an! o*ser#a)ions.

7 Purpose o )he la* plus )he pro)o'ol mus) *e en)ere! *eore la*

7 4ea)l" an! le$i*l" en)er "our ins)rumen) se) up, !a)a, i$ures,e)'. in "our no)e*oo.

7 a* en)r" mus) *e 'omple)e! *eore lea#in$ or )he !a".

7 Su$$es)ion: Brin$ "our 'ameras )o )ae 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

• Ci x Vi = Cf x Vf

• Percent solution

- W/V – ! "aCl

- V/V – #$! %t&'



Microscopy



Microscope

• Must accomlis tree tas*s.

– Magnification

– +esolution

– Visi,ility



Compound Microscope components

=en)erin$ s're+s

Conenser iris leer



Lenses

• ocus ligt rays at a secific lacecalle te focal oint

• Distance ,et0een center of lens

an focal oint is te focal lengt

• 1trengt of lens relate to focallengt

– sort focal lengt ⇒more

magnificationi$ure 2.2



Path of Light



Sub-stage Condenser

• 2aters ligt from te ligtsource an concentrates it intoa cone of ligt tat illuminateste secimen 0it uniformintensity oer te entire fiel ofie0

• %ac time an o,3ectie iscange4 te su,-stageconenser is a3uste toroie te roer ligt conefor te numerical aerture ofte ne0 o,3ectie.

• 5s most imortant in securingcorrect illumination4 contrast4an et of fiel.

tt6//micro.magnet.fsu.eu/rimer/



Objective Lens

• 7e o,3ectie as seeral ma3or functions6 – Must gater te ligt coming from eac of te arious arts or oints

of te secimen.

– Must ,e a,le to reconstitute te ligt coming from te arious ointsof te secimen into te arious corresoning oints in te image.

– 1oul ,e correcte for serical an cromatic a,errations - 8cromatic

• Color fringes aroun te secimen

• 1traigt lines aear cure

– 1oul ,e correcte for curature of fiel - Prouce flat fiel – lan

– Dry o,3ectie lenses – 1canning4 lo0 o0er an ig o0er.

– &il immersion lens.




Ocular Lens

• 7e eyeiece seres to furter magnify te real image ro3ecte,y te o,3ectie.

• 7e eyeiece can ,e fitte 0it scales4 mar*ers or ointers(often referre to as reticle) in suc a 0ay tat te image of

tese inserts can ,e suerimose on te image of tesecimen.

• 5nteruillry istance a3ustment ial an eye iece ioter – 8llo0s you to ie0 0it ,ot eyes simultaneously – 8llo0s you to focus image for eac eye ineenently

• Correct for near an far sigteness ,ut not astigmatism.

• 1oul ,e acromatic an soul ,e flat fiel.




Ocular Lens

tt6//000.microscoe-eot.com/ret9coose.as



7otal Magnification

Power o Objective Power o Ocular

tt6//000.cas.muoio.eu/m,i-0s/microscoes/Magnification.tml

Plate magnification = si:e of ra0ing (mm) ; actual si:e (<m)



Microscopic !esolution

• 8,ility of a lens to searate or istinguis small o,3ects tatare close togeter

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

– 1maller te limit of resolution4 te ,etter resole te image



NA = n sine θ

n ? rera')i#e in!e o )he me!ium *e)+een

)he o*8e')i#e lens an! )he 'o#er slip ;air, +a)er, oil<

@ ? hal )he 'one an$el o li$h) en)erin$ )he o*8e')i#e

'on)rolle! *" 'on!enser iris




Lenses and Light !eraction

• igt is refracte (,ent) 0en assing from one

meium to anoter

• +efractie inex

– a measure of o0 greatly a su,stance slo0s te elocity

of ligt

• Direction an magnitue of ,ening is etermine

,y te refractie inexes of te t0o meia forming

te interface



Oil Immersion

era')ion a) )he air- 'o#erslip in)era'e 'auses some ra"s )o

miss )he o*8e')i#eA Those ra"s 'an rea'h )he o*8e')i#e i oil is use!.

&il - 8ir

Coer sli1ecimen

1lie

tt6//en.0i*ieia.org/0i*i/&il9immersion



7 4earl" )ransparen) o*8e')s 'an *e !ii'ul) )o see

7=on)ras) - !ieren'e in appearan'e *e)+een par)s o an

o*8e') an! )he *a'$roun!

7=on)rol )he in)ensi)" o )he li$h) sour'e

7=lose )he 'on!enser ris re!u'es )he 'one an$le poor l.r.

7S)ain !ieren) par)s o )he 'ell

7D"es - eul$en s)ain or D4, DP ;luores'en) !"e or

D4<, =oomasie *lue s)ains pro)eins, luorophores.

