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

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Jobin Yvon (JY) JY is a World leader in Optical Spectroscopy founded in 1819 in ParisJY 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 GlobeSupplier of Scientific Instrumentation and Custom Diffraction Gratings used in the detection, measurement, and analysis of light around the Globe Introduction of the Saccharimeter Introduction of the Polarimeter 1923 Logo

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

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

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

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Ground State Electrons S 1 excited state S 2 excited state Absorbance energy Fluorescence =10 -9 s Absorption= s Nonradiative dissipation Light Absorption and Fluorescence

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

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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)

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Fluorolog-Tau3 Picosecond Lifetime System

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

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

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

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

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( ) M = Frequency Domain Transform Principle

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

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450 W Xe V 300 nm blaze 1200 g/mm V V r exit slit iris slit pockelspolarizer UV-VIS: R928 = nm 500 nm blaze 1200 g/mm grating NIR: = nm 1000 nm blaze 600 g/mm grating

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

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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 )

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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 N and D are the numerator and denominator terms

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

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

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Data Analysis in Frequency Domain Compare the calculated values to actual data Calculate the reduced 2 value and residuals Interpret physical significance of the results

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Data Analysis in Frequency Domain Calculation of 2 and the residuals of the fit: where d and dm are the errors of the measurement, and is the number of degrees of freedom.

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Multiexponential decay in Frequency Domain

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Data Fitting – wrong model will be clearly evident Mixture of two fluorophores will not fit to single exponential decay model!

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Mixtures and multicomponent decays on the SPEX Tau3 This data is clearly not single exponential, we need to increment the model

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Mixtures and multicomponent decays

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Mixtures and multicomponent decays on SPEX Tau3 Adding a second component greatly improves the fit – and is justified statistically

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Fluorescence Polarization

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SPEX Fluorescence Anisotropy Measurements in Steady State Anisotropy - measure polarized emission Uses polarizers in excitation and emission paths Measure vertical (V) and horizontal (H) intensities Calculate from these intensity measurements

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

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V HV H x z y x y I VV I VH I HH I HV Anisotropy r= Grating Factor G= I VV I VH - +2 GG x x

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Anisotropy: steady state

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Steady State Anisotropy: the Perrin equation r 0 is the fundamental anisotropy (at zero time), is the (average) fluorescence lifetime, and is the (average) rotational correlation time

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[Protein], M Anisotropy, r Monomers: Small Rapid rotation Unhindered Low anisotropy Depolarized Dimers: Larger Slow rotation Hindered by Viscosity High anisotropy Polarized

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Anisotropy decay (TREA) in frequency domain Excitation is always V polarized –Pockels cell or laser provide this Measures the differential phase of emission diff = V - H (this may be in L or T format) Also measures the RMA: –Ratio of the Modulated Amplitudes –Corrected for all instrument response terms, including PMT frequency dependent gain differences and G factor VERY RAPID DATA COLLECTION

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Anisotropy Decay - Data Analysis where the terms represent rotational correlation times. Frequently the preexponential terms are normalized to the fundamental anisotropy, becoming fractional contributions:

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TREA of perylene in oil

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Excitation Anisotropy Spectrum of Perylene in Oil

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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 D 2h rotor, can we see different modes? –what can the Fluorolog-Tau3 show us is happening? –(Assume isotropic solvent - oil in this case)

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TREA of perylene - using a spherical rotor model Wrong Model! (3 Reasons)

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TREA of perylene - using anisotropic rotor model Correct Model!

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Anisotropy Decay - TREA Modeling

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

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Our Line of Jobin Yvon-Spex Spectrofluorometers FMAX3 World’s Most Sensitive Self-ContainedFluorometer 5000UTCSPCFlagshipTau3 World’s Most Reliable FrequencyFluorometer FLLOG3 World’s Most Sensitive ModularFluorometer SkinSkan Skin-SurfaceFluorometer Fluoromap 1 micron Spatial Resolution Lifetime Microscope

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