HORIBA JobinYvon Inc., Leading the 21 st Century in Time-Resolved Fluorescence Instrumentation

1. HORIBA JobinYvon Inc., Leading the 21st Century in Time-Resolved Fluorescence Instrumentation Dr. Adam M. Gilmore Applications Scientist Fluorescence Division HORIBA Jobin Yvon Inc. Edison, NJ USA

2. Jobin Yvon (JY) 1923 Logo • JY is a World leader in Optical Spectroscopy founded in 1819 in Paris • Supplier of Scientific Instrumentation and Custom Diffraction Gratings used in the detection, measurement, and analysis of light around the Globe 1848 - Introduction of the Saccharimeter 1882 - Introduction of the Polarimeter

3. JY-Horiba Divisions Emission Spectrometry Gratings / OEM Optical Spectroscopy Fluorescence Raman Spectroscopy Forensic Thin Films

4. Fluorescence Group“The World’s Most Sensitive Instruments” • Specializing in research grade fluorescence detection • Steady state and time-resolved (ps to hrs) • All reflective optics • No chromatic aberration • High throughput (S/N>5000) • Modular and self-contained instruments • Thousands Operating Worldwide

5. Fluorescence: a type of light emission • First observed from quinine by Sir J. F. W. Herschel in 1845 Yellow glass of wine Em filter > 400 nm 1853 G.G. Stoke coined term “fluorescence” Blue glass Filter Church Window! <400nm Quinine Solution

6. Light Absorption and Fluorescence Absorption=10-15 s S2 excited state Absorbance energy S1 excited state Nonradiative dissipation Fluorescence =10-9 s Ground State Electrons

7. 1 0.8 0.6 t=1/e=37% I(t) 0.4 0.2 0 0 500 1000 time, ps What is a Fluorescence Lifetime? Random Decay Back to Ground State: Each Molecule Emits 1 Photon Population of Molecules Excited With Instantaneous Flash

8. Why measure lifetimes? • Absolute quantities- not merely ratios or time-averaged intensities • A snapshot of the excited state behavior • Largely independent of sample concentration and absorbance cross-section-in contrast to- steady state • Dynamic information-rotation-correlation times, collisional quenching rates and energy transfer processes • Additional dimensions for fluorescence data – increased specificity, sensitivity and selectivity • Lifetime senses local molecular environment (e.g. polarity, pH, temperature, electrostatics etc)

9. Fluorolog-Tau3 Picosecond Lifetime System

10. Groove spacing Classical Ruled Diffraction Grating Diffracted light cone Slit Diffracted light Normal to groove face Blaze angle Reflex angle Grating normal Incident light

11. Grating Information • Groove Density  Higher Resolution • Blaze Wavelength (Angle) Peak Efficiency • Rule Of Thumb: 2/3 l to 2 l • Slit (Bandpass) Determines Resolution • 1200g/mm • Reciprocal Linear Dispersion 4 nm/mm

12. Stray Light Reduction: Front Face Accessory Avoid Specular Reflectance at 45°: Collect at 22.5° Ref diode Grating 1 Swing away Mirror: 22.5° Grating 2 Front Face Solid Sample Excitation

13. Stray Light Reduction II: Front Face Accessory Avoid Specular Reflectance off solid samples: Collect at 22.5° Ref diode Grating 1 Grating 2 Front Face Solid Sample Excitation

14. ( ) = M ( ) Frequency Domain Transform Principle f

15. FFT: Fast Fourier Transform Rf+Df f M Fluorolog-Tau3: Multifrequency Fluorometer X Rf Spectracq MHz Df=cross correlation frequency R928P PMT amp SLAVE Rf + Df MASTER Rf amp 450W cw xenon filter Pockels Cell sample reference sample turret

16. 450 W Xe 300 nm blaze 1200 g/mm exit slit iris NIR: 9170-75=950-1700 nm 1000 nm blaze 600 g/mm grating pockels polarizer slit UV-VIS: R928 = 250-850nm 500 nm blaze 1200 g/mm grating r V V V

17. Mirror Mirror Tau-3-Fluoromap Olympus BX51 Lens Pinhole, d=0.1-3 mm Microscope lens (f = 180mm) <15 mm Dichroic mirror Mirror Lens Objectives Ex-Mono 1 mm mapping XYZ-Scanning Stage Mirror Mirror Pockels Cell Em-Mono Triax 320 CCD PMT

