HORIBA JobinYvon Inc., Leading the 21st Century in Time-Resolved Fluorescence Instrumentation Dr. Adam M.
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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 1923 Logo Jobin Yvon (JY) • 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 JY-Horiba Divisions Gratings / OEM Emission Spectrometry Optical Spectroscopy Fluorescence Raman Spectroscopy Thin Films Forensic 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 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 Light Absorption and Fluorescence Absorbance energy Absorption=10-15 s S2 excited state S1 excited state Fluorescence =10-9 s Ground State Electrons Nonradiative dissipation What is a Fluorescence Lifetime? Random Decay Back to Ground State: Each Molecule Emits 1 Photon 1 I(t) 0.8 0.6 t=1/e=37% 0.4 0.2 0 0 Population of Molecules Excited With Instantaneous Flash 500 time, ps 1000 Why measure lifetimes? Absolute quantities- not merely ratios or timeaveraged 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) Fluorolog-Tau3 Picosecond Lifetime System Classical Ruled Diffraction Grating Diffracted light cone Slit Diffracted light Normal to groove face Blaze angle Reflex angle Grating normal Incident light Groove spacing 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 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 Stray Light Reduction II: Front Face Accessory Ref diode Avoid Specular Reflectance off solid samples: Collect at 22.5° Grating 1 Grating 2 Front Face Solid Sample Excitation 1.5 1.5 M ( ) ( ) Frequency Domain Transform Principle 0.5 0.5 00 tanf tf -0.5 -0.5 100 1010 100 10 100 10 100 10 100 Frequency, MHz Frequency, MHz Frequency, MHz Frequency, MHz Frequency, MHz 1 11 0.9 0.9 0.9 0.8 0.8 0.8 0.7 0.7 0.7 0.6 0.6 0.6 0.5 0.5 0.5 0.4 0.4 0.4 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.1 00 00 0 1000 1000 1000 1000 1000 M M M M M f f f TIME TIME 1 2t M2 f 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1000 f M 909090 808080 707070 606060 505050 404040 303030 2020 20 2020 10 10 10 1010 00 00 0 11 11 1 M 10 100 Frequency, MHz -1.5 -1.5 1 90 80 70 60 50 40 30 20 10 0 -1 -1 1 AMPLITUDE AMPLITUDE 11 = M f Spectracq MHz FFT: Fast Fourier Transform amp SLAVE Rf + Df Rf+Df X Rf Df=cross correlation frequency R928P PMT MASTER Rf 450W cw xenon amp filter Pockels Cell sample reference sample turret Fluorolog-Tau3: Multifrequency Fluorometer 450 W Xe 300 nm blaze 1200 g/mm exit slit iris pockels polarizer NIR: 9170-75=950-1700 nm 1000 nm blaze 600 g/mm grating slit r V V V UV-VIS: R928 = 250-850nm 500 nm blaze 1200 g/mm grating Mirror Mirror Tau-3-Fluoromap Olympus BX51 Lens Pinhole, d=0.1-3 mm Microscope lens (f = 180mm) <15 mm Dichroic mirror Mirror Lens Objective s 1 mm mapping Ex-Mono XYZ-Scanning Stage Mirror Mirror Em-Mono PMT Triax 320 CCD Pockels Cell 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) Data Analysis in Frequency Domain • If the time domain response expression is given by I(t), it will have sine and cosine transform expressions: N I (t ) sin (t ) dt 0 I (t ) dt 0 D I (t ) cos(t ) dt 0 I (t ) dt 0 in which N and D are the numerator and denominator terms Data Analysis in Frequency Domain • For the sum of exponentials model, the sine transform is: it i2 N 2 2 ( 1 t ) i t i i i and the cosine transform is: it i2 D 2 2 ( 1 t ) i t i i i Data Analysis in Frequency Domain • From the sine and cosine transforms, one calculates, at each frequency, the expected phase and magnitude terms: N fc , tan D 1 mc, N D 2 2 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 Data Analysis in Frequency Domain • Calculation of c2 and the residuals of the fit: c 2 1 (fc , f ) (mc , m ) df dm 2 where df and dm are the errors of the measurement, and is the number of degrees of freedom. 2 Multiexponential decay in Frequency Domain Mixtures and multicomponent decays on the SPEX Tau3 This data is clearly not single exponential, we need to increment the model Mixtures and multicomponent decays Mixtures and multicomponent decays on SPEX Tau3 Adding a second component greatly improves the fit – and is justified statistically Fluorescence Polarization 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 <r> from these intensity measurements Anisotropy and Polarization Polarized emission with polarized excited light P= I║ - I ┴ y I║ + I ┴ x I║ - I ┴ r = I + 2I ║ ┴ P= z 3r 2+r ; r= Photoselection 2P 3- P Anisotropy r= - x IVV IHH +2 x IVH IHV H V Grating Factor G= G H zx x V yy Steady State Anisotropy: the Perrin equation r 1 r0 t 1 f r0 is the fundamental anisotropy (at zero time), t is the (average) fluorescence lifetime, and f is the (average) rotational correlation time 0.5 Dimers: Larger Slow rotation Hindered by Viscosity High anisotropy Polarized 0.4 Anisotropy, r Monomers: Small Rapid rotation Unhindered Low anisotropy Depolarized 0.3 0.2 0.1 0 0 20 40 60 [Protein], mM 80 100 120 Fluorescence Emission Dipole Long wavelength absorption (430nm) TREA of perylene in oil Short wavelength absorption (256nm) Excitation Anisotropy Spectrum of Perylene in Oil 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) TREA of perylene - using anisotropic rotor model Correct Model! Anisotropy Decay - TREA Modeling 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 researchdevelopment • 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… 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 FLLOG3 Fluorometer World’s Most Sensitive Modular Fluoromap Fluorometer 1 micron Spatial Resolution Lifetime Microscope Thanks for Your Attention! 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