Copyright © 2010 ISS, Inc. All Rights Reserved. Ewald Terpetschnig Ph.D. Newly designed Fluorescent...

Copyright © 2010 ISS, Inc. All Rights Reserved.

Ewald Terpetschnig Ph.DEwald Terpetschnig Ph.D..

Newly designed Fluorescent Probes & Labels Newly designed Fluorescent Probes & Labels enabling Lifetime-Based Sensorsenabling Lifetime-Based Sensors

Newly designed Fluorescent Probes & Labels Newly designed Fluorescent Probes & Labels enabling Lifetime-Based Sensorsenabling Lifetime-Based Sensors

Analytica 2010Analytica 2010


Two Ways To Measure Fluorescence LifetimeTwo Ways To Measure Fluorescence Lifetime


Time-DomainTime-Domain

It = e – t/

TCSPC

Excitation with pulse -Measurement of photons time-correlated

Time-to-amplitude Converter


Frequency DomainFrequency Domain

Phase Shift

Demodulation

tan =

EX

EM

(AC/DC)

(AC/DC)M

21

1

)(M


Reactive group for covalent attachment to biomoleculesReactive group for covalent attachment to biomolecules

Lifetime-sensitive probe Lifetime-sensitive probe

Good overall brightnessGood overall brightness

Fluorescence Lifetime LabelsFluorescence Lifetime Labels

300 350 400 450 500 550 600 650 700 750 8000

50000

100000

150000

200000

250000

Flu

ore

sce

nce

Ext

inct

ion

coe

ffici

ent

[M

-1 c

m-1]

Wavelength [nm]

High chemo- and photostabilityHigh chemo- and photostability


Lifetime-Sensitive Cyanine Dye: Lifetime-Sensitive Cyanine Dye: Seta-633Seta-633Lifetime-Sensitive Cyanine Dye: Lifetime-Sensitive Cyanine Dye: Seta-633Seta-633

Study interaction between low-molecular-weight Study interaction between low-molecular-weight analytes and proteinsanalytes and proteins Fluorescence lifetime as the readout parameterFluorescence lifetime as the readout parameter

Model-System:

Biotinylated label with anti-biotin antibodyBiotinylated label with anti-biotin antibody

Bioconjugate Chem. 20092009, 20, 1907–1912


Measurement PrincipleMeasurement Principle

Lifetime of labeled species changes upon binding to proteinLifetime of labeled species changes upon binding to protein

Lifetime Lifetime – More robust parameter compared to intensity – More robust parameter compared to intensity –– Independent of probe concentration Independent of probe concentration

–– Unaffected by probe volume and to Unaffected by probe volume and to some extend some extend photobleaching photobleaching

Antibody-Antigen-Complex

AbAb AbAb


Comparison With Commercially Available DyesComparison With Commercially Available Dyes

Cyanine dyes like Cy5 and Alexa 647 exhibit smaller Cyanine dyes like Cy5 and Alexa 647 exhibit smaller lifetimelifetime changes (factor < 2) upon binding to proteinschanges (factor < 2) upon binding to proteins

Seta-633 exhibits a 10-fold lifetime change upon binding Seta-633 exhibits a 10-fold lifetime change upon binding to BSA and a 6-fold change with IgGto BSA and a 6-fold change with IgG


Synthesis of D-Biotin ConjugateSynthesis of D-Biotin Conjugate


Competitive ImmunoassayCompetitive Immunoassay

Titrate fixed [Seta-633-biotin] concentration withTitrate fixed [Seta-633-biotin] concentration with

increasing amounts of [Anti-biotin] and record phase-increasing amounts of [Anti-biotin] and record phase-

modulation datamodulation data


Free analyte replaces labeled analyte – lifetime changeFree analyte replaces labeled analyte – lifetime change

FD measurements – phase angle changes at single frequencyFD measurements – phase angle changes at single frequency

Competitive Binding StudyCompetitive Binding Study

FD measurements – accuracy of measurement of FD measurements – accuracy of measurement of = 0.1 deg= 0.1 deg


Ratiometric MeasurementRatiometric Measurement

Lifetime of Tracer determines MW of Analyte in FPALifetime of Tracer determines MW of Analyte in FPA

Fluorescence Polarization Fluorescence Polarization

Short Lifetime – Low MW Analyte Short Lifetime – Low MW Analyte Long Lifetime – High MW AnalyteLong Lifetime – High MW Analyte


Fluorescence Polarization

A Race between Emission and Molecular MotionFluorescence Polarization

A Race between Emission and Molecular MotionPolarized light excitesPolarized light excitesmolecules aligned withmolecules aligned withplane of polarizationplane of polarization

flfl<<<<rotrotflfl>>>>rotrot

Excited molecules remain aligned.Excited molecules remain aligned.Fluorescence is polarized.Fluorescence is polarized.

Orientation of excited molecules Orientation of excited molecules randomizes. Fluorescence is depolarized.randomizes. Fluorescence is depolarized.


How is Polarization measured?How is Polarization measured?


