Selected measurements with FluoTime 300 Sebastian Tannert, Peter Kapusta, Felix Koberling, Manoel Veiga, Steffen Rüttinger Uwe Ortmann, Matthias Patting, Marcus Sackrow, Michael Wahl, Rainer Erdmann 12th of October 2016, Moscow Copyright of this document belongs to PicoQuant GmbH. No parts of it may be reproduced, translated or transferred to third parties without written permission of PicoQuant GmbH., 2014
Fluorescence and TCSPC principles Components of a FluoTime 300 Time resolution and sensitivity Software for acquisition Software for analysis Examples of measurements 54
Multi-exponential Fit and Average Lifetime t t t F(t) = A1*exp(-t/ 1) + A2*exp(-t/ 2) + A3*exp(-t/ 3) + B A1 (amplitude) A2 (amplitude) A3 (amplitude 3) t1 =2.8 ns t2 =33 ns t3 =3.2 ms t A1* 1 (intensity) t A2* 2 (intensity) Amplitude Intensity 99% 71% 0.6% 4% 0.4% 25% t A3* 3 (intensity) 3.8 ns B (background) 84 ns [ns] 55
Global Analysis (Example: A TRES Measurement) t 56
Applications/Performance Decay Associated Spectra Tryptophan O OH 1.0 t1 = 360 ps t2 = 2.5 ns t3 = 7.4 ns Steady-state emission spectrum Normalized amplitude 0.8 HN 0.6 0.4 NH2 Data acquisition time: 12 min Number of histograms: 31 Global data analysis 3-exponential model 0.2 Sample: Tryptophan 17 µm A @ 290 nm = 0.064 Excitation: 10 MHz pulsed 290 nm LED Detection: 310-460 nm by 5 nm steps 0.0 320 340 360 380 400 420 440 460 Wavelength [nm] 58
Foundations of Fluorescence Polarization Fluorophores have an absorption and emission transition dipole moment. Excitation is most efficient, when the absorption dipole transition moment is parallel to the excitation polarization. The emission of a molecule has the polarization of the emission dipole transition moment. For most fluorophores, excitation and emission dipole transition moments are close to parallel. For more detailed explanations, see: J. Lakowicz, Principles of Fluorescence Spectroscopy, Springer 2006 61
What is Fluorescence Anisotropy? I II I r= I II + 2 I rr ==Anisotropy Anisotropy IIII == Intensity Intensity (polarization (polarization parallel parallel to to that that of of excitation) excitation) II II == Intensity Intensity (polarization (polarization perpendicular perpendicular to to that that of of excitation) excitation) Microscope set-up Spectrometer set-up Perpendicular polarization 2 (axial polarization relative to detector) Perpendicular polarization 2 (not reaching detector) Dichroic mirror Excitation Excitation Parallel polarization Perpendicular polarization 1 Rotatable polarizer Detector Polarizing beamsplitter Parallel polarization Parallel detector Perpendicular polarization 1 Perpendicular detector 62
Fluorescence Depolarization Origins Rotational diffusion Mol. 1 Mol. 2 Mol. 3 Time Energy transfer HOMO-FRET 63
Rotational Diffusion: The Perrin-Equation r0 =1+ τ =1+ 6 D τ θ r ηv θ= (R T ) rr0 == Fundamental Fundamental anisotropy anisotropy 0 rr == Measured anisotropy Measured anisotropy ττ == Fluorescence Fluorescence lifetime lifetime == Rotational correlation Rotational correlation time time DD == Rotational Rotational diffusion diffusion coefficient coefficient hh == Viscosity Viscosity VV == Volume Volume (incl. (incl. hydration hydration shell) shell) RR == Gas constant Gas constant TT ==Temperature Temperature The rotational diffusion time can be measured with fluorescence decay measurements only, if it is not significantly longer than t. Longer rotational diffusion times can be measured with dead-time-free fluorescence correlation spectroscopy. For more detailed explanations, see: J. Lakowicz, Principles of Fluorescence Spectroscopy, Springer 2006 64
