P. Kaltofen
14/09/2010
Rendere visibile l‘invisibile
La Fluorescenza come rivelatore di interazioni molecolari
Principi
La Fluorescenza è una forma particolare di luminescenza e può essere
descritta come un processo di tre passaggi :
1) Eccitazione della molecola
Assorbimento di un fotone E=h.ν
νEX – comporta uno stato di eccitazione
elettronica della molecola
2) Fluorescence Lifetime (tempo medio tra eccitazione ed emissione)
Cambiamenti conformazionali, interazioni ambientali, trasferimento di carica
intermolecolare nell’ordine di nanosecondi)
3) Emissione di Fluorescenza (dis-eccitazione della molecola)
Bressanone GNB 2010
1
P. Kaltofen
Principi
Quantum Yield (Φ
Φ)
…è la frazione di energia ceduta in forma di emissione
Φ=
Numero di fotoni emessi
Numeri di fotoni assorbiti
La resa quantica è considerato come misura dell'efficienza con cui la luce
assorbita produce un effetto (in questo caso fluorescenza).
Principi
Simplified Jablonski Diagram
Bressanone GNB 2010
2
P. Kaltofen
Principi
Stoke´s shift
…è la differenza (in lunghezza d‘onda o in unità di frequenza) tra i massimi di
banda dello spettro di eccitazione ed emissione di un certo fluoroforo)
STOKES’ SHIFT
E xc it a t io n
E m is s io n
40000
Fluorescence emission
45000
Fluorescence excitation
50000
35000
30000
25000
20000
15000
10000
5000
0
400
450
W a v e le 5n0g0 th
550
Fluorescence – Principles – Optical Filters
Excitation Filter
LARGE
Emission Filter
Stokes Shift
Bressanone GNB 2010
3
P. Kaltofen
Fluorescence – Principles – Optical Filters
Excitation Filter
SMALL Stokes Shift
Emission Filter
Crosstalk, filter overlap
Fluorescence – Principles – Optical Filters
Excitation Filter
SMALL Stokes Shift
Emission Filter
Crosstalk prevented
by choosing appropriate
Filter-pairs
Bressanone GNB 2010
4
P. Kaltofen
Tecniche ed Applicazioni
1)
Intensità di Fluorescenza
● Nucleic Acid Stain: PicoGreen ds DNA Quantification
● Fluorescent Ca2+ Indicator Fura-2
● Cell based Assay
● Biosensori cellulari
Fluorescence – Application
Nucleic Acid Stain: PicoGreen ds DNA Quantification
Pico Green binds dsDNA and can be detected by means of fluorescence. It is
much more sensitive than the common DNA-absorbance technique.
Em = 535 nm
Em = 535 nm
www.probes.com
Bressanone GNB 2010
5
P. Kaltofen
Fluorescence – Application
Cell based assays
There is a big variety of different
assays to detect physiological
and metabolic parameters of
living cells, for example the well
known PI (Propidium Iodide)
stain.
PI is a non permeable dye, which
only passes the membrane of
necrotic cells. As a result it stains
the DNA of dead cells but not
those of living cells (Ex 540, Em
620), therefore it is used to
determine the viability.
Confocal images of human lung cancer;
green is the microtubule network (part of the
cell cytoskeleton) stained with an FITC-antibody to tubulin.
The red is the nucleus stained with PI.
http://oceanexplorer.noaa.gov
14/09/2010
Infinite 200 – Basic Measurement Techniques
FLUORESCENCE RESONANCE ENERGY
TRANSFER - FRET
Bressanone GNB 2010
6
P. Kaltofen
FRET (Förster or Fluorescence Resonance Energy Transfer)
Fluorescence Intensity
emitted fluorescence
FRET – energy transfer as de-excitation process for the “first” fluorochorme
energy transfer
energy transfer
FRET - Principles
X nm
Z nm
Y nm
D
A
Energy Transfer
DONOR
ACCEPTOR
Ex1 / Em1
Ex2
Ex2 / Em2
Em2
Em1
Em1 ~ Ex
Ex2
2
Bressanone GNB 2010
7
P. Kaltofen
FRET - Principles
In the case of close proximity (10-100 Å) a donor molecule can transfer energy
to an acceptor molecule WITHOUT EMISSION OF A PHOTON.
X nm
Z nm
Y nm
D
10-100 Å
A
Energy Transfer
DONOR
ACCEPTOR
1Å = 0,1nm
FRET - Applications
● Analysis of protein folding and changes in conformation (a):
Bressanone GNB 2010
8
P. Kaltofen
FRET - Applications
● Analysis of all kinds of interactions (protein-protein (b), proteinnucleic acids, nucleic acids-nucleic acid, receptor-ligand, etc.)
14/09/2010
Infinite 200 – Basic Measurement Techniques
TIME RESOLVED FLUORESCENCE - TRF
Bressanone GNB 2010
9
P. Kaltofen
Time Resolved Fluorescence (TRF) - Principles
● better: “Time gated Fluorescence” or “Time delayed Fluorescence”
● Fluorophores of the Lanthanoid series (Lanthanoid Ions); most
popular is the Europium Ion (Eu3+)
● shows a better sensitivity than FI because of...
1) a large Stoke´s shift
2) a long fluorescence decay time (~ms)
TRF - Principles
Bressanone GNB 2010
10
P. Kaltofen
TRF - Principles
Excitation Filter
LARGE
Emission Filter
Stokes Shift
No crosstalk with Lanthanoids!
