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Microscopia in fluorescenza

BUSBhttp://www.zeiss.de/C12567BE0045ACF1/Contents-Frame/10BBB27317CDCE32C125752000466B8F

Fluorocromi comuni

Fluorochromes are stains, somewhat similar to the better-known tissue stains, which attach themselves to visible or sub-visible organic matter. These fluorochromes, capable of absorbing and then re-radiating light, are often highly specific in their attachment targeting and have significant yield in absorption-emission ratios.

Absorbance

ln (Io/I) = σnd (Beer –Lambert law)

Io = light intensity entering cuvetteI=light intensity leaving cuvetteσ : absorption cross sectionn moleculesd = cross section (cm)

or

ln (Io/I) = α C d (Beer –Lambert law)

a=absorption coefficientC = concentration

• Converting to decimal logs and standardizing quantities we get

Log (I0/I) = εcd = A

Now e is the decadic molar extinction coefficientA = absorbance or optical density (OD) a dimensionless

quantity

d

n molecules

σ – absorption cross section

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http://www.olympusmicro.com/primer/techniques/fluorescence/fluorescenceintro.html

Diagramma di Jablonski

Fluorescenza

FluorescenceStokes Shift

– is the energy difference between the lowest energy peak of absorbance and the highest energy of emission

495 nm 520 nm

Stokes Shift is 25 nmFluoresceinmolecule

Fluo

resc

ence

Int

ensi

ty

Wavelength

Fluorescence Excitation Spectra

Intensity related to the probability of the

event

Wavelengththe energy of the light absorbed

or emitted

Timescale Range for Fluorescence Processes

Transition Process Rate Constant Timescale(Seconds)

S(0) => S(1) or S(n)

Absorption (Excitation) Instantaneous 10-15

S(n) => S(1) Internal Conversion k(ic) 10-14 to 10-10

S(1) => S(1) Vibrational Relaxation k(vr) 10-12 to 10-10

S(1) => S(0) Fluorescence k(f) or Γ 10-9 to 10-7

S(1) => T(1) Intersystem Crossing k(pT) 10-10 to 10-8

S(1) => S(0)Non-Radiative

RelaxationQuenching

k(nr), k(q) 10-7 to 10-5

T(1) => S(0) Phosphorescence k(p) 10-3 to 100

T(1) => S(0)Non-Radiative

RelaxationQuenching

k(nr), k(qT) 10-3 to 100

http://www.olympusmicro.com/primer/techniques/fluorescence/fluorescenceintro.html

Fluorescence

The longer the wavelength the lower the energy

The shorter the wavelength the higher the energye.g. UV light from sun causes the sunburn

not the red visible light

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Parameters

• Extinction Coefficient– ε refers to a single wavelength (usually the absorption maximum)

• Quantum Yield– Qf is a measure of the integrated photon emission over the fluorophore

spectral band

• At sub-saturation excitation rates, fluorescence intensity is proportional to the product of ε and Qf

Number of emitted photonsNumber of absorbed photonsφ=

Fluorescent Microscope

Dichroic Filter

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

EPI-Illumination

Eccitazione della fluorescenza

Excitation Sources

Excitation Sources

LampsXenonXenon/Mercury

LasersArgon Ion (Ar)Krypton (Kr)Violet 405Helium Neon (He-Ne)Helium Cadmium (He-Cd)Krypton-Argon (Kr-Ar)

Lampade al mercurio o allo xenon

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Lampada al tungsteno – alogene(campo chiaro)

Light Sources - Lasers

• Argon Ar 353-361, 488, 514 nm

• Violet Diode 405 nm• Krypton-Ar Kr-Ar 488, 568, 647 nm• Helium-NeonHe-Ne 543 nm, 633 nm• He-CadmiumHe-Cd 325 - 441 nm(He-Cd light difficult to get 325 nm band through some optical systems)

Laser Abbrev. Excitation Lines

Sorgenti laser per eccitazione di fluorescenza(microscopio confocale) Microscopio in fluorescenza

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html

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Obiettivo

Importanza dell’apertura numerica dell’obiettivo

Percorso ottico dell’eccitazione e dell’emissione nel microscopio

http://www.microscopy.fsu.edu/primer/techniques/fluorescence/fluorhome.htmlhttp://www.jic.ac.uk/microscopy/more/images/5_4.gif

Importanza dell’apertura numerica dell’obiettivo

Cubetti con filtro di eccitazione, dicroico e di sbarramento

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Cubetto tipico Eccitazione & Emissione

Tipi di filtro per selezionareeccitazione e/o emissione Band-pass filter

Sistemi di eccitazione per un solo fluorocromo

Eccitazione nell’UV

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Eccitazione nel BluEmissione solo Verde

Filter set Zeiss 10

Eccitazione nel BluEmissione del Verde al Rosso

Filter set Zeiss 9

Eccitazione nel Verde

Sistemi di eccitazione simultaneaper due fluorocromi

Eccitazione doppia Un altro tipo di eccitazione doppia

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Sistemi di eccitazione simultaneaper tre fluorocromi

Eccitazione tripla

DIC + Fluorescenza

Fluorocromi

Excitation - Emission Peaks

Fluorophore EXpeak EMpeak

% Max Excitation at488 568 647 nm

FITC 496 518 87 0 0Bodipy 503 511 58 1 1Tetra-M-Rho 554 576 10 61 0L-Rhodamine 572 590 5 92 0Texas Red 592 610 3 45 1CY5 649 666 1 11 98

Note: You will not be able to see CY5 fluorescenceunder the regular fluorescent microscope becausethe wavelength is too high.