Rotation of Proteins For proteins can be approximated: hh == viscosity viscosity VV == volume volume (incl. (incl. hydration hydration shell) shell) RR == gas constant gas constant TT == temperature temperature M = molecular M = molecular weight weight vvspec == specific protein volume, specific protein volume, typically typically 0.73 0.73 ml/g ml/g spec hh == hydration hydration ηv ηm θ= = (v spec +h) (R T ) RT For room temperature, the rotational correlation time of a protein can be calculated: Rotational correlation time [ns] 100 without Hydration with 0.23ml/g Hydration 80 60 Prerequisite: globular protein shape vspec = 0.73 ml/g T = 298 K h 1 cp 40 20 0 0 100 200 Molecular weight [kda] 65
Applications/Performance Global Data Analysis Dynamic Anisotropy Direct anisotropy calculation without reconvolution 0.4 Sample: Coumarin 6 in ethylene glycol Excitation: 20 MHz pulsed 440 nm laser, V pol Detection: 510 nm, 3 nm bandwidth, H, V, M pol Data acquired by a script macro Anisotropy 0.3 Results of global reconvolution fitting 0.1 0.30 Steady-state anisotropy calculated from kinetic parameters 4.5 4.0 0.20 3.5 0.15 3.0 0.10 2.5 0.05 2.0 0.00 Fluorescence lifetime t Rotational correlation time, 1.5 10 15 20 25-0.05 30 Temperature [ C] 35-0.1 0.25 40-0.10 Steady-state anisotropy Fluorescence lifetime [ns] Rotational correlation time [ns] 0.2 0.0 5.0 1.0 10 C 25 C 40 C 0 2 4 6 8 10 12 14 Time [ns] global χ2 = 1.007 r0 = 0.380 ± 0.001 G = 1.06 ± 0.01 @ 510 nm 69
Spectrally Resolved Measurements can Separate Contributions from Different Layers Structure of the Quantum Well Sample 735 nm 650 nm Τav. ~ 2 ns Τav. ~ 25 ps τi, max. ~ 0.2 ns τi, max. ~ 11 ns 860 nm Τav. ~ 15 ps τi, max. ~ 1.3 ns 595 nm Illumination Al0.4Ga0.6As GaAsP (quantum well) GaInP Al0.4Ga0.6As n-gaas FluoTime 300 exc = 595 nm Laser, 10 MHz det = 630 nm - 900 nm (monochromator) + add. 620LP-filter Sample courtesy of Andrea Knigge, Ferdinand-Braun-Institut, Berlin, Germany Buschmann et al., J. Appl. Spectr., 80, 449-457 (2013) 70
Upconversion particles Deep Tissue Label for IR excitation with VIS emission TEM Image ETU: Energy transfer upconversion [1] 1 um Sample: NaYF4:Yb/Er with silica coating Size: 20 nm Activator ion, e.g. Er3+ Sensitizer ion, e.g. Yb3+ Samples courtesy of: Prof. Nyokong, Edith Antunes, Rhodes University, South Africa [1] F. Auzel, Chem. Rev. 2004, 104, 139-173 71
Steady State Emission 120000 NaYF4:Yb/Er Intensity [a.u.] 100000 80000 Sample: NaYF4:Yb/Er in acetonitrile Size: 20 nm, silica coating Excitation source: LDH-D-C-980 Excitation wavelength: 980 nm, CW, 180 mw Emission bandwidth: 4 nm Detector: PMA-C-192M 60000 40000 20000 0 510 520 530 540 550 560 570 Wavelength [nm] 72
Burst Excitation for Phosphorescence burst mode: 8500 shots, 100 ps pulse width, 980 nm Intensity [Counts] 1000 Sample: NaYF4:Yb/Er in acetonitrile Size: 20 nm, silica coating Excitation source: LDH-D-C-980 Excitation wavelength: 980 nm, CW, burst mode Emission bandwidth: 10.4 nm Detector: PMA-C-192M Detection wavelength: 540 nm 100 t = 113+-3 ms 10 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Time [ms] 73
Singlet Oxygen Generation Mechanism of photosensitization ISC S1 T1 1 A F P O2 1270 nm Energy Transfer S0 Photosensitizer 3 O2 Oxygen 74
Singlet Oxygen in Photodynamic Therapy 1. Intravenous injection 4. Introduction of laser light 2. PS transport and accumulation 3. Selective tissue retention 5. Tumor tissue necrosis/apoptosis 75
