1 / 27

Ewald Terpetschnig Ph.D .

Newly designed Fluorescent Probes & Labels enabling Lifetime-Based Sensors. Analytica 2010. Ewald Terpetschnig Ph.D. Two Ways To Measure Fluorescence Lifetime. Time-Domain. I t = a e – t/ t. TCSPC. Excitation with pulse - Measurement of photons time-correlated.

vince
Download Presentation

Ewald Terpetschnig Ph.D .

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Newly designed Fluorescent Probes & Labels enabling Lifetime-Based Sensors Analytica 2010 Ewald Terpetschnig Ph.D.

  2. Two Ways To Measure Fluorescence Lifetime

  3. Time-Domain It = a e– t/t TCSPC Excitation with pulse - Measurement of photons time-correlated Time-to-amplitude Converter

  4. Frequency Domain tan= Phase Shift Demodulation

  5. Fluorescence Lifetime Labels • Reactive group for covalent attachment to biomolecules • Lifetime-sensitive probe • Good overall brightness • High chemo- and photostability

  6. Lifetime-Sensitive Cyanine Dye: Seta-633 • Study interaction between low-molecular-weight • analytes and proteins • Fluorescence lifetime as the readout parameter Model-System: • Biotinylated label with anti-biotin antibody Bioconjugate Chem. 2009, 20, 1907–1912

  7. Measurement Principle Ab Ab Antibody- t1 t2 Antigen-Complex • Lifetime of labeled species changes upon binding to protein • Lifetime – More robust parameter compared to intensity –Independent of probe concentration –Unaffected by probe volume and to some extend photobleaching

  8. Comparison With Commercially Available Dyes • Cyanine dyes like Cy5 and Alexa 647 exhibit smaller lifetimechanges (factor < 2) upon binding to proteins • Seta-633 exhibits a 10-fold lifetime change upon binding to BSA and a 6-fold change with IgG

  9. Synthesis of D-Biotin Conjugate

  10. Competitive Immunoassay • Titrate fixed [Seta-633-biotin] concentration with increasing amounts of [Anti-biotin] and record phase- modulation data

  11. Competitive Binding Study • Free analyte replaces labeled analyte – lifetime change • FD measurements – phase angle changes at single frequency • FD measurements – accuracy of measurement of f = 0.1 deg

  12. Fluorescence Polarization • Ratiometric Measurement • Lifetime of Tracer determines MW of Analyte in FPA • Short Lifetime – Low MW Analyte Long Lifetime – High MW Analyte

  13. Fluorescence Polarization A Race between Emission and Molecular Motion Polarized light excites molecules aligned with plane of polarization tfl>>rot tfl<<rot Excited molecules remain aligned. Fluorescence is polarized. Orientation of excited molecules randomizes. Fluorescence is depolarized.

  14. How is Polarization measured?

  15. Polarization (P) = Iv - Ih / Iv+ Ih Anisotropy (r) = Iv - Ih / Iv + 2 Ih P = 3 r / 2 + r r = 2 P / 3 -P

  16. Measurement of High-MW-Analytes in FPIA tfl ~ 300 ns tfl = 4ns HSA = 40 ns

  17. Commercially available Lifetime Dyes: tfl= 26 ns SeTau-425 tfl= 9 ns TG-404 • Lifetime tfl of the label is a measure for MW of the • species that can be measured in a FPIA • For best results lifetime tfl should be in the range of rotational correlation time  of antigen that is labeled

  18. Fluorescence Sensing in Cells • Loading of Cells with Florescent Probes cannot be controlled • Fluorescent Probes will accumulate differently at various locations in the cell Quantitative Measurements: • Ratiometric Measurements • FLIM (Fluorescence Lifetime Imaging)

  19. How to Choosethe Correct Fluorescent Probe • Measurement Mode • Qualitative or quantitative measurements. • Ion-probes showing spectral shifts - radiometric measurements • Are light source available? • Dissociation Constant (Kd) • Calibration: Kd of probe is dependent on pH, temperature, viscosity, ionic strength etc……. • Indicator Form • Influences cell loading and distribution of the probe • Salts and dextran-conjugates - microinjection, electroporation, • AM-esters - passively loaded and cleaved by intracellular • esterases

  20. Seta-650-pH Dual-Ratiometric pH-Measurements in Cells • Absorption (653 nm) and emission (671 nm) in the red region • Excitation and emission ratiometric probe • pKa in the physiolocical pH-range Anal. Biochem. 390, 136–140 (2009)

  21. pH-Sensing in Cells • Excitation and Emission Ratiometric Measurements • Excitation-ratiometric measurement λem = 710 nm • Emission-ratiometric measurement • λex = 590 nm

  22. FLIM pH-Measurements in Cells • Seta-650-pH probe also shows pH- sensitive lifetime changes • Phase angle changes ~ 16 deg between protonated and deprotonated forms at 100 MHz • pH-Label applicable for FLIM

  23. pH-Sensing in Cells - Phagocytosis • Seta-650-pH labeled E.Coli is internalized in Cell • Inside Cell pH of certain compartments is lower pH ~ 5 • Fluorescence Intensity or Lifetime will reflect these pH changes and are utilized as parameters for visualization Anal. Biochem. 390, 136–140 (2009)

  24. Acknowledgment: Y. Povrozin ISC, Kharkov, Ukraine A. Tatarets ISC, Kharkov, Ukraine L. Patsenker ISC, Kharkov, Ukraine and Seta BioMedicals, Urbana IL, USA

More Related