Material Source:Pawley: Handbook of Confocal Microscopy

Probes for ProteinsFITC 488 525PE 488 575APC 630 650PerCP™ 488 680Cascade Blue 360

450Coumerin-phalloidin 350 450Texas Red™ 610 630Tetramethylrhodamine-amines 550

575CY3 (indotrimethinecyanines) 540 575CY5 (indopentamethinecyanines) 640

670

Probe Excitation Emission

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• Hoechst 33342 (AT rich) (uv) 346 460• DAPI (uv) 359 461• POPO-1 434 456• YOYO-1 491 509• Acridine Orange (RNA) 460 650• Acridine Orange (DNA) 502 536• Thiazole Orange (vis) 509

525• TOTO-1 514 533• Ethidium Bromide 526 604• PI (uv/vis) 536 620• 7-Aminoactinomycin D (7AAD) 555 655

Probes for Nucleic Acids DNA Probes• AO

– Metachromatic dye• concentration dependent emission• double stranded NA - Green• single stranded NA - Red

• AT/GC binding dyes– AT rich: DAPI, Hoechst, quinacrine– GC rich: antibiotics bleomycin, chromamycin

A3, mithramycin, olivomycin, rhodamine 800

Specific Organelle Probes

BODIPY Golgi 505 511NBD Golgi 488 525DPH Lipid 350 420TMA-DPH Lipid 350 420Rhodamine 123 Mitochondria 488 525DiO Lipid 488 500diI-Cn-(5) Lipid 550 565diO-Cn-(3) Lipid 488 500

Probe Site Excitation Emission

BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazoleDPH – diphenylhexatriene TMA - trimethylammonium

Probes for Oxidation States

• DCFH-DA (H2O2) 488 525• HE (O2

-) 488 590• DHR 123 (H2O2) 488 525

Probe Oxidant Excitation Emission

DCFH-DA - dichlorofluorescin diacetateHE - hydroethidine 3,8-Phenanthridinediamine, 5-ethyl-5,6-dihydro-6-phenyl-

DHR-123 - dihydrorhodamine 123 Benzoic acid, 2-(3,6-diamino-9H-xanthene-9-yl)-, methyl ester

DCFH-DA: 2',7'-dichlorodihydrofluorescein diacetate (2',7'-dichlorofluorescin diacetate; H2DCFDA)

C24H16Cl2O7

C21H21N3

C21H18N2O3

Fluorocromi Alexa 488 e 594

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Image-Gallery/Image-Detail.1199.html

Immunofluorescence analysis of mesenchymal stem cells using Zymed Ms anti-Stro-1 (Cat. No. 39-8401) and Gt anti-Mouse-Alexa Fluor 488 (Cat. No. A21042) (green). Tubulin is stained with phalloidin-Alexa 594 (red) and nuclei are stained with DAPI (blue). Sample is mounted in ProLong Gold antifade reagent.

Fluorocromizzazione del DNA con Ioduro di Propidio

Immunocytochemistry/ Immunofluorescence -beta Catenin antibody (ab2365)

http://www.abcam.com/beta-catenin-antibody-ab2365.html#ab2365_1.jpg

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Hoechst 33258 /DNA

Nuclei fluorocromizzati con Hoechst

ACRIDINE ORANGEOther Probes of Interest

• GFP - Green Fluorescent Protein– GFP is from the chemiluminescent jellyfish Aequorea

victoria– excitation maxima at 395 and 470 nm (quantum

efficiency is 0.8) Peak emission at 509 nm– contains a p-hydroxybenzylidene-imidazolone

chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence

– Major application is as a reporter gene for assay of promoter activity

– requires no added substrates

pH Sensitive Indicators

• SNARF-1 488 575

• BCECF 488 525/620440/488 525

Probe Excitation Emission

SNARF-1: Benzenedicarboxylic acid, 2(or 4)-[10-(dimethylamino)-3-oxo-3H- benzo[c]xanthene-7-yl]-BCECF: Spiro(isobenzofuran-1(3H),9'-(9H) xanthene)-2',7'-dipropanoic acid, ar-carboxy-3',6'-dihydroxy-3-oxo-

C27H20O11

C27H19NO6

Probes for Ions

• INDO-1 Ex350 Em405/480• QUIN-2 Ex350 Em490• Fluo-3 Ex488 Em525• Fura -2 Ex330/360 Em510

INDO-1: 1H-Indole-6-carboxylic acid, 2-[4-[bis[2-[(acetyloxy)methoxy]-2- oxoethyl]amino]-3-[2-[2-[bis[2- [(acetyloxy)methoxy]-2-oxoetyl]amino]-5- methylphenoxy]ethoxy]phenyl]-, (acetyloxy)methyl ester [C47H51N3O22 ]

Indo-1

FLUO-3: Glycine, N-[4-[6-[(acetyloxy)methoxy]-2,7- dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2- oxyethyl]amino]-5- methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-, (acetyloxy)methyl ester

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Fluorescence Microscope withColor Video (CCD) 35 mm Camera

Cameras and emission filters

Color CCD camera does not need optical filters to collect all wavelengths but if you want to collect each emission wavelength optimally, you need a monochrome camera with separate emission filters shown on the right (camera is not in position in this photo).

Camera goes here

Cooled color CCD camera

Ethidium

PE

cis-Parinaric acid

Texas Red

PE-TR Conj.

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser Lines

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Bella & informativa Tumori e fegato studiati con tecniche di fluorescenza