Polarization (P) = IPolarization (P) = Ivv - I - Ih h / I/ Ivv+ I+ Ihh

Anisotropy (r) = IAnisotropy (r) = Ivv - I - Ihh / I / Ivv + 2 I + 2 Ihh

P = 3 r / 2 + rP = 3 r / 2 + r

r = 2 P / 3 -Pr = 2 P / 3 -P


fl fl ~ 300 ns ~ 300 ns flfl = 4ns = 4ns)/ (1

rr 0

HSAHSA = 40 ns = 40 ns

Measurement of High-MW-Analytes in FPIAMeasurement of High-MW-Analytes in FPIA


For best results lifetime For best results lifetime flfl should be in the range of should be in the range of

rotational correlation time rotational correlation time of antigen that is labeled of antigen that is labeled

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

160

180

200

220

240

260

280

300

320

340

K7-545-BSA — conjugate

Pol

ariz

atio

n [m

P]

[ANTI-BSA / K7-545-BSA]

flfl = = 26 ns26 nsSeTau-425SeTau-425

TG-404TG-404 flfl = 9 ns= 9 ns

Lifetime Lifetime flfl of the label is a measure for MW of the of the label is a measure for MW of the species that can be measured in a FPIAspecies that can be measured in a FPIA

Commercially available Lifetime Dyes:Commercially available Lifetime Dyes:Commercially available Lifetime Dyes:Commercially available Lifetime Dyes:


Loading of Cells with Florescent Probes cannot be Loading of Cells with Florescent Probes cannot be controlled controlled

Ratiometric MeasurementsRatiometric Measurements

Fluorescence Sensing in CellsFluorescence Sensing in Cells

FLIM (Fluorescence Lifetime Imaging)FLIM (Fluorescence Lifetime Imaging)

Fluorescent Probes will accumulate differently at Fluorescent Probes will accumulate differently at various locations in the cell various locations in the cell

Quantitative Measurements:Quantitative Measurements:


Measurement ModeMeasurement Mode Qualitative or quantitative measurements. Ion-probes showing spectral shifts - radiometric measurements Are light source available?

How to Choose the Correct Fluorescent ProbeHow to Choose the Correct Fluorescent Probe

Dissociation Constant (KDissociation Constant (Kdd)) Calibration: Kd of probe is dependent on pH, temperature,

viscosity, ionic strength etc…….

Indicator FormIndicator Form Influences cell loading and distribution of the probe Salts and dextran-conjugates - microinjection, electroporation, AM-esters - passively loaded and cleaved by intracellular esterases


pKa in the physiolocical pKa in the physiolocical pH-rangepH-range

Absorption Absorption (653 nm)(653 nm) and and emission emission (671 nm)(671 nm) in the in the red regionred region

Seta-650-pH Dual-Ratiometric pH-Measurements in Cells

Seta-650-pH Dual-Ratiometric pH-Measurements in Cells

Excitation and emissionExcitation and emission ratiometric proberatiometric probe

Anal. Biochem. Anal. Biochem. 390390, , 136–140 (2009)136–140 (2009)


Excitation and Emission Ratiometric Excitation and Emission Ratiometric MeasurementsMeasurements

Excitation-ratiometric measurement Excitation-ratiometric measurement λλemem = 710 nm = 710 nm

pH-Sensing in Cells pH-Sensing in Cells

Emission-ratiometric measurement Emission-ratiometric measurement λλexex = 590 nm = 590 nm


Seta-650-pH probe also shows Seta-650-pH probe also shows pH- sensitive lifetime changespH- sensitive lifetime changes

Phase angle changes ~ 16 degPhase angle changes ~ 16 deg between protonated and between protonated and deprotonated forms at 100 MHzdeprotonated forms at 100 MHz

FLIM pH-Measurements in Cells FLIM pH-Measurements in Cells

pH-Label applicable for FLIM pH-Label applicable for FLIM

6 7 8 90

2

4

6

8

10

12

14

16

100 MHz

pKa = 7.35

Ph

ase

An

gle

Ch

an

ge

[

de

g]

Square-650-pH

pH


Seta-650-pH labeled E.Coli Seta-650-pH labeled E.Coli is internalized in Cellis internalized in Cell

Inside Cell pH of certain Inside Cell pH of certain compartments is lower pH ~ 5compartments is lower pH ~ 5

pH-Sensing in Cells - Phagocytosis pH-Sensing in Cells - Phagocytosis

Fluorescence Intensity or LifetimeFluorescence Intensity or Lifetime will reflect these pH changes and will reflect these pH changes and are utilized as parameters for are utilized as parameters for visualization visualization

Square-650-pH labeledE.Coli

Exterior CellpH ~ 7.4

Interior PhagocyteCell pH ~ 7.0-7.2

Phago-lysosome

pH ~ 5

Phago-lysosome

pH ~ 5

Anal. Biochem. Anal. Biochem. 390390, , 136–140 (2009)136–140 (2009)


Acknowledgment:Acknowledgment:

Y. Povrozin Y. Povrozin ISC, Kharkov,ISC, Kharkov, Ukraine Ukraine A. Tatarets A. Tatarets ISC, KharkovISC, Kharkov, Ukraine, Ukraine L. PatsenkerL. Patsenker ISC, KharkovISC, Kharkov, Ukraine and , Ukraine and

Seta BioMedicalsSeta BioMedicals, Urbana IL, USA, Urbana IL, USA

Copyright © 2010 ISS, Inc. All Rights Reserved. Ewald Terpetschnig Ph.D. Newly designed Fluorescent...

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