Principles of fluorescent

probes

Anna Dubiel

Warsaw, 17.05.2011

Plan of presentation

-> introduction in fluorescence microscopy-> basics of fluorescence-> properties of fluorescent probes-> cell probing:

> DNA> membranes> tubulin> mitochondria> indicators for Ca2+

> pH-> summary

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Disadvantages of optical microscopy- Usual microscopes working at visible

spectral range have magnification coefficient up to 1 500x

- Microscopes for ultra-violet have magnification coefficient up to 3500x

- Majority of cell components are transparent and colourless

- Dyes used in traditional microscopy often are not selective and paint whole cell

3

Advantages of fluorescent microscopy

- Higher sensitivity

- Using visible or near IR spectral range

- Dyes which are specific for subcellular components, proteins or ions

- Observation of cell division

- 3D

4

Jabłoński diagram

Basics of fluorescence

Aleksander Jabłoński1898 – 1980Profesor of

Nicolaus Сoperniсus University in Torun

IC +

VR

IC +

VR

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Basics of fluorescence

Absorption:

Fluorescent quantum yield:

Jabłoński diagram

Brightness:

Stokes shift:

IC +

VR

IC +

VR

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Fluorescence microscope :

7

Dyes:

- High absorption

- High fluorescent quantum yield

- Water solubility

- Affinity to a particular part of the cell

- Chemical and photostability

- Stability in cell conditions

- Low cytotoxicity

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Dyes for imaging:

O

COOH

N+N

O

COOH

OHO

Rhodamine dyes Fluoresceines

fluoresceineλabs=489nmλem=534nm

ϕf=0,73ε= 92 300 M-1cm-1

J. Mater. Chem., 2009, 19, 2018–2025

rhodamine Bλabs=542nmλem=579nm

ϕf=0,50ε= 106 000 M-1cm-1

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Dyes for imaging :

N+

N

Cyanines

Angew. Chem. Int. Ed. 2009, 48, 299 –303Chem. Phys. Lett., 1978, 54, 159-163

Cy3 λabs=546nmλem=571nm

ϕf=0,05ε= 271 000M-1cm-1

O OH2N

Coumarins

coumarin 440λabs=354nmλem=434nm

ϕf=0,73ε= 23 500M-1cm-1

Chem. Med. Chem. 2010, 5, 103 – 11710

Dyes for imaging :

BODIPY

4,4-difluoro-4-bora-3a,4a-diaza-s-indaceneλabs=499nmλem=535nm

ϕf=0,93ε > 80 000 M-1cm-1

J. Org. Chem. 2009, 74, 5719–5722

N N

B

FF

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DNA detection

DAPIΛabs=358nmλem=461nm

ϕf=20%

The Molecular Probes Handbook; A Guide to fluorescent Probes and Labeling technologies; Eleventh Edition

Ewa M. Goldys, Fluorescence Aplications in Biotechnology and

the Life Science; Wiley-Blackwell; 2009

Cy5-dUTPλabs=649nmλem=670nm

ϕf=28%

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Probes for membranes

ß-BODIPY FL C5-HPAλabs=504nmλem=511nm

ε= 79 000 M-1cm-1

Fluorescein DHPEλabs=496nmλem=519nm

ε= 88 000 M-1cm-1

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Probes for tubulin

BODIPY FL vinblastineλabs=503nmλem=510nm

ε= 83 000 M-1cm-1

Oregon Green 488 Taxolλabs=494nmλem=522nm

ε= 80 000 M-1cm-1

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Probes for mitochondia

MitoTracker Red CMXRos λabs=578nmλem=599nmε = 116 000 M-1cm-1

MitoTracker Green FMλabs=490nmλem=516nmε = 119 000 M-1cm-1

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Indicators for Ca2+

Calcium Orangeλabs=549nmλem=575nmε = 80 000 M-1cm-1

Fura Redλabs=473, 436nmλem=670, 655nmε = 29 000, 41 000 M-1cm-1

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pH indicators

O

COOH

O+HOH

O

COOH

OHO O

COO-

OHO O

COO-

O-O

kationpH<2

cz. neutralnapH=3,3

monoanionpH=5,5

dianionpH>8

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5-(and-6)-carboxy SNARF-1λabs=548, 576nmλem=587, 635nmε = 27 000, 48 000 M-1cm-1

Summary

Fluorescence microscopy:

-> An essential tool in biology and

the biomedical sciences

-> Based on fluorescence

fenomena

-> Use fluorescent probes

Aplication of fluorescence

microscopy:

1.Detection and determination of

the proteins localization in cell and

tissue

2.Examination changes of ions

concetration

3.Diagnostic of diseases

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Thank you for your attention

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