fluorescence applications and intro to luminescence
TRANSCRIPT
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Fluorescence Applications
& an introduction to Luminescence
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Fluorescence & LuminescenceFluorescence & Luminescence
What is the difference
- Enzyme
- Triggering
reagent
Light
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Standard fluorescence Ex. DNA quantitation
FRET (fluorescence resonance energy transfer)
Accessibility of molecules, distances (= quenching)
Flash Fluorescence Ex. Calcium uptake
Follow and quantitate fast events
TRF (time resolved fluorescence)
Background free
TR-FRET (TRET)
Combination of FRET and TRF FP (fluorescence polarization)
Mobility of molecules
>> To Luminescence
Different type of applications
FluorescenceFluorescence
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FluorescenceFluorescence Standard fluorescent applications
EnzymeAntibody
Direct quantitation (NADH, NADPH)
GFP Molecular Biology
Fluorescent substrates (enzyme activitymonitoring, Fluorescent ELISA )
Immuno-fluorescence.
Labeled antibodies used to locate orquantitate antigens in complex molecular
environments (cells) Labeling (DNA, protein quantitation)
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FluorescenceFluorescence
FRET
Fluorescence Resonance Energy Transfer
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FluorescenceFluorescence
FRET: principle
2 fluorescent molecules(here blue and green)
One is the donor (blue)
One is the acceptor (green)
When both are very close to eachother, FRET occurs. The emissionof the donor is reduced and theemission of the acceptor isincreased.
Allows to quantitate interactions ata molecular level.
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Bi-labeled fluorophor (FRET)
The primer and the labelhybridize with target DNA
During the Taq polymeraseextension step, the label isdestroyed, FRET disappears
Real time quantitative PCRamplification follow-up
FRET
Primer
Target DNA
Label
FRET
Fluorescence
FRET : Example of applicationTaqMan
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FluorescenceFluorescence
Receptor / ligand binding
Detection of nucleic acid hybridization
Membrane fusion assays
Distribution and transport of lipids
Protein folding
FRET can also be used for binding assay, as it also detects distance
changes at the molecular level, but the assays are more difficult to
design than FP assays. FRET assays use two fluorescent labels, so
labeling is more complex and more problematic.
Limitations of FRET: Background due to non-specific excitation of
Acceptor (1) and residual emission of Donor in the Acceptors
emission range (2). Limited assay window and sensitivity.
FRET: Typical applications
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FluorescenceFluorescence
FRET: reader requirements
Highly sensitive fluorometer
Ability to use 2 filter sets in the same run (donor and acceptor
fluorescence monitoring) Filter based systems faster than monochromator based readers
Compatible with all BioTeks Fluorometers
For more detailed information on FRET and TR-FRET please referto the presentation Introduction_FRET_and_TR-FRET_240406.ppt
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FluorescenceFluorescence Flash fluorescence
Calcium uptake, and why you need injectors
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FluorescenceFluorescenceIntracellular Calcium
Ca2+
Calcium is a very important intracellular component (2nd messenger)
It plays a role in a lot of cellular events : extensively studied
The regulation of the intracellular level of Calcium is very complex
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F F
F F
F
F
F F
+
CA++CA++
Fast kinetic events
Requires to read just after the injection of the test molecule
Injection
Intracellular Calcium : example
CA++CA++
FluorescenceFluorescence
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Standard : emission levelchanges with [Ca++]
Fluo-4
Calcium Green-1
Calcium Green-2 Fluo-3
Calcium Orange
F
F
F
F
F
F
FF
+CA++CA++
F
F
F
F
F
F
FF
+CA++CA++
Ratio-metric : emission or excitationWL shifted
Indo-1
Fura-2
Intracellular Calcium : two type of dyes
FluorescenceFluorescence
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FluorescenceFluorescence
Intracellular Calcium: standard dye Fluo-4
0.00
20.00
40.0060.00
80.00
100.00
120.00
350 450 550 650Wavelength (nm)
Relative
Excitation/Emission
Excitation
Emission
Excitation 485/20 Emission 530/20
Emission level increases / decreases with [Ca++]
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FluorescenceFluorescenceIntracellular Calcium: standard dye Fluo-4
Fluo-4 with CaCl 2 Injection
0
5000
10000
15000
20000
25000
30000
0 2 4 6 8 10 12 14 16 18
Time (sec)
Fluorescenc
e 5 ul
10 ul
20 ul
40 ul
no dispense
Fluo-4: emission increases with the quantity of calcium injected.
