FluoTime 300 for Time-Resolved and Steady-State Spectroscopy

Transcription

1 FluoTime 300 for Time-Resolved and Steady-State Spectroscopy Christian Litwinski, Sebastian Tannert, Alexander Glatz, 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

2 PicoQuant GmbH Our location WISTA-MG Technology Park Adlershof The Brandenburg Gate Pulsed Diode Lasers Time-resolved Confocal Microscopes & LSM upgrade kits Fluorescence Lifetime Spectrometers The PicoQuant Team Photon Counting Instrumentation 2

3 PicoQuant GmbH Pulsed Diode Lasers Time-resolved Confocal Microscopes & LSM upgrade kits Photon Counting Instrumentation Fluorescence Lifetime Spectrometers 3

4 PicoQuant Around the World Active Reseller Network PicoQuant GmbH Kawa.ska Opton Laser Intl PicoQuant Photonics North America Inc Crisel Instruments Srl Mexitek TechnoInfo ETSC Technology SIMCO Group DongWoo Optron Japan Laser Corp. Jinsung Laser AST Instruments Pretek Co. EINST Technology Quantum Tech Lastek Pty Ltd Bioanalítica 4

5 FluoTime 300 High-end scientific grade fluorescence spectrometer For beginners and experts Time-resolved and steady-state measurement modes Modular design 5

6 Outline Fluorescence and TCSPC principles Components of a FluoTime 300 Time resolution and sensitivity Software for acquisition Software for analysis Examples of measurements 6

7 Fluorescence and TCSPC principles Components of a FluoTime 300 Time resolution and sensitivity Software for acquisition Software for analysis Examples of measurements 7

8 Fluorescence Photocycle A = Photon absorption F = Fluorescence (emission) P = Phosphorescence S = Singlet state T = Triplet state IC = Internal conversion ISC = Intersystem crossing Sn S2 energy IC S1 ISC A F T1 IC ISC P S0 Polarization anisotropy Fluorescence observables T2 IC Wavelength Lifetime Intensity electronic ground state fluorophore Fluorescence Lifetime: average time that a molecule remains in the fluorescent state excited state? hν t F = 1 / (kf + kic + kisc) How fast is the photocycle? typ. ps (10-12 s) to ns (10-9 s) hν light source detector ground state 8

9 Time-Correlated Single Photon Counting Fluorophore Excited state h Detector h Light source stop Ground state start For TCSPC we need: A defined start of the experiment pulsed excitation; each pulse is a new start A defined stop of the experiment photon counting detector A fast stopwatch to measure time difference between start and stop 9

10 TCSPC Principle In principle with a stop watch: 1. Start the clock with a laser pulse. 2. Stop the clock with the first photon that arrives at the detector. 3. Reset the clock and wait for next start signal. Photon emission is a stochastic process! laser pulse laser pulse photon 1 e.g. 3.4 ns laser pulse no photon photon 2 e.g. 4.7 ns Repeat this time measurement very often and count how many photons have arrived after what time, i.e. build a histogram. Time axis is not continuous, but divided into time bins. 10

11 Photon Counting Time Histogram Only one molecule at a time tested, but each time another one Counts After many many time measurements: Histogram = intensity decay (ergodic system) Time 11

12 Photon Counting Time Histogram After many many time measurements: Histogram = intensity decay (ergodic system) Only one molecule at a time tested, but each time another one Averaged ensemble measurement Counts F(t) = A exp (-t / t) Time 12

13 Photon Counting Time Histogram After many many time measurements: Histogram = intensity decay (ergodic system) F(t) = A1 exp (-t / t1) + A2 exp (-t / t2) Counts Exponential fit information on the shape of the decay Only can be done when the adquisition has finished Time 13

14 Laser Repetition Rate 14

15 ps TCSPC Dead time: Time required for the electronics to place the photon in the histogram with ps accuraccy (several ns) True decay Maximum of one photon counted per excitation cycle If count rate is similar to excitation rate, this to overestimation of early photons (pile-up distortion) Pile-up distortion (quicker!) To obtain the true decay keep count rate < 2 % with respect to the excitation rate laser pulse lost + pileup lost Dead-time 15

16 ns TCSPC Only nanosecond accuracy but in practice no dead-time ( < 2.5 ns) Ideal to measure Phosphorescence (ms ms) and steady-state spectra Count rates can be much higher than the excitation rate Xe lamp / laser burst All counted!! All counted!! In practice no dead time!! 16

