Fluorescence Applications and Intro to Luminescence

7/28/2019 Fluorescence Applications and Intro to Luminescence

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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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FRET

Fluorescence Resonance Energy Transfer


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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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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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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++



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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



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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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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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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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FP

Fluorescence Polarization


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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



Vertically polarized


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FluorescenceFluorescenceFP: principle

Non polarized

Depolarization

Non polarized



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)

Fluorescence Applications and Intro to Luminescence

Documents

Transcript of Fluorescence Applications and Intro to Luminescence