Fluorescence Quenching, Lecture notes of Biological Systems

Download Fluorescence Quenching and more Lecture notes Biological Systems in PDF only on Docsity! Physikalisch-chemisches Praktikum I Fluorescence Quenching – 2016 Fluorescence Quenching Summary The emission of light from the excited state of a molecule (fluorescence or phospho- rescence) can be quenched by interaction with another molecule. The stationary and time-dependent observation of such processes reveals insight into the deactiva- tion mechanisms of the excited molecule and can be used for monitoring distance and orientation changes between different parts of biomolecules. In this experiment you will record fluorescence spectra of different dyes and measure the fluorescence intensity after adding quencher molecules at different concentrations. Fluorescence lifetimes are derived from a Stern-Volmer analysis of this data. Contents 1 Introduction 2 1.1 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2 Singlet and Triplet States . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 Deactivation Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.4 Fluorescence Decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.5 Energy transfer and assisted relaxation . . . . . . . . . . . . . . . . . . . . 5 1.6 Stern-Volmer Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.7 Estimating the quenching rate . . . . . . . . . . . . . . . . . . . . . . . . . 7 2 Experiment 8 2.1 Fluorescence Spectrometers . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.2 Dye Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.3 Experimental Tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 Data Analysis 10 4 Appendix 11 A Lifetime determination via phase shift measurements 11 B Sample Preparation 12 B.1 For Stern-Volmer plot (25 ml flasks, all values in ml): . . . . . . . . . . . . . . . . . . . . . . . . . 12 B.2 For viscosity dependent measurements (25 ml flasks, all values in ml): . . . . . . . . . . . . . . . . . . . . . . . . . 12 C The FL Winlab Software 13 D Viscosity of Water Glycerol Mixtures and other useful values 13 Page 1 of 14 Physikalisch-chemisches Praktikum I Fluorescence Quenching – 2016 1 Introduction 1.1 Fluorescence When a molecule absorbs light in the visible or ultraviolet range of the spectrum, it is excited from the electronic ground state to an excited state. From there it can return to the ground state by releasing the absorbed energy in the form of heat and by radiation in the visible or near-infrared spectral range. The emitted light is called fluorescence (or phosphorescence if the excited state is a triplet state, see below). Fluorescence can be detected with very high sensitivity even from single molecules and it is used in a large number of chemical and biochemical applications. Sensitive fluorescence detection relies on the fact that the emitted light usually has a longer wavelength than the intense light used for excitation, which can therefore be suppressed by filters or monochromators. This difference between absorption and fluorescence wavelength (maxima) is also known as Stokes shift and can be understood in the following way: in addition to the change of electronic structure absorption can also lead to the excitation of vibrational levels, which requires more energy or light of shorter wavelength. In some molecules like benzene, this leads to a distinct pattern (vibrational progression) in the absorption spectrum, as shown on the left hand side of Figure 1. In solution, the vibrational energy is very quickly dissipated by collisions with the solvent and the molecule adopts a new equilibrium configuration from where emission takes place. Emission can again populate excited vibrational states, this time however, in the electronic ground state (right hand side of Figure 1). In contrast to the excitation process, the energy gaps are now smaller, leading to a shift of the fluorescence to longer wavelength. Absorption Fluorescence S0 S1 Figure 1: Absorption and emission of light in the case of benzene (left) and schematically for two shifted potential energy surfaces (right). The excitation of vibrational levels leads to a blue shift in absorption and a red shift in emission. 