17 Fluorescence and TCSPC principles Components of a FluoTime 300 Time resolution and sensitivity Software for acquisition Software for analysis Examples of measurements 17

18 Fully equiped FluoTime 300 Technical Overview Optional 2nd detector Standard detector TCSPC modules Emission monochromator Excitation monochromator Motorized slit Polarizer Filter wheel Emission attenuation ns Flashlamp Photodiode Excitation attenuation Steady-state Xenon arc lamp Laser input Sample holder 18

19 Sample Compartment Standard holder Cuvettes Stirring Temperature setting by external chiller Front face holder Solids Single cuvette holder temperature stabilized -20 to 100 C Cuvettes Stirring Cryostat 2.3 to 500 K Cuvettes Solids Four cuvette changer temperature stabilized -20 to 100 C Cuvettes Stirring 20

20 Sample Compartment Standard holder Cuvettes Stirring Temperature setting by external chiller Front face holder Solids Single cuvette holder temperature stabilized -10 to 100 C Cuvettes Stirring Cryostat 2.3 to 500 K Cuvettes Solids Four cuvette changer temperature stabilized -10 to 100 C Cuvettes Stirring 21

21 Pulsed Light Sources Broad spectral band covered by exchangeable laser heads and LEDs with: Spectral bandwidths Laser: 3-5 nm (vis) LEDs: nm Pulse width Laser: down to 50 ps LEDs: down to 500 ps Fixed wavelength (UV to IR) Selectable repetition frequencies, up to 80 MHz Single channel or multichannel system (multicolor experiments) Compact, maintenance-free and high performance-to-cost 22

22 Pulsed Light Sources LDH Series Laser Diode Heads Wavelengths: 375 to 1990 nm Pulse width down to 50 ps Optional dual mode: CW and pulsed operation Optional fiber coupling PLS Series Pulsed LEDs Wavelengths from 245 to 600 nm Pulse width < 1 ns Optional bandpass filter 23

23 LDH-FA Series Key Specifications LDH-P-FA mw output power < 80 ps pulse width Collimated output 1 to 80 MHz repetition rate LDH-P-FA-530B > 4 mw output power < 100 ps pulse width Emits from fiber 10 khz to 80 MHz repetition rate LDH-P-FA-530L / XL 20 mw or 200 mw output power < 100 ps pulse width Collimated output (fiber coupling optional) 1 to 80 MHz repetition rate LDH-P-FA mw output power < 100 ps pulse width Collimated output (fiber coupling optional) 1 to 80 MHz repetition rate Further fiber amplified wavelengths: 1060 and 1530 nm 24

24 Fiber Amplified UV Laser for Pulsed 266 nm Excitation seed laser fiber amplifier 1062 nm two stage SHG 266 nm, < 100 ps pulses variable repetition rates from 1 MHz to 80 MHz 25

25 Solea Tunable Picosecond Laser Gain-switched laser diode Pump Pump Yb:Fiber Pump Yb:Fiber Yb:Fiber Seed Pre-amplifier Pre-amplifier Main amplifier PCF Delivery fiber Photonic Crystal Fiber for spectral broadening Wavelength selection with automatic filter Typical pulse width < 150 ps Spectral range: nm (ECO mode) nm (BOOST mode) Emission from a polarization maintaining singlemode fiber with FC/PC connector Externally triggerable Low noise < 3 % rms 26

26 Fast, Sensitive and Compact Photomultiplier Assemblies PMA Series: optimal price/performance ratio Photo-Cathode Quantum Efficiency [%] PMA 165 PMA 182 PMA Wavelength [nm] Fast: 200 ps transit time spread (all models) Sensitive: up to 40% quantum efficiency in blue, extended red-sensitive version available Compact: 12V DC supply, thermo-electrical cooling as an option, no more HV supply or cooling water PMA 182: best compromise for spectral range and sensitivity 27

27 Special Detectors: MCP, Hybrid PMT, NIR MCP: the fastest photon detector today (25 ps TTS) but less sensitive than a standard PMT Hybrid PMT: new detector type, fusion of PMT and SPAD technology. Fast (120 ps response), sensitive (up to 45% QE), but smaller active area NIR-PMT: large thermal noise, but the only alternative for single photon counting in NIR on spectrometers (large active area). MCP (Hamamatsu) 100 Photo-Cathode Quantum Efficiency [%] MCP Hybrid-PMT NIR PMTs Wavelength [nm] Hybrid PMT NIR-PMT (Hamamatsu) Courtesy from Hamamatsu 28

