07physiological Probes

8/9/2019 07physiological Probes

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Announcements and Outline

Sequential scanning reports duetoday.

Midterm exam is next Monday, Feb.26.

Ater paper discussion Mostly multiple c!oice test on t!e

lecture content.

"ro#ect proposals are also due nextMonday. "ro$ide%

&roup members 'up to () Organism

"robe *uestion

+!ecouts during -A time next /ee'see sc!edule)

0e$ie/ using grading rubric

A. "aper 1iscussion

. Fluorescent "robes

. 3uclear stains

2. 4on indicators(. Organelle probes

5. Membrane probes

. -racers

+. 7ab% Onion

epit!elium as asource o li$e cells.

1. -A -ime% re$ie/or c!ecout


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+!ecout times

0e$ie/ c!eclist and practice &roup '-uesday)

Amy 8%88


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Anot!er pro#ect idea

>ou mig!t consider a ?teac!ing@ pro#ect instead o a?researc!@ pro#ect.

For example, prepare your o/n cryostat section o

mouse idney or intestine and do a demonstration ocorrect controls or double or triple labeling. acground control% no label leed


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+ontrols or 1ouble 7abeling

Background control:specimen /it!outsecondary antibody or luoroc!rome

+ontrols or autoluorescence

Bleed-through controls:specimens labeled /it!eac! luoroc!rome separately. -o determinemaximum gain beore bleed


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. Fluorescent "robes

Molecular "robes '4n$itrogen) ///.probes.com +atalog contains t!ousands o luorescent probes,

/it! $aluable tec!nical inormation.

+onocal Microscopy 7istser$er -o subscribe, send message ?subscribe conocal@

to listser$Dub$m.cc.bualo.edu/it! not!ing int!e sub#ect line, /it!out t!e quotation mars.

Arc!i$e%!ttp%listser$ascu.bualo.eduarc!i$esconocal.!tml
http://www.probes.com/mailto:[email protected]://listserv/ascu.buffalo.edu/archives/confocal.htmlhttp://listserv/ascu.buffalo.edu/archives/confocal.htmlhttp://listserv/ascu.buffalo.edu/archives/confocal.htmlhttp://listserv/ascu.buffalo.edu/archives/confocal.htmlmailto:[email protected]://www.probes.com/


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Fluorescein


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+ontrolling "!otobleac!ing% "roperties o

"opular Antiade 0eagents

Antifade ReagentComments

p


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-etramet!ylr!odamine '-04-+)

-raditional second dye in double


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+ommon luoroc!romes used or

cell stainingFluorochrome Excitation (nm) Emission (nm) Color

DA!

"oechst ##$%&

Fluorescein

Rhodamine

'exas Red

C$

C#

(Amersham)BOD! des(*olecular ro+es)

AlexaFluor des(*olecular ro+es)

#%&

#%$

,%

%%$

%.

,$

%%/

0arious

0arious

,.1

,.1

%$%

%2/

.$/

%1/

%2/

0arious

0arious

Blue

Blue

3reen

Red-orange

Red

3reen

Red

0arious

0arious


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AlexaFluor 1yes

Eig!er quantum yield, lo/er p!otobleac!ing, narro/er excitation spectra, etc.


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3ucleic Acid "robes

4ame Excitation Emission Comments

Acridine orange %// %$. (+roads5ectrum)

'aken u5 + li6ecells7 es5eciallacidic 6esicles

ro5idium iodide %#. .12 Dead8fixed cells

Ethidium +romide %1& .12 Dead8fixed cells7higher +ackground

90 des DA!7"oeschst ##$%&

#%&8#%$ ,.18,.1 emi-5ermeant85ermeant

to des

(*olecular ro+es)

0arious 0arious Cell 5ermeant

tox des(*olecular ro+es)

,,%8%/,8%,2; others ,2/8%$#8%2/; others "ighl im5ermeant

Ot!ers listed on p. 2 o textboo.


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Maintaining !ealt!y cells or li$e

imaging +ell structure and integrity

Jarious imaging c!ambers may be necessary

-emperature control

-emperature control c!ambers

pE control

uers

O2, +O2le$els

A concern in enclosed c!ambers

+ontrol o Osmolarity

A concern in open c!ambers


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"!ysiological "robes

'Fluorescent 4on 4ndicators) -!ese molecular probes speciically bind an ion,

/!ic! causes a c!ange in eit!er t!e intensityor

wavelength of emissiono t!e probe.