7Various 'ell s)ru')ures )ae up !ieren) !"es *sor* li$h)

!ieren)l"

7 Vi)al s)ains 8anus $reen

Visibility



i$ure 2.15'

Escherichia coli

Visibility



"he Light Microscope

• @ses ligt to illuminate a secimen

• Prouces a ar* image against a ,rigter ,ac*groun

• 8re comoun microscoes

– image forme ,y action of ≥A lenses

• 'as seeral o,3ectie lenses – Parfocal an arcentral

• Many tyes – ,rigt-fiel microscoe

– ar*-fiel microscoe

– ase-contrast microscoe



7e Pase-Contrast Microscoe

7 Light microscopy suited for

transparent specimens, unstained

tissue

7 Enhances the contrast betweenintracellular structures haing slight

differences in refractieinde!

7 "ifferences in refractie inde! conertedto differences in intensity#

7 E!cellent way to obsere

liing cells



Contrast Due ToInterference

Constructie interference Destructie interference



luorescence Microscoy

• @ses luoroores.

• 8llo0 examining fixe tissue

as 0ell as lie cells.

• 8llo0s for locali:ation stuies

• Multicolor images are seeral

single color imagescom,ine

• +e – 8ctin (Palloiin)

2reen – Microtu,ules (57C

con3ugate 8,)

Blue – D8P5 la,ele "uclei

• Comuteri:e imageetection



Det of fiel

• 'iger at lo0er magnifications – 7e entire secimen aears focuse

• o0er at 'iger magnifications – 1ecimen can only ,e focuse in one focal lane 0ile te

focal lanes a,oe an ,elo0 are out of focus



Confocal Microscoy

• 5n orinary ligt microscoy4 an o,serer ie0s te secimen at ifferentets ,y canging te osition of te o,3ectie lens ,y rotating tefocusing *no,. – Different arts of te secimen go in an out of focus – 1ecimen as ifferent leels of focus oesnt allo0 for a cris image

• Confocal microscoe rouces an image of a tin lane situate 0itina tic*er secimen. – 8c?uires in focus images – &tical sectioning

• 'ig resolution images 0it limite et of focus

• 7ose images are suer imose to rouce a tree imensionalimage.

• 5mage ?uality is enance ,ecause image information from multileets is not suerimose.



+e fluorescent anti,oy as staine D"8 in te nuclues

2reen fluorescent anti,oy is ,oun to a telomerase ,ining rotein.

&tical sections of east nucleus – $.Emm tic*



%lectron Microscoy

• 'ig magnifications – #4$$$4$$$

• 'ig limit of resolution – l.r. = $.#> / ".8. – nm

• 1%M an 7%M – 5mage from electrons tat ae ,ounce off or

transmitte troug te o,3ect.

Light microscope

S#M

"#M



Pietting

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



Ioler 5llumination

• ocusing of fiel iris at te lane of o,3ect. – %enly 5lluminates fiel of ie0.

• Bring u su,stage conenser all te 0ay

• &en fiel iris an conenser 5ris all te 0ay

• @sing #$J o,3ectie focus te secimen

• ocus eac ocular for ifferences in ision of te t0o eyes

• Close fiel 5ris almost all te 0ay

• o0er te su,stage conenser until a sar images ofleaes of fiel iris aears

• Center image of fiel iris using centering scre0s• 8fter its centere4 oen fiel iris until ligt fills te fiel ofie0

• +emoe an eyeiece4 close te iris until A/E te iameter of,ac* focal lane of o,3ectie is fille 0it ligt.



Cali,ration of te ocular ruler



7oay• Cali,rate te ocular ruler

– or KJ4 #$J4 A$J an K$J – 2ra o,3ectie s. <m in eac ocular unit . Wats te relationsi ,et0een temL 'o0

many <m e?ual an ocular unit uner oil immersion (erie from te lot)L

• &nion %iermis – &,sere 0it an 0itout te ioine stain – ,rigt fiel microscoy – Measure te lengt of #$ ifferent cells. – Measure #$ ifferent nuclei – Calculate mean an stanar eiation using M1 %JC%

– Dra0/otogra a tyical onion eiermal cell (a,el4 late magnification)

• %loea leaf – "& 1785"5"2 – @er surface facing you. – Determine te tic*ness of eloea leaf EJ

• "ote te line iision on te fine focusing *no, as te uer surface 3ust comes into focus an again0en te lo0er surface 3ust goes out of focus.

– &,sere cytolasmic streaming an etermine rate of cyclosis• 7ime re?uire to trael lengt of cell ; lengt of te cell (o it for as many cells as ossi,le)

– Dra04 la,el an inclue late magnification

• Pase contrast microscoy – "& 1785"5"2 – Ma*e sure te annular iaragm matces te ase o,3ectie. – oo* at onion eiermal cells an eloea leaf. Wats more clear tan ,efore – Cee* Cells – Dra04 la,el4 inclue late magnification.



Resolution --eyes ~0.1mm --light microscope

~0.2 µm




$% &'% $(%

lab1 microscopy (1)

Documents