Optical Spectroscopy. Steady State and Time Dependent Fluorescence Measurements. Kai Wen Teng. October 8 th PHYS 403 Fall 2013

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1 Optical Spectroscopy Steady State and Time Dependent Fluorescence Measurements Kai Wen Teng October 8 th 2013 PHYS 403 Fall 2013

2 EM Spectrum of molecules Rotational Energy Infrared Vibrational Energy Near Infrared Electronic Energy Visible and Ultra-Violet r v Diagram from Diagram by Robert Clegg in Photosynth Res Aug- Sep;101(2-3):

3 Types of Fluorescent Molecules Synthetic Organic: Fluorescein Semiconductor Nanocrystal: Crystals: Naturally Occuring: Ruby and assorted mineral From mineralman.net Fluorescent Nanodiamonds Fluorescent Proteins: Green Fluorescent Protein Image from Zrazhevskiy et al Nano Lett., 2010, 10 (9), pp DOI: /nl

4 Perrin-Jablonski energy diagram (S0, S1 and S2 transitions) Alexander Jablonski Diagram by Ulai Noomnarm in Photosynth Res Aug-Sep;101(2-3):

5 Absorption (S 0 -S 1 ) l? Beer-Lambert s Law log(i 0 ) log(i) cl Extinction coefficient: Concentration

6 Steady State Measurements: Absorbance One of the very first commercially available instrument that measures absorbance was the Beckman DU Machine nowadays that utilizes diffraction grating and diode array detector can acquire an absorbance spectra in less than 10 seconds.

7 Organic dye: Fluorescence (S 1 -S 0 ) Solvent Effect: Ruby:

8 Time-Dependent Fluorescence: Fluorescence Lifetime Fluorescence Lifetime: The average amount of time a molecule stays in excited state Probability of being in the excited state kf = rate constant for leaving excited state while emitting a photon ki = rate constant for leaving excited state through other means (ie. Dynamic quenching, Energy Transfer, etc) Excited State kf ki ki Diagram by Robert Clegg in Photosynth Res Aug-Sep;101(2-3): Fluorescence Lifetime: i 1 ki Lifetime is sensitive to other decaying pathway present!

9 Measuring the Depletion of the excited state * * kf kt t # x # xo e # F * x k Intensity that you = measure [x] * KF is rate constant of fluorescence Intensity measured is proportional to the # of molecules in the excited state!

10 Measuring Lifetime: Time Domain nsec What do you need? -Collect signal fast enough -Fitting

11 Measuring Lifetime: Frequency Domain E(t) = E o + Eωcos ωet + φe F(t) = F + F cos ω t + φ - φ o ω E E tan ω E F F 1 ω o M = = Eω Eo 1+ ωτ Mod 2 What do you need? -Intensity modulators -Synchronization

12 Samples Described by Multiple Lifetimes I(t) = i a e i -t τ i Some Examples: -ECFP (Enhanced Cyan Fluorescent Protein (FP)) -mcherry -Teal FP a τ F(t) = E a τ + E cos(ω t - (φ - φ )) i i o i i ω 2 E i E i i 1+ (ωeτ i ) You still can only measure M,φ E α F(t) = 1+ ω i cos(ω 2 E t - (φ i - φ E )) Fo Eo i 1+ (ωeτ i ) F(t) = 1+ F o E E ω o Mcos(ω t - (φ - φ)) E i

13 Mixing is used in commercial instruments [G(t)*F(t)]= DC+ Terms with frequencies GE 2 ω ω + M (ω E,ω G,ω E +ω G cos((ωg - ω E )t + φg - φ E + φ) ) Images from Molecular Fluorescence by Bernard Valeur

14 AOMs Intensity Modulator Annals of the New York Academy of Sciences Vol. 158 pp , modulation frequency limited by resonance frequency of the acoustooptic cell -variations in the intensity modulation caused by temperature

15 Pockels Cell Biophysical Journal Vol. 44 (1983) pp Hecht Optics

16 Directly Modulated Diode System in ESB Laser Diodes -> (405nm,436nm,473nm,635nm,690nm,780nm,830nm) LEDs -> (280nm,300nm,335nm,345nm,460nm,500nm,520nm)

17 ISS SLM Phoenix Upgrade Original System New Upgrades PMT Housing New Acquisition Box Stepper Motor Controller -photon counting -Measures nanosecond lifetime

18 Champaign, Illinois Domain of FD FLIM Robert Clegg UIUC Full-Field FLIM Enrico Gratton UIUC Scanning Confocal FLIM (FLIMBox) Beniamino Barbieri ISS Inc. Commercialization of FD FLIM

19 Msin(φ) The Polar Plot Msin(φ) φ Mcos(φ) Mcos(φ) Vectors on the Polar Plot R = Mcos(φ)x ˆ+ Msin(φ)y ˆ R=αiR i + α jrj R= α M cos(φ )+ α M cos(φ ) xˆ i i i j j j + α M sin(φ )+ α M sin(φ ) yˆ i i i j j j -movement of the vector depends on the emitted intensity of each species (i)

20 Msin(Ф) Emission Spectral Analysis on the SLM Msin(Ф) Fluorescein 4.3ns Measured Data (60MHz) RhodB 1.7ns Simulated Data (60MHz) Fluorescein 4.3ns RhodB 1.7ns Mcos(Ф) Mcos(Ф) Measured Spectrum Wavelength (nm)

21 Intensity Intensity Intensity Quantum Yield/FRET D FRET R A D A Wavelength Donor Exc. Donor Em. Acceptor Exc. Acceptor Em. D A Wavelength Wavelength Quantum Yield of Energy Transfer = Rate of Energy Transfer Total deexcitation rate 1 1 DA D = 1 = 1 D DA D

22 Top of cell: Intensity Msinφ Top of cell: Lifetimes Msinφ Optical Sections Rendered by Lifetime 0µm 0.5µm 1.0µm 1.5µm 2.0µm 2.5µm 3.0µm 3.5µm 4.0µm 4.5µm 5.0µm 5.5µm 6.0µm 6.5µm 7.0µm 7.5µm 8.0µm 8.5µm 9.0µm 9.5µm 10.0µm 10.5µm 4.5µm ns ns 1 4 ns 3 ns 4 ns 3 ns 2 #1 #2 0 Mcosφ Images courtesy of John Eichorst Mcosφ 1.0