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FluorescenceFluorescenceIntracellular Calcium: ratio-metric dye Indo-1
0
20
40
60
80
100
120
250 350 450 550
Wavelength (nm)
Relative
Excitation/Em
ission
Ex/zero Ca
Ex/high Ca
Em/zero Ca
Em/high Ca
Excitation at 360/40 nm
Emission measured at 410/10 and 485/20
The ratio 410 / 485 is monitored
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FluorescenceFluorescenceIntracellular Calcium: ratio-metric dye Indo-1
Indo-1 Fluorescence with Calcium Changes
0
0.2
0.4
0.60.8
1
1.2
1.4
0 5 10 15 20 25
Time (sec)
Ratio
EGTA injection
Calcium Injection
Indo-1: monitoring the 410 / 485 ratio
Ratio measurement allows to correct for signal drift
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FluorescenceFluorescenceIntracellular Calcium: reader requirements
Reagent injector
Quick signal monitoring (fast events)
Simultaneous follow-up of several emission wavelengths (ratio-
metric dyes), filter based systems much faster than monochromator
based systems
Injectors are available for all our Multi-Detection readers
Works perfect with all BioTek Multidetection readers
For more detailed information please refer to the presentation
Fluorescence Calcium Flux.ppt
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FluorescenceFluorescence
TRF
Time Resolved Fluorescence
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FluorescenceFluorescenceTRF: Principle
Time (s)
In
te
n
s
ity
0 2
Pulse of excitation light
25-125 s
Reading
Reading window
Standard fluorescence (standard dyes, auto-fluorescence)
Rare Earth fluorescence>> No backgroun d l ight>> No backgroun d l ight
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FluorescenceFluorescence
H3C
H3C
Eu3+
CH3
CH3
N
N
N
N
N
N
Stokes shift
TRF: Rare Earth
Rare earth are heavy atoms of the periodic
table (uranium and plutonium are part of this
group)
TRF uses Lanthanide Chelates
Long-lived Fluorescence (10-1000
sec) instead of nano seconds for
standard fluorescence
Large Stokes Shift
Narrow Emission Bandwidth
>> Easy c ol lect ion o f emit ted l ight
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FluorescenceFluorescenceTRF: applications
Why Time Resolved Fluorescence? If you excite them with a pulsed light source (e.g. Xenon Flash), wait (e.g. 20 s)
and read, all short-lived background fluorescence is gone at the time of the
measurement, and the excitation light is off.
Low Background Fluorescence
Works well in the presence of analytical interference:
Scattered light
Short-lived background fluorescence
Cell lysate assays (highly auto-fluorescent samples)
But!
Relatively low signal
May require the use of enhancers
Still expensive reagents and instrumentation
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FluorescenceFluorescenceTRF: reader requirements
Flash excitation of the sample
Fast data collection after excitation
TRF is working perfectly with Synergy 2
you will need to work with the High Xenon Flash Lamp
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TR-FRET / TRET
Time Resolved Fluorescence Energy Transfer
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H3C
H3C
CH3
CH3
N
N
N
N
N
N
Eu3+
Time (s)
Intens
ity
0 2 25-125 s
Reading
TR-FRET: Time Resolved Compounds
Time-Resolved compounds provideextremely low background comparedto conventional fluorescent dyes.
They can be used as Donors in
FRET assays. These assays arecalled TR-FRET or TRET assays.
HTRF (Homogeneous TimeResolved Fluorescence) is a
trademarked assay platform (Cisbio)based on TR-FRET.