+!anges in concentration o t!e ion can t!ereore be

monitored.

-!ere are indicators or most ions, but +a2Kand pE

are most commonly used.

Salt or dextran orms must be microin#ectedL ester

deri$ati$es can be taen up by cells /!ere t!ey are

con$erted to impermeant orm.


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lue +a2K4ndicators% Fluo


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+a2K0elease 1uring S!rimp 9gg Acti$ation

From 7indsay et al. '::2). 9xtracellular Mg2K4nduces an

4ntracellular +a2Ka$e 1uring Oocyte Acti$ation in t!e

Marine S!rimp Sicyonia ingentis. 1e$. iol. 2%:5


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"roblems /it! *uantitati$e Single

a$elengt! Fluorescence 4maging +ompartmentaliIation o probe in

intracellular organelles.

Cne$en loading o dye in t!e cell.

4nstrumentation noise

Sample geometry 't!icness)

Solution% ratio imagingo t!e dye


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CJ +a2K4ndicators% 4ndo


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CJ +a2K4ndicators% Fura


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Free +a2K+oncentration in a "urin#e

3euron rom 9mbryonic Mouse +erebellum

3eurons /ere loaded /it!

ura


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1ual lue +a2K4ndicators%

Fluo


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Frog Olactory ulb 3eurons

7abeled /it! Fluo


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-!e pE 4ndicator S3A0F


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Organelle "robes

'arget7 4ame Excitation Emission Comments

*itochondria

*ito'racker des ,/8%2&8%%1 %1.8%8%2.

*' 3reen accumulates inmitochondria regardless of*B 5otential7 Red andOrange in acti6emitochondria< aldehdefixa+le

3olgi

BOD! F=and 'R

C%-ceramide

%/%8%& %117 .$/8.12 'R is +etter for dou+lela+eling7 since no greenemission

=sosomes

=so'racker

=soensor

0arious

0arious

0arious

0arious

Endo5lasmic reticulum

DiOC.7 Di!C.

Fluorescent Brefeldin A

,&,8%, %/18%.%

DiO and Di! also stainmem+ranes generall

!nhi+itor of 5rotein trans5ort

Ot!ers listed on p. 225


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1ouble


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&olgi Apparatus and

7ysosomesViable MDCK cells sequentially stained

with !D"#$ %& C5'ce(amide)

&ys*+(ac,e( -ed D.D'//and *echst

331582 (een'lu*(escent !D"#$ %&

C5'ce(amide l*calied t* the *li

aa(atus) (ed'lu*(escent &ys*+(ac,e(-ed stain accumulated in the lys*s*mes

and blue'lu*(escent *echst 33158 dye

stained the nuclei2


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Membrane "robes

Cseul or tracing

cell ates /it!out

microin#ecting 1i4 'red, 1 on rig!t)

1iO 'green)

Many ot!ers

a$ailable rom

Molecular "robes


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+ell -racers

4ame Ex Em Comments

Cell'racker 0arious 0arious ermeant7 then attaches to cellular5roteins7 aldehde fixa+le

=ucifer ello> ,&& %//-.//

ello>

Common ga5 ?unction @ neuronaltracer7 aldehde fixa+le

*em+rane 5ro+es 0arious 0arious Can la+el + touching de crstalagainst cell

Fluorescentdextrans

0arious 0arious Reuire microin?ection


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7ucier yello/ and gap #unctions

E22 lucie(

yell*w

ie( than a

uncti*n cut*


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"romotor constructs

ot! can be used as re5orters for 5romoter constructs.For example, transorm an embryo /it! 13A coding orpromoteren!ancer or gro/t! actor used to &F" to loo

or tissues t!at normally express t!e gro/t! actor.

E # %# D.9

%# m-.9

%# (*tein

%

+%


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&F" Fusion "roteins

-o determine sub


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+. 7ab% Onion epit!elium prep

'1ailey et al. 288GL Murp!y, 288)

+ut a small square o a layer rom t!e onion using a raIor blade. Cse a orceps to peel o t!e t!in epit!elium on t!e inner surace o t!e

onion layer. "ut t!e epit!elium on a microscope slide, co$er /it!


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07physiological Probes