1.2 Singlet and Triplet States If we describe the electronic states of a molecule using simple molecular orbital theory, absorption of light at longest wavelength corresponds to a transition of an electron from the highest occupied orbital to the lowest unoccupied orbital (HOMO→ LUMO transi- tion). There are two different possibilities for this excitation: The two electrons, which Page 2 of 14 Physikalisch-chemisches Praktikum I Fluorescence Quenching – 2016 lifetimes are on the sub-nanosecond timescale, even more involved experimental methods are needed. In this practical course you will use an indirect method for determining nanosecond lifetimes, which relies on a further deactivation process which is discussed in detail below: 1.5 Energy transfer and assisted relaxation Excited state deactivation by energy transfer is illustrated in Figure 4, depicting the HOMO and LUMO spin configurations. The photo excited molecule, called donor, starts in the S1 configuration and has a larger gap between HOMO and LUMO than the accep- tor molecule, which is initially in the S0 ground state. As the donor returns to the ground state, the acceptor is promoted to the excited state. There are two different mechanisms by which this energy transfer can take place: donor (S1) acceptor (S0) donor (S0) acceptor (S1) Förster HOMO LUMO HOMO LUMO Dexter Figure 4: Changes in spin configuration of HOMO and LUMO during energy transfer. Förster mechanism: Charge fluctuations in donor and acceptor can influence each other over distances of the order of 10 nm if they occur near resonance of an electronic transition in both molecules (transition dipole interaction). The probability of energy transfer in this case is proportional to the overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor and decreases with donor-acceptor distance R like 1/R6. This mechanism is responsible for the transfer of energy from the light-collecting antenna complexes to the reaction centre in natural photosynthesis. Dexter mechanism: When donor and acceptor come sufficiently close for their or- bitals to overlap, the excited electron of the donor can be transferred to an unoccupied orbital of the acceptor. At the same time, an electron of the acceptor moves to the HOMO of the donor. This process is only effective for donor-acceptor distances smaller than 15Å. A common variant of this process is triplet quenching, when the donor is initially in the T1 state. The excited states of typical quenchers like I− are usually too high in energy for efficient resonant excitation transfer from dyes that emit in the visible, however, there can still be directed electron transfer from one molecule to another.[1] During reductive quenching the quencher transfers an electron to the excited molecule, which stops to fluoresce. This Page 5 of 14 Physikalisch-chemisches Praktikum I Fluorescence Quenching – 2016 is a very important step in many photocatalytic reactions, for example for solar fuel production.[2] Heavy elements can also quench fluorescence by strongly enhancing the rate of in- tersystem crossing (i.e. the change from triplet to singlet states or vice versa) and this relaxation mechanism is therefore known as the External heavy atom effect. The nearby heavy atom only favors the spin flip in the originally excited molecule but is not excited itself as in the Förster or Dexter mechanisms. Apart from the reactive channels, which may require a second molecule as a reac- tion partner, all the deactivation processes shown in Figure 2 are first order processes, meaning that they are independent of concentration. Transfer of excitation or electrons to another molecule or the external heavy atom effect, on the other hand, are strongly concentration-dependent second order processes. They are not only very important de- activation mechanisms in many biological systems, the quenching of the excited state by another molecule can also be used for the determination of short fluorescence lifetimes by relatively simple means. 1.6 Stern-Volmer Method We can gain information about the fluorescence lifetime and excited state deactivation by introducing quenchers (heavy ions or acceptor molecules) and observing the fluorescence intensity as a function of their concentration. To see how this is possible we build a rate model for the concentration of molecules in the fluorescing excited state S1 and use the following notations: [M ] Concentration of the fluorescing molecule in the ground state [M∗] Concentration of the molecule (donor) in the fluorescing S1 state [Q] Concentration of the quenching molecule (acceptor in the ground state) Neglecting the possibility photo chemical reactions, the following processes contribute to a change of [M∗] (compare Figure 2): Photo excitation M →M∗ kabs[M ] Fluorescence M∗ →M kf [M∗] Non-fluorescent relaxation (intramolecular) M∗ →M (or other excited state) knf[M ∗] Quenching (intermolecular) M∗ +Q→M +Q (or Q∗) kq[M ∗][Q] The fraction of excited molecules at any time is usually very small (unless intense pulsed light sources are used), so [M ] ≈ const. Defining Iabs = kabs[M ] we obtain the following differential equation for the excited state population: ∂[M∗] ∂t = Iabs − (kf + knf + kq[Q]) [M∗] (2) Without quencher molecules ([Q] = 0) the equation becomes even simpler: ∂[M∗] ∂t = Iabs − (kf + knf) [M∗] (3) Page 6 of 14 Physikalisch-chemisches Praktikum I Fluorescence Quenching – 2016 Under continuous irradiation a stationary state is quickly established and the excited state population [M∗] is constant (∂[M∗]/∂t = 0). An important quantity for the determination of different reaction rate constants is the fluorescence quantum yield Φ: Φ = number emitted photons number absorbed photons = rate of emission rate of absorption (4) In our notation, the rate of absorption is Iabs = kabs[M ] and the rate of emission is kf[M ∗]. With the help of equations 2 and 3 under stationary conditions (∂[M∗]/∂t = 0), we obtain for the quantum efficiencies with and without quencher molecules: ΦQ = kf kf + knf + kq[Q] (5) and Φ0 = kf kf + knf (6) The ratio of the quantum yields is equal to the ratio of the observed emission intensities without and with the quencher molecules: I0 IQ = Φ0 ΦQ = kf + knf + kq[Q] kf + knf = 1 + 1 kf + knf kq[Q] (7) Inserting the fluorescence lifetime in the absence of the quencher molecules (equation 1) we obtain the final result (also known as Stern-Volmer equation): I0 IQ = 1 + τfkq[Q] (8) 1.7 Estimating the quenching rate The Stern-Volmer equation allows us to determine the product τfkq from the slope of a plot of I0/IQ − 1 against the quencher concentration [Q]. In order to extract the fluorescence lifetime of the excited molecules we thus need to know the quenching rate kq. To estimate it, we assume that the quenching process is diffusion-limited. This means that it is much less likely that molecule and quencher come close to each other than that they interact (or exchange electrons as a donor/acceptor pair) once they actually meet. In other words, quenching (by deactivation or electron transfer) would take place much more often, if quencher and molecule were to meet more frequently. kq is then the given by the rate at which M∗ and Q meet. For two (equal) solid spheres in a solution of viscosity η this second order diffusion rate is given by (see e.g.[3]) kdiff = 8RT 3η (9) where R=8.314 JK−1mol−1 is the gas constant, T is the temperature in Kelvin and η is the viscosity in kg m−1 s−1. If quenching is diffusion-limited, kq ≈ kdiff should be inversely proportional to the viscosity of the solution. Note that usually only the heavy atom effect or excitation transfer of the Dexter type (electron exchange) are diffusion limited because Förster energy transfer can take place over a much longer distance. Page 7 of 14 Physikalisch-chemisches Praktikum I Fluorescence Quenching – 2016 • From the absorption data, deduce the range of possible excitation wavelengths and record the fluorescence spectra of the same solutions with the commercial fluores- cence spectrometer. Adjust the slit width of the excitation and detection monochro- mators to achieve good signal to noise without saturation (see Appendix). • Use the simpler setup (with appropriate filters if necessary) to record the fluorescence intensities IQ of the samples with different quencher concentration. There is a turning knob and a switch with two positions at the detector. In one position of the switch, the knob can be adjusted until the output voltage is minimal (0). Flip the switch after adjustment and do not touch the knob anymore. The voltage you read is now proportional to the fluorescence intensity. Before and after each measurement of a quencher solution, also record the fluorescence intensity of the dye-only solution to obtain I0 under identical conditions for normalization. • Repeat each measurement 2-3 times. Estimate the range of the signal fluctuations and try to obtain a mean value. • Prepare three solutions with identical dye concentrations and increasing amounts of glycerol. Then prepare identical solutions all with the same quencher concentration (see the second table in the appendix). • Measure the fluorescence intensity ratios of the water/glycerol solutions with and without quencher (average over several readings). • Dispose of all dye solutions in the aqueous waste container. First collect all the alkaline fluorescein solutions