Time Correlated Single Photon Counting (TCSPC): Examples Prof. C. Altucci Corso di Fisica Atomica Molecolare e Spettroscopia a.a Laurea Magistrale in Fisica PhD School in Physics 1

What do we mean by Fluorophore “lifetime”? Absroptiona dn emission are relative to populations of molecular species. Generally, single molecule properties are deduced by the measured ensemble response. For a population of excited fluorophores we can write the rate equation: describing the variation per unit time of the number of molecules in the excited state at the time t 2

The solution is: The lifetime  is equal to k -1 The lifetime is then the time needed for the excited molecules to decay at 1/e (36.8%) of the initial value. t=0 is the end of the excitation pulse. 3

The decay constant k stands for the sum of the constants related to all possible decay channels. k = k f + k i + k x + k ET + …= k f + k nr k f fluorescence decay, k i internal conversion decay, k x inter-system crossing decay, k ET energy-transfer decay, k nr all non-radiative decays 4

Non-radiative processes: Isolated molecules in “gas-phase” undergo only internal conversion or inter-system crossing In the condensed phase additional mechanisms have to be accounted for due to interactions with the micro-environment: chemical reactions in the excited state, energy transfer, … Lifetime of Tryptophan in proteins vary from ~0.1 ns to ~8 ns Coumarine in ethanol has a 4 ns lifetime Isoalloxazine in water has a 4.5 ns lifetime 5

The radiative lifetime  r = k f -1 is nearly constant for a given molecule The overall fluorescence lifetime  = k -1 = (k f + k nr ) -1 depends on the micro-environment surrounding through k nr. Quantum yield for fluorescence: It is proportional to fluorescence lifetime The addition of a further decay path, non-radiative, increases k nr and decreases  and thus QY. The fluorescnce intensity is proportional to n*(t), I (t) = k f n*(t) 6

The Stokes shift consists in the fluorescence to occur with a photon energy smaller (larger wavelength) than that of the excitation radiation The emission spectra or substantially independent on the excitation wavelength. The excess energy is rapidly dissipated ( s). The mirror rule. 7

Diffusion times in solution. The excess energy is quickly dissipated ( s): x 2 =2D. For example: for oxygen in water D  2.510 -5 cm 2 /s, if the fluorephore lifetime is 10 ns we obtain x  70 Å which is comparable with the thickness of a biological membrane or with the linear size of a protein. Absorption spectroscopy gives info of the ground state structure. Fluorescence/Emission spectroscopy gives info on the excited state structure. 8

Let’s focus on TCSPC. How to measure the fluorescence lifetime? Molecules are excited by a short pulse (ideally a  function ) at t = 0. The intensity of the fluorescence decay is usually measured by the Time Correlated Single Photon Counting (TCSPC) Time domain The real fluorescence decay is a convolution with the excitation pulse profile The system response function i REF is typically measured as the response to the “direct” excitation pulse. The measured fluorescence decay decadimento di fluorescenza misurato ism a convolution the overall system response 9

Steady-state and time-resolved regimes While steady-state fluorescence measurements are simple, nanosecond time-resolved measurements typically require complex and expensive instrumentation. Given the relationship between steady-state and time-resolved measurements, what is the value of these more complex measurements? It turns out that much of the molecular information available from fluorescence is lost during the time averaging process. 10

Time domain I(t) parameters are usually obtained by a non-linear best-fit combined to a deconvolution. The deconvolution is not required when the excitation pulse is much shorter than the lifetime to measure and/or when we do not need a high accuracy in the lifetime determination. 11

Mono-exponential decay Multi-exponential (at least two distinct lifetimes) A similar analysis is performed in case of multi-dim. Decay to extract the lifetimes τ i and the weights α i. Increasing the number of parameters in the fitting procedure reults in increasing the risk of numerical artifacts (more than 3 lifetimes are not recommended). Alternatively, the Method of Maximum Entropy can be used to analyze distributions of lifetimes. Esposito R., Altucci C., Velotta R., Analysis of Simulated Fluorescence Intensities Decays by a New Maximum Entropy Method Algorithm, Journal of Fluorescence, 23, , 2012 Mean lifetime – the time that a molecules spends in its excited state as an average over the molecular ensemble 12

Time Correlated Single Photon Counting (TCSPC) Measurement of start-stop times in time-resolved fluorescence measurement with TCSPC. Histogram of start-stop times in time-resolved fluorescence measurement with TCSPC. Simple experimental set-up for fluorescence decay measurements with TCSPC. 13

14 Glucose Oxidase Red: FAD cofactors bound deep inside the enzyme. FAD (Flavine Adenine Dinucleotide) * The active site where glucose binds just above the FAD. This enzyme, like many other proteins, is covered with carbohydrate chains, shown in green. It is produced by a mould species, the Aspergillus Niger 60 x 52 x 37 Å

15 Flavin Adenine Dinucleotide (FAD) Flavine Adenine “Open” configuration “Stacked” configuration Fluorescent site

16 Glucose + GOD (FAD + )  gluconic acid + GOD (FADH 2 ) (1) Reaction induced by Glucose GOD (FADH 2 ) + O 2  H 2 O 2 + GOD (FAD + ) (2) Unlike other enzymes GOD needs an external agent (O 2 ) to complete the cycle (this allows one to control the reduced FAD concentration) Glucose oxidation and FAD reduction

17 Experimental set-up Microscope objective Diode laser = 404 nm rep. rate= 40 MHz = 1 mW width=80 ps IRF~120 ps Bandpass FWHM:10nm Sample (stirring cell) GODGOD+Glu

18 GOD Effect of Glucose GOD+GLU 1 mM GODFAD in oxidized form GOD+GLU FAD in reduced form (due to Glucose)

19 Free FAD Lifetime (conformational effects) GOD+glu GOD The experiment with acid and basic buffers shed light on the FAD conformation in GOD!

20 Estimation of the features for sensors (preliminary results in sol-gel) Assuming Michaelis-Menten behaviour

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A Model System for DNA-Protein cross-link Dynamics and Photocyclization of 5-benzyluracil Dynamics and Photocyclization of 5-benzyluracil take a brick from DNA side (Uracil) and a part from the protein side (benzene) and study their interaction and dynamics induced by UV light. 5,6-benzyluracil (5,6BU) 5-benzyluracil (5BU) UV LASER 258 nm Sun, G.; Fecko, C. J.; Nicewonger, R. B.; Webb, W. W.; Begley, T. P., “DNA-protein cross-linking: model systems for pyrimidine-aromatic amino acid cross-linking.” Org. Lett. 2006, 8, 681–3 Another example: 5-Benzyluracil 22

TCSPC Results τ 2 = 1.6 ± 0.2 ns τ 1 = 50 ± 5 ps Decay Constants: Fitting function: 23