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340 nm 620 nm 633 nm 665 nm
TR-compoundDonor
Standard dyeAcceptor
(ms) (ns)
340 nm 665 nm
FRET
TR-compound
DonorStandard dye
Acceptor
(ms)
TR-FRET: theory
When excited, the TR Donoremits light over a few
milliseconds.
When excited directly, the
Acceptor emits light over a
few nanoseconds.
When FRET occurs, the
energy transfer occurs over afew milliseconds, thus the
emission of the Acceptor
occurs in the same time-
frame.
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667 nm
(1)667 nm
(2)
550 nm 565 nm 550 nm
Donor Acceptor
Crosstalk issue in
standard FRET
TR-FRET: benefits compared to FRET
Recall crosstalk problem with FRET:
Some direct excitation of the Acceptor at Donor excitationwavelength
Some Donor emission appears in Acceptor emission channel
TR-FRET eliminates problem 1, because at the time ofmeasurement (delayed after excitation), the unwanted emission ofthe free Acceptor (in the ns time scale after excitation) is gone.
TR-FRET reduces assay background compared to FRET.
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TR-FRET: limitations
TR fluorescent dyes are not very bright (low quantum yield). Theyare not efficient Donors compared to standard FRET Donors. This
limits the benefit of having a reduced background level.
Background due to residual emission of Donor in the Acceptors
emission range.
Complex labeling, relatively complex assay development
Lanthanide labeling can be expensive.
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FluorescenceFluorescenceTR-FRET: reader requirements
Highly sensitive fluorometer Ability to use 2 filter sets in the same run (donor and acceptor
fluorescence monitoring)
Filter based systems faster than monochromator based readers
Flash excitation of the sample Fast data collection after excitation
TRF is working perfectly with Synergy 2
you will need to work with the High Xenon Flash Lamp
For more detailed information on FRET and TR-FRET please refer
to the presentation Introduction_FRET_and_TR-FRET_240406.ppt
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FluorescenceFluorescence
FP
Fluorescence Polarization
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FluorescenceFluorescence
Non polarized Vertically polarized
Vertical polarizer Vertical polarizer
Non polarized Vertically polarized
Vertical polarizer Horizontal polarizer
FP: principle
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FluorescenceFluorescenceFP: principle
Non polarized
Vertically polarized
Non polarized
Vertical polarizer Vertical polarizer
Vertical polarizer Horizontal polarizer
Vertically polarized
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FluorescenceFluorescenceFP: principle
Non polarized
Depolarization
Non polarized
Vertical polarizer Vertical polarizer
Vertical polarizer Horizontal polarizer
Depolarization
Level of depolar ization depends on rotat ion speed
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FluorescenceFluorescenceFP: applications
PolarizedLight
RapidRotation
Light isde-polarized
Small Molecule (up to 1 kDa) rotate 360 in 10-10 seconds
PolarizedLight
SlowerRotation
Lightremainspolarized
Large Complex (large proteins) rotate 360 in 10-8 seconds
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FluorescenceFP: applications
50
0
100
150
200
Polarization(mP)
10310210110-2 10-1 100
Receptor Conc. (nM)
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FP: Binding Assays
Labeled
positive control
Receptor
(drug target)Non-labeled
drug candidate
Question: is the drug
candidate (Q) able to bind tothe target?
Assay:
The target is pre-bound to
a labeled positive control. If the drug candidate has
high affinity for the target it
will displace the control:
polarization changes.
Low
Polarization
High
Polarization
FP is a great tool to study these binding events and is used extensively in research
labs (understand the fundamentals of molecule interactions) and screening labs
(screen for drug candidates).
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Fluorescence
A mirror system is required to direct light to the sample, as fibers cantcarry polarized light
Fibers depolarize light. You cant use a fiber to carry polarized light
Mirrors preserve light polarization
FP works perfectly with the Synergy 2 reader
For more information regarding FP please refer to the presentation:
Fluorescence_Polarization_on_the_Synergy2_150606.ppt
FP: reader requirements
Fiber optics
Mirror
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Fluorescence
FP: applications
FP shares with FRET and TR-FRET another big advantage: itis an homogeneous technology (also known as mix and read
technology). There is no need to have a wash step in an FP
assay (unlike ELISA assays for example).