in a beaker and neutralize them. 3 Data Analysis • Generate combined plots of normalized absorption and fluorescence spectra. • Calculate the exact quencher concentrations in all solutions. • Form the ratios of quenched and unquenched fluorescence intensity (I0/IQ− 1) and plot them against the quencher concentrations [Q] (Stern-Volmer equation 8). • Determine the product τfkq from the gradient of these plots (with errors and units). • Assuming that quenching is diffusion-controlled (kq = kdiff) calculate the quenching rate constants kq using equation 9 (ηH2O≈ 10−3 kg m−1s−1). • Compute the fluorescence lifetime τf and the experimental uncertainty. • Plot (I0/IQ − 1) for the three water/glycerol mixtures as a function of viscosity. The viscosity of water/glycerol mixtures[4] can be read off Figure 7 in the appendix or calculated with the corresponding Mathematica CDF program. Do your results support the assumption that quenching is diffusion controlled? Page 10 of 14 Physikalisch-chemisches Praktikum I Fluorescence Quenching – 2016 Fluorescence Excitation/scatter Exponential decay out of phase in phase time (a. u.) Figure 6: Time modulation of the excitation light (blue) and the fluorescence signal (orange). When the lifetime τ is a significant fraction of the modulation period 2π/ω, the fluorescence signal is shifted in time. Bottom: the detector multiplies the signal with a reference signal (black) and yields a time-integrated output. 4 Appendix A Lifetime determination via phase shift measure- ments Unless we make use of a pulsed laser, it is very difficult to turn off the excitation light fast enough to observe the fluorescence decay directly. It is, however, possible to modulate a light source - in particular modern LEDs - at 10-50 kHz frequencies. As illustrated in Fig. 6, this is slower than a typical decay time. Since the detector sees the sum of all the fluorescence signals excited at different moments in time, the fluorescence light is slightly time-shifted with respect to the excitation (or scattered) light. This shift becomes larger with increasing fluorescence lifetime. Mathematically, the detected intensity is the convolution of the cosine-modulated excitation (blue) and the exponential fluorescence decay (green): S(t, ω, τ) = ∫ ∞ −∞ (1 + cosωt0) Θ[t− t0] τ exp[ −(t− t0) τ ]dt0 = 1 1 + τ 2ω2 (cosωt+ τω sinωt) (10) A very sensitive way of measuring the time-shift is to multiply the fluorescence signal S with a reference signal which is changing between positive and negative at the modulation frequency, as shown by the black lines in Fig 6. When the sign change of this reference occurs at the maxima and minima of the modulation cosine (bottom left in Fig. 6), integration yields the out of phase signal Iout of phase = ∫ π ω 0 S(t, ω, τ)− ∫ 2π ω π ω S(t, ω, τ) = τ 4 1 + τ 2ω2 (11) Page 11 of 14 Physikalisch-chemisches Praktikum I Fluorescence Quenching – 2016 This signal should be zero when τ = 0, so we can use scattered excitation light to properly adjust the delay of the reference signal. When we now change the switch on the detector, the reference signal is time-shifted by exactly a quarter period (bottom right in Fig. 6)and we record the in phase signal: Iin phase = − ∫ π 2ω 0 S(t, ω, τ) + ∫ 3π 2ω π 2ω S(t, ω, τ)− ∫ 2π ω 3π 2ω S(t, ω, τ) = 1 ω 4 1 + τ 2ω2 (12) The ratio of these two signals is simply Iout of phase Iin phase = ωτ = 2πfτ (13) where f is the frequency at which the light source is modulated. The procedure for measuring the fluorescence lifetime of a dye molecule is thus rather straightforward: • Remove any filters and replace the sample with a piece of paper in order to record the scattered excitation light. • Turn the potentiometer knob until the out of phase detector output is zero. • Measure the in phase and the out of phase signals of your sample. • Measure the modulation frequency f = ω/(2π) by connecting an oscilloscope or frequency meter to the BNC output of the detector. • Calculate ωτ = Iout of phase/Iin phase and hence determine τ . B Sample Preparation B.1 For Stern-Volmer plot (25 ml flasks, all values in ml): Sample label 1a 1b 1c 1d 1e 0.1 M KI (in 0.1M KOH) 0 2 5 7 10 burette and weigh Fluorescein (in 0.1M KOH) 10 10 10 10 10 pipette 0.1M KOH 15 13 10 8 5 fill to level B.2 For viscosity dependent measurements (25 ml flasks, all values in ml): sample 2a 2b 2c 3a 3b 3c Fluorescein (in 0.1M KOH) 10 10 10 10 10 10 pipette 0.1 M KI (in water) 0 0 0 5 5 5 pipette Glycerol 0 5 10 0 5 10 burette Water 15 10 5 10 5 0 fill to level Page 12 of 14