28 Precise Picosecond Timing, TCSPC & MCS HydraHarp 400 PicoHarp 300 Up to 8 independent detector channels Independent sync channel Down to 1 ps base resolution 2 independent channels Down to 4 ps base resolution TimeHarp 260 Either 25 ps ( PICO version ) or 1 ns ( NANO version ) base resolution One or two independent detector channels Independent sync channel 29

29 Precise Timing, ps and ns TCSPC TimeHarp 260 P Two modes of operation PICO mode 25 ps base resolution Dead time < 25 ns Fluorescence decay measurements Long Range (LR) mode 2.5 ns base resolution No pile-up distortion!! Dead time < 2.5ns Seady-State measurements and Phosphorescence decays 30

30 Fluorescence and TCSPC principles Components of a FluoTime 300 Time resolution and sensitivity Software for acquisition Software for analysis Examples of measurements 33

31 Analysis of a Decay Curve Convolved data t Dec t = IRF t Fluor t t d t 0 IRF Reconvolution or Tailfit Background Experimental noise i= Dec i i= Weighting 1 1 = i Deci Weighted residuals Least squares fitting (c2 = sum2) Distrib. randomly around 0 34

32 Temporal Resolution The IRF The Instrument Response Function (IRF) width depends mainly on three factors: Pulse width of the laser (t ) p Dispersion in the grating (t ) g Temporal uncertainty of the detector (t ) d Timing uncertainty of the TCSPC electronics (t ) e These factors are combined in the Instrument Response Function. Short IRF means that short lifetimes can be resolved. IRF = + Approximation: + + IRF τ + τ + τ + τ 2 p 2 g 2 d 2 e The IRF is dominated by the slowest component! Ideal combinations are therefore components with similar timing uncertainties! 35

33 Applications/Performance Decay Standard Erythrosin B Intensity [Counts] Sample: Erythrosin B in water 100 nm Excitation: 80 MHz pulsed 532 nm Laser pulse width 72 ps FWHM Detection: 550 nm, M pol Decay Fit IRF Weighted residuals Standard blue sensitive PMT: PMA165 System IRF: 200 ps τ = 89 ± 4 ps χ2 = 1.08 Published reference value: 89 ± 3 ps Time [ns] Boens N. et al., Analytical Chemistry, Vol.79, p (2007) 36

34 FluoTime 300 Detection Sensitivity Raman Signal Raman scattering of solvent is always present. Pure water is a clearly defined substance. Signal to noise ratio is a good parameter to evaluate the performance of a spectrometer. Sample: Sample: Water Water sealed sealed cuvette cuvette Excitation source: 300 W Excitation source: 300 W Xe Xe lamp lamp Excitation wavelength: 350 Excitation wavelength: 350 nm nm Excitation Excitation and and emission emission bandwidth: bandwidth: 55 nm nm Step size: 0.5 nm Step size: 0.5 nm Acquisition Acquisition time time per per point: point: 11 ss No No polarizers polarizers or orfilters filters Detector: PMA-C-175 Detector: PMA-C-175 nm = cps nm = cps Best case S/N = : Corrected Intensity [cps] Signal Background S / N ratio = Background Corrected Intensity [cps] Wavelength [nm] Wavelength [nm] 37

35 FluoTime 300 Detection Sensitivity Fluorescein Dilution Series Steady-State pm Fluorescein Blank Fluorescein Fluorescein (blank corrected) Corrected intensity [kcounts] Corrected intensity [kcounts] Sample: Fluorescein solution in basic buffer Excitation source: 300 W Xe lamp Excitation λ: 478 nm Excitation and emission bandwidth: 4 nm Spectral step: 1 nm Acquisition time: 1 s per point No Polarizers or filters Detector: PMA-C pm Fluorescein Blank Fluorescein Fluorescein (blank corrected) 520 Wavelength [nm] Wavelength [nm] pm Fluorescein Blank Fluorescein Fluorescein (blank corrected) Corrected intensity [kcounts] Corrected intensity [kcounts] Wavelength [nm] Peak count [cps] pm Fluorescein Blank Fluorescein Fluorescein (blank corrected) x Excellent linear dependence till 1pM Concentration [pm] Wavelength [nm] Sample nm, solvent pm Fluorescein