Homogeneous assays are very desirable in screening labs for
the following reasons:
Easier automation (no 384/1536 washer needed)
Higher throughput (less steps)
No complex timing requirement like for ELISA assays
For that reason, homogeneous technologies have been used
as assay platforms for screening assays.
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LuminescenceLuminescenceReminder
-Enzyme
-Triggering reagent
Principle :
10-3 milli m
10-6 micro 10-9 nano n
10-12 pico p
10-15 femto f
10-18
atto a10-21 zepto z
10-24 yocto y
10-x unit symbol
Units :
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LuminescenceLuminescenceReminder: reading principle
Excitation
Emission fiber
No need for an excitation system
Even the emission fiber is notalways required
Quite often using photon countingPMT (instead of analogical PMTfor fluorescence)
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LuminescenceLuminescenceWhy is a fluorometer less sensitive in luminescence?
Photon counting is superior at low levels
But is limited at higher levels (too sensitive)
V
V
t
t
Low level
t
V
V
t
High level
Photon counting (digital)
Analogic
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Digital Photon Integration (used by BioTek Multidetection readers)
Luminescence
Photon counting
Photon integration
V
t
(Clarity)
V
t
(Synergy HT)
(FLx800)(Synergy2)
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LuminescenceLuminescenceWhy is a fluorometer less sensitive in luminescence?
Excitation & Emission fibers Emission fibers only
Fiber optic bundle
Some Luminometers do not have fiber optics
Difference between a fluorescent and a luminescent
bundle :
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LuminescenceLuminescenceDifferent type of applications
Glow Luminescence
Long lasting (minutes to hours)
Injectors not required
Similar to color development incubation Flash Luminescence
Very rapid (seconds)
requires fluid injectors
BRET (bioluminescence resonance energy transfer) Accessibility of molecules, distances (= quenching)
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LuminescenceLuminescence Glow applications
Luminescence vs. Time
0
20000
40000
60000
80000
100000
120000
0 10 20 30
Time (minutes)
RLU
Typical kinetic profile:
Applications include:
Reporter Gene Assays ATP Assays
Cytotoxic i ty / Cel l pro l iferat ion
DNA Probe Assays
PCR Quantification
Immunoassays
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LuminescenceLuminescence Flash applications
Aequorin Kinetics
0
10000
20000
30000
40000
50000
60000
70000
8000090000
100000
0 5 10
Time (sec)
Luminescence
Typical kinetic profile:
Applications include:
Flash ATP assays Calc ium moni tor ing
React ive oxyg en species
(ROS) ass ay
Some luci fer ine / luc i ferase
assays
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Bioluminescence Resonance Energy Transfer
Luciferase
Coelenterazine Coelenteramide
blue
Green
Fluorescent
Protein
greenEnergy Transfer
BRET : similar to FRET
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LuminescenceLuminescenceReader requirements
High sensitivity, works with all Multidetection readers from BioTek
Injectors for some applications
Ask us: some applications have been tested in the US
Dual luciferase from Promega: working
(application note available) Luminescent substrates : working (tested with 1,2 dioxetane
substrate)
If we have no background with a specific application, you should try it
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Fluorescence & LuminescenceFluorescence & LuminescenceSummary
High number of applications
Dont hesitate to contact BioTek, we are here to help, and have an
excellent scientific team in the US, for advanced applications
As you know, our readers will accommodate any standard
fluorescent assay with high sensitivity and powerful data reduction
In addition, our SynergyHT and Synergy2 instruments have a
monochromator based detection unit for any kind of absorbance
assay
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Fluorescence ApplicationsFluorescence Applications
For more information, visit our website
www.biotek.com
Recommended website for fluorescence
http://probes.invitrogen.com
(Invitrogen, formerly Molecular Probes)