Singlet Oxygen Spectra 50000 H2TPP in H2O H2TPP in acetone Intensity / counts 40000 30000 20000 10000 0 1220 1240 1260 1280 1300 1320 1340 Wavelength / nm Sample: H2TPP in water/acetone Excitation source: LDH-P-C-405 Excitation wavelength: 405 nm Emission bandwidth: 4 nm Step size: 1 nm Acquisition time per point: 0.5 s No polarizers or filters Detector: H01033-45, 2008 76
Singlet Oxygen Phosphorescence Decay burst mode: 500 shots, 100 ps pulse width, 405 nm 10000 Counts Sample: H2TPP in water Excitation source: LDH-P-C-405 Excitation wavelength: 405 nm, burst mode Emission bandwidth: 10.4 nm 834L long pass filter Detector: H01033-45 Literature: τδ = 3.7 μs [1] td = 3.4 ± 0.3 µs 1000 0 10 20 30 40 50 Time [µs] [1] S. Nonell et al., Molecules 2013, 18, 2712-2725 77
Pervoskite S. D. Stranks et al., Science, 342 (2013), p.341 78
Time-resolved Emission Spectra on a Microscope Stage Confocal microscope coupled to time-resolved fluorescence spectrometer. Excitation beam Laser head Spatially resolved Time-Resolved Emission Spectra (TRES) Lifetime imaging with spectral selection TRES-imaging Detection Confocal micrsocope MicroTime 100 Time-resolved fluorescence spectrometer FluoTime 300 79
TRPL Imaging can Reveal the Defect Structure of CIGS Material Intensity Image Sample: CIGS layer on glass MicroTime 100 Objective: LCPlan N 20x, 0.45 IR Excitation: exc = 530 nm, 3MHz, 5µW Major Dichoic T825 DCXRT Detection: > 1064 nm t-spad coupled via a 600µm MM-fiber Image size: 500 x 500 pixel, 230 x 230µm (images displayed are binned 2x2) Time/Pixel: 2ms Intensity + Lifetime Image tav., Int = 5.3 ns tav., Int = 3.0 ns the CIGS structure shows areas with low luminescence on the microscopic level the luminescence lifetime at these areas is decreased Data courtesy of Christian Wolf, PVcomB, Helmholtz Center Berlin, Institute Competence Center Berlin, Germany 80
Time-resolved Emission Imaging of CIGS Material 30µm Sample: CIGS layer on glass MicroTime 100 coupled to FluoTime 300 Objective: LCPlan N 20x, 0.45 IR Excitation: exc = 530 nm, 3MHz Major Dichoic T825 DCXRT Detection: NIR PMT-detector (Typ H10330A45) Spectral selection: via monochromator set between 1150nm and 1220nm with 21.6nm spectral bandwidth, step width 10nm Image size: 300 x 300 pixel, 150 x 150µm Time/Pixel: 1ms, acquisition time/frame 6.5min Decays can be analyzed for all recorded wavelength at all locations In this example, the quenched areas are observable at the same positions over the complete wavelength range scanned. All areas show a similar wavelength dependency, therefore quenching is not associated with large spectral shifts Data courtesy of Christian Wolf, PVcomB, Helmholtz Center Berlin, Institute Competence Center Berlin, Germany 81
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PicoQuant Events Worldwide th Feb 28-Mar 02, 2017 9 European Short Course on Time-resolved Microscopy and Correlation Spectroscopy Berlin, Germany Sept, 2017 Berlin, Germany 23nd International Workshop on Single Molecule Spectroscopy and Super-resolution Microscopy in the Life Sciences Nov 7-10, 2016 Berlin, Germany 14th European Short Course on Principles and Applications of Time-resolved Fluorescence Spectroscopy Feb/Sep Berlin, Germany SymPhoTime Training Day throughout the year Science in your lab, Series of events organized by PicoQuant along with a local research institute worldwide hands-on hands-on Hands-on/ demo Hands-on/ demo www.picoquant.com/events 83
Acknowledgement Thank You for your attention and interest! Thanks to all my colleagues at PicoQuant! 84