principles of fluorescent probes anna dubiel warsaw, 17.05.2011
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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
2
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
5
Basics of fluorescence
Absorption:
Fluorescent quantum yield:
Jabłoński diagram
Brightness:
Stokes shift:
IC +
VR
IC +
VR
6
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
8
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
9
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%
12
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
13
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
14
Probes for mitochondia
MitoTracker Red CMXRos λabs=578nmλem=599nmε = 116 000 M-1cm-1
MitoTracker Green FMλabs=490nmλem=516nmε = 119 000 M-1cm-1
15
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
16
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
18
Thank you for your attention
19
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