36 FluoTime 300 Detection Sensitivity Fluorescein Dilution Series Time-Resolved M Lifetime [ns] Count rate [cps] 1000 Counts pm 250 pm 100 pm 33 pm 10 pm IRF Concentration [pm] Time [ns] Sample: Fluorescein in basic buffer Excitation source: LDH-D-C-470 Excitation rep. rate: 20 MHz Excitation wavelength: 470 nm Detection wavelength: (512 ± 5.4) nm Detection polarizer: 54.7 (magic angle) Detector: PMA-C-175 Measurement time: 1 minute only!!! t = ns Concentration [pm] At 10 pm Fluorescein and below, the signal is strongly dominated by scattering. Fit model: - 10 pm pm: single exponential with a scattering term pm - µm: single exponential Literature lifetime value (4 ns) was obtained down to 33 pm. 39

37 Quantum Yield Measurements Using an Integrating Sphere Detector Monochromator The integrating sphere consists of a broadband reflecting material, that ideally reflects all wavelengths evenly The idea is to distribute all light introduced of present in the cell as evenly as possible Excitation light 40

38 FluoTime 300 Integrating Sphere EtOH Rh 6G Photons Abs. = EtOH - Rh 6G Photons Emit. = Rh 6G - EtOH Counts Counts 1400 Counts Counts water Ru(bpy)3 Photons Abs. = water - Ru(bpy)3 Photons Emit. = Ru(bpy)3 - water Wavelength [nm] Wavelength [nm] Wavelength [nm] Excitation source: Xe lamp Excitation wavelength: nm Excitation and emission bandwidth: 2 nm Step size: 1 nm Acquisition time per point: 0.1 s Detector: HPD Wavelength [nm] Ru(bpy)3 / H2O(aerated) C-153 / MeOH Rh 6G / EtOH Literature Measured

39 Fluorescence and TCSPC principles Components of a FluoTime 300 Time resolution and sensitivity Software for acquisition Software for analysis Examples of measurements 42

40 Data Acquisition Wizards Standard measurements for a single sample with reasonable fluorescence Steady-state measurements Excitation spectrum Excitation anisotropy spectrum Excitation spectrum time course Emission spectrum Emission anisotropy spectrum Emission spectrum time course Emission intensity time trace Time-resolved measurements Fluorescence decay Dynamic anisotropy Decay time course Time-resolved emission scan 43

41 Wizards Guiding through the measurement Sample and initial conditions Optimization step Measurement steps 44

42 Driving it to the cutting edge / Full Control Customized Measurements 45

43 Let the FluoTime 300 Work For You - Scripting - 46

44 Organizing Your Data The Workspace Sample oriented Raw data & analysis results bundled All known information stored Reorganization of data easy 47

45 Plotting & Calculations Zooming Normalizing Data readout ASCII export Image export Built-in mathematics 48

46 Fluorescence and TCSPC principles Components of a FluoTime 300 Time resolution and sensitivity Software for acquisition Software for analysis Examples of measurements 49

47 Decay Data Analysis: FluoFit Decay analysis Tailfit IRF reconvolution Global analysis Model functions Exp. decay Gauss distribution Lorenztian distr. Stretched exp. Dyn. anisotropy Error analysis Support plane Bootstrap Analysis report 50

48 Error Estimation: Support Plane Analysis How far can a parameter be dragged from the best fit without a significant change occurring in the c2? ASE: c2 calculated with the other params. kept fix Supp. plane analysis: c2 calculated with the other params. fitted 51

49 Error Estimation: Bootstrap Analysis Parameter errors estimated from a series of simulated data sets Simulated data sets are subsets of the original experimental data set Visualization: correlation plots of parameter pairs ( 2D histogram ) Each fit of a simulated data set produces a single point in the plot Cloud of points show the correlations between parameters: a) Unstructured, axis parallel ellipsoids: parameters are uncorrelated b) Tilted structures: parameters are correlated 52

50 PicoQuant Support 69

51 PicoQuant Events Worldwide th Feb 28-Mar 02, 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 70

52 Acknowledgement Thank You for your attention and interest! Thanks to all my colleagues at PicoQuant! 71