18. Hallmarks of Frequency Domain • Rapid, robust data collection, no worry about pulse pileup as single-photon techniques • True differential technique, no deconvolution of IRF • Economical 10 ps resolution with common cw sources Xenon lamps and lasers (strong, affordable UV source!) • Intuitive numerical data interpretation • Compatible with global analysis (separate complex decays)

19. Data Analysis in Frequency Domain • If the time domain response expression is given by I(t), it will have sine and cosine transform expressions: in which Nw and Dw are the numerator and denominator terms

20. Data Analysis in Frequency Domain • For the sum of exponentials model, the sine transform is: and the cosine transform is:

21. Data Analysis in Frequency Domain • From the sine and cosine transforms, one calculates, at each frequency, the expected phase and magnitude terms:

22. Data Analysis in Frequency Domain • Compare the calculated values to actual data • Calculate the reduced c2 value and residuals • Interpret physical significance of the results

23. Data Analysis in Frequency Domain • Calculation of c2 and the residuals of the fit: where df and dm are the errors of the measurement, and n is the number of degrees of freedom.

24. Multiexponential decay in Frequency Domain

25. Mixtures and multicomponent decays on the SPEX Tau3This data is clearly not single exponential, we need to increment the model

26. Mixtures and multicomponent decays

27. Mixtures and multicomponent decays on SPEX Tau3Adding a second component greatly improves the fit – and is justified statistically

28. Fluorescence Polarization

29. SPEX FluorescenceAnisotropy Measurements in Steady State • Anisotropy - measure polarized emission • Uses polarizers in excitation and emission paths • Measure vertical (V) and horizontal (H) intensities • Calculate <r> from these intensity measurements

30. Anisotropy and Polarization z Polarized emission with polarized excited light P = r = P = ; r = I║ -I┴ y I║ + I ┴ x I║ -I┴ Photoselection I║ + 2I ┴ 3r 2P 2 + r 3- P

31. z y x x y Anisotropy r= Grating Factor G= IVV IVV IHH - x IVH IVH IHV +2 x G G H V H V

32. Steady State Anisotropy: the Perrin equation r0 is the fundamental anisotropy (at zero time), t is the (average) fluorescence lifetime, and f is the (average) rotational correlation time

33. 0.5 0.4 0.3 Anisotropy, r 0.2 0.1 0 0 20 40 60 80 100 120 m [Protein], M Dimers: Larger Slow rotation Hindered by Viscosity High anisotropy Polarized Monomers: Small Rapid rotation Unhindered Low anisotropy Depolarized

34. TREA of perylene in oil

35. Excitation Anisotropy Spectrum of Perylene in Oil

36. TREA of perylene in oil • Fun questions: • does perylene rotate like a sphere? • or like a disk? • how can we tell? • We know perylene is a D2h rotor, can we see different modes? • what can the Fluorolog-Tau3 show us is happening? • (Assume isotropic solvent - oil in this case)

37. TREA of perylene - using anisotropic rotor model Correct Model!

38. Anisotropy Decay - TREA Modeling

39. Current Hot Topics and Applications for JY-IBH Instruments • Nanoparticles: quantum dots, nanotubes for physical and molecular research • Semiconductor PL: QC and applied LED and LD research-development • FRET-resonance energy transfer (FRET): distance and orientation of donors-acceptors • Green, red and yellow fluorescent proteins (FPs):in vivo molecular markers • Fluorescence Lifetime Microscopy: ultrastructural and biochemical characterization at 1 micron x-y-z resolution • Photosynthesis: natural, engineered and artificial systems • Drug- and Protein-design: ligand binding, anisotropy, molecular beacons • Etc…

40. Our Line of Jobin Yvon-Spex Spectrofluorometers FMAX3 World’s Most Sensitive Self-Contained Fluorometer 5000U TCSPC Flagship Tau3 World’s Most Reliable Frequency Fluorometer SkinSkan World’s Most Sensitive Skin-Surface Fluorometer FLLOG3 World’s Most Sensitive Modular Fluorometer Fluoromap 1 micron Spatial Resolution Lifetime Microscope

41. Thanks for Your Attention! Visit Our Websites: www.ibh.co.uk www.jobinyvon.com