Intensity
TRF - Principles
Elapsed Time
Excitation
AutoFluorescence
Fluorescein Emission
------ Measurement ----Europium Emission
Bressanone GNB 2010
11
P. Kaltofen
14/09/2010
Infinite 200 – Basic Measurement Techniques
TR – FRET
TR-FRET Assays - Principles
Time Resolved-Fluorescence Resonance Energy Transfer
TR-FRET
Y nm
Z nm
X nm
Eu, Tb = DONOR
long-lived emission!
D
A
Allophycocyanine (XL-665),
fluorescein, TAMRA = ACCEPTOR
Bressanone GNB 2010
12
P. Kaltofen
TR-FRET Assays - Principles
● Donor: long-lived emission in the range of
µs (300 µs to 1 msec)
● Delay (lag) time: 100 - 400 µs
● Integration time: 400 – 2000 µs
TR-FRET Assays - Principles
Characteristics of TRF Assays:
● Time-delayed signal detection: elimination of short-lived fluorescent
background caused by cells, screening compounds, plates,
reagents, etc.
● Low background and high signal-to-noise ratios
● ratio values – no dependency on filling volume and dye
concentration
Bressanone GNB 2010
13
P. Kaltofen
14/09/2010
Infinite 200 – Basic Measurement Techniques
FLUORESCENCE POLARIZATION - FP
Fluorescence Polarization (FP) - Principles
FP – Working with Linear Polarized Light
Polarization of a transverse
wave (such as light) describes
the direction of oscillation in the
plane perpendicular
to the direction of travel.
Bressanone GNB 2010
14
P. Kaltofen
Fluorescence Polarization (FP) - Principles
Generation of Polarized Light
Polarizer
Fluorescence Polarization (FP) - Principles
FP Detection
Light source
Ex-Filter
Sample
Em-Filter
Polarizer
Bressanone GNB 2010
Polarizers
Detection of
Parallel and
perpendicular
parallel and
perpendicular
signal
15
P. Kaltofen
Fluorescence Polarization (FP) - Principles
Excitation light polarization
FP provides information on molecular orientation and mobility.
High FP
Low FP
Ex
Em
Fluorescence Polarization (FP) - Applications
FP is a powerful tool for studying molecular interactions
● Receptor-ligand binding studies
● Antibody-antigen binding
● Enzyme assays (protease)
● Protein-DNA interactions
● DNA hybridization
● Measurements of membrane fluidity
NOTE: SUFFICIENT CHANGE IN MOLECULAR WEIGHT NECCESSARY!!!
Detailed information on FP applications will be presented
in the “Advanced Applications” lesson of the Infinite F 500 training!
Bressanone GNB 2010
16
P. Kaltofen
Infinite™ 200 Pro Series
16/09/2010, p 34
Infinite 200 Series Reader Platform
Infinite
Infinite M200Pro
200Pro
Monochromator
Bressanone GNB 2010
or
Infinite
Infinite F200Pro
or filter based
17
P. Kaltofen
16/09/2010, p 35
infinite
infinite 200 Series Reader Platform
ִ Offers all main detection techniques:
• FI top / bottom
• FRET
• TRF
• FP for Infinite
Infinite F200
• Luminescence (Flash & Glow)
• Dual color luminescence
• Absorbance (cuvette for M200
M200)
200)
• ExcitationExcitation-, emissionemission-, absorbanceabsorbance-scan
ִ Up to 384 well plates
ִ Injector option
16/09/2010, p 36
Infinite
Infinite M200 – Available Modules
ִFI top (incl TRF)
ִFI bottom
ִSpectrally Enhanced PMT
ִAbsorbance
ִCuvette port for Abs
ִPhoton counting luminescence & Dual color
ִInjectors
ִTemperature control
ִNanoQuant Plate
Bressanone GNB 2010
18
P. Kaltofen
16/09/2010, p 37
infinite
infinite F200 – Available Modules
ִFI top incl TRF
ִFI bottom incl TRF
ִSpectrally Enhanced PMT
ִAbsorbance
ִFluorescence Polarization
ִPhoton counting luminescence & Dual color
ִInjectors
ִTemperature control
ִNanoQuant Plate
16/09/2010, p 38
Fluorescence Intensity – Filter slide
Tool for Filter Exchange
StopStop-Ring
Emission Filter Positions
Filter
Polarizer
ID Chip
Excitation/Absorbance Filter Positions
Bressanone GNB 2010
19
P. Kaltofen
16/09/2010, p 39
infinite
infinite 200 Series Reader Platform – Injector System
ִ
Injector option for fast kinetics
• Available for infinite M200 & F200
• Inject and read: Lumi, FI, Abs
ִ
Dispense function replaces pipette
ִ
User convenient: Wash/Prime procedures possible
w/o PC via buttons on injector module
ִ
Metal free coating of injector needles
ִ
Distance of needles < Ø 384 well
ִ
Opaque Teflon tubing & lid
ִ
Minimized dead volume
Tecan Group AG is making all efforts to include accurate and upup -toto-date information into this presentation. Yet, it
cannot be ruled out that omissions or errors might have occurred. Therefore, Tecan Group AG cannot make any
representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in
this presentation. Changes in this presentation can be made any time without notice. All mentioned trademarks are
protected by law. For technical details and detailed procedures of the specifications provided in this document please
contact your Tecan representative. © Tecan Trading AG, Switzerland, all rights reserved.
Thank you for your attention
Bressanone GNB 2010
20