Fluorescence Spectroscopy Steady State and Time Dependent Fluorescence Measurements Kai Wen Teng PHYS 403 Fall 15
EM Spectrum of molecules Rotational Energy Infrared Vibrational Energy Near Infrared Electronic Energy Visible and Ultra-Violet r v Diagram from http://www.lbl.gov Diagram by Robert Clegg in Photosynth Res. 2009 Aug- Sep;101(2-3):181-94.
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. 2010 Nano Lett., 2010, 10 (9), pp 3692-3699. DOI: 10.1021/nl1021909
Perrin-Jablonski energy diagram (S0, S1 and S2 transitions) Alexander Jablonski Diagram by Ulai Noomnarm in Photosynth Res. 2009 Aug-Sep;101(2-3):181-94.
O.D O.D Absorption (S 0 -S 1 ) l Beer-Lambert s Law log(i 0 ) log(i) cl? Extinction coefficient: Concentration Caffeine
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.
Organic dye: Fluorescence (S 1 -S 0 ) Solvent Effect: Ruby: http://micro.magnet.fsu.edu http://www.bio.davidson.edu
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. 2009 Aug-Sep;101(2-3):181-94. Fluorescence Lifetime: i 1 ki Lifetime is sensitive to other decaying pathway present!
The Bolognian Stone http://www.isbc.unibo.it/files/10_se_bostone.htm MarcAntonioCellio (1680) representing the light emission of heated barite
First mention of lifetimes? The Bologna stone, when placed in the sun attracts the rays, and retains them so long as to give light a considerable time after it is removed into the dark. Goethe The Sorrows of Werter
Lastusaari et al. 2011
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!
Measuring Lifetime: Time Domain nsec What do you need? -Collect signal fast enough -Fitting
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
Samples Described by Multiple Lifetimes I(t) = i a e i -t τ i Some Examples: -ECFP (Enhanced Cyan Fluorescent Protein (FP)), FPs -Ruby Rhodamine Mixture -Crystals 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 one 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
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
Example of Heterodyning Lifetime Imaging With Your Phone 440 nm LED Fiber Optic Video Recording (30 fps) 30.5 Hz Ruby Function Generatior 2s/cycle = 0.5 Hz [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) )
AOMs Intensity Modulator Annals of the New York Academy of Sciences Vol. 158 pp 361-376, 1969 -modulation frequency limited by resonance frequency of the acoustooptic cell -variations in the intensity modulation caused by temperature
Pockels Cell Biophysical Journal Vol. 44 (1983) pp 315-324 Hecht Optics
Directly Modulated Diode System in ESB Laser Diodes -> (405nm,436nm,473nm,635nm,690nm,780nm,830nm) LEDs -> (280nm,300nm,335nm,345nm,460nm,500nm,520nm)
ISS SLM Phoenix Upgrade Original System New Upgrades PMT Housing New Acquisition Box Stepper Motor Controller -photon counting -Measures nanosecond lifetime
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
Msin(φ) The Polar Plot Msin(φ) 0.5 0.5 0 φ Mcos(φ) 0 1.0 1.0 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)
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 1.0 0.8 0.6 0.4 0.2 0 480 500 520 540 560 580 Wavelength (nm)
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 (E) Rate of Energy Transfer Total deexcitation rate 1 1 DA D = 1 = 1 D Theoretically: DA D
Applications of Fluorescence in Biology
Msinφ Msinφ Fluorescence Lifetime Imaging on Live Cells Top of cell: Intensity Top of cell: Lifetimes 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 Intensity @ 4.5µm 0.6 2 ns 0.6 2 ns 1 4 ns 3 ns 4 ns 3 ns 2 #1 #2 0 Mcosφ 0 1.0 Images courtesy of John Eichorst Mcosφ 1.0
Center of the distribution can be determined in ~1.5 nm accuracy if #N is more than 10^4 Single Molecule Fluorescence Imaging
Super Resolution Fluorescence Imaging http://www.microscopyu.com/articles/superresolution/stormintro.html Huang et al Science,2008