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Absorbance-detected electron spin echo spectroscopy of non-radiative triplet states in zero field
Volume 140, number 6
CHEMICAL PHYSICS LETTERS
23 October 1987
ABSORBANCE-DETECTED ELECTRON SPIN ECHO SPECTROSCOPY OF NON-RADIATIVE TRIPLET STATES IN ZERO FIELD. AN APPLICATION TO THE PRIMARY DONOR OF THE PHOTOSYNTHETIC BACTERIUM RHODOBACTER SPHAEROIDES R-26 E.J. LOUS and A.J. HOFF Departmentof Biophysics,Huygens Laboratoryof the State University,P.O. Box 9504,230O RA Leyden, The Netherlands Received 30 July 1987
Electron spin echoes of triplet states in zero field were optically detected using the singlet ground-state absorbance. The technique has been applied to reaction centers of the photosynthetic bacterium RhodobactersphaeroidesR-26. The spin-spin relazation time T, of the triplet state of the primary electron donor P (a bacteriochlorophyll dimer) was 1.16 k 0.05 us, independent of temperature in the range I .2-2.1 K.
1. Introduction The measurement of electron spin coherence of photoexcited triplet states is a valuable tool to gain information on the dynamics of spin relaxation processes [ l-41. A special attraction of photoexcited systems is that the detection of coherence can be carried out by optical means. Usually this is done via the phosphorescence of the excited triplet state. Following the observation of zero-field transitions of triplet states in non-phosphorescent molecules by monitoring the fluorescence [ 51, we demonstrated several years ago that spin coherence may also be observed via the fluorescence [6]. In these experiments spin coherence was converted into a population difference (the observable in optical detection methods) by an additional x/2 probe pulse. Using this technique, which was independently suggested by Brenner [ 71, we studied the T2relaxation of triplet states of porphin free base in n-octane and of reaction centers of a photosynthetic bacterium. The phase memory time T2of a system is a sensitive probe of its structure and environment. This is especially of interest for the photoactive reaction centers in photosynthesis. The current notion is that at least in photosynthetic bacteria, the reaction center triplet state is located on a dimeric (bacterio) chlorophyll molecule [ 8-101. Hence thermally 620
induced transitions between the exciton components of the dimer state are expected to influence the phase memory time [2]. From this temperature dependence one might derive the interaction between the dimer components. Optically detected electron spin echo (ESE) spectroscopy of triplet states in zero field has, compared to high-field ESE, many advantages for measuring T2 with high accuracy, since (a) microwave excitation and optical detection are decoupled, leading to a better time resolution, (b) hyperfme interactions are quenched to first order, leading to a less modulated decay trace, (c) the sensitivity is enhanced because in zero field the resonance line of a randomly oriented system is not broadened by the anisotropic electron spin dipolar interaction. Up to now, however, efforts to measure T2in the non-phosphorescent bacterial reaction centers by optically detected spin echoes have been frustrated by the low sensitivity of the fluorescence-detection method employed [ 61. Recently, we have shown that for non-phosphorescent systems with low quantum yield of fluorescence, zero-field resonance of the triplet state may be observed via the singlet ground-state absorbance with a sensitivity that is one to two orders greater than when fluorescence is used [ 11,121. In this communication we report on the application of absorbance-detected magnetic resonance ( ADMR) to the
0 009-2614/87/$ 03.50 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
Volume 140, number 6
CHEMICAL PHYSICS LETTERS
study of spin coherence. We show that electron spin echoes can be observed with good sensitivity at temperatures where helium is superfluid. The phase memory time Tz of the triplet state of bacterial reaction centers was found to be virtually independent of temperature in the range 1.2-2.1 K, suggesting that T, is still determined by interactions of the triplet spin with the nuclear system rather than by thermally activated transitions between two exciton states.
23 October 1987
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---i
5 300
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I-
2. Experimental Reaction centers of Rb. sphaeroides R-26 were prepared as described [ 121. Reduction of the primary electron acceptor was carried out by adding 10 mM ascorbate to a buffer-66% glycerol suspension and freezing in the light. Illumination at low temperature then produces the triplet state on the dimeric primary electron donor P by a radical recombination reaction. The concentration of the triplet state 3P is monitored by measuring the absorbance of the primary donor at 890 nm, at which wavelength the triplet state has little absorption, The absorbance of a sample (OD = 0.15 at 890 nm) was monitored with a single-beam apparatus as in refs. [ 11,121, in which the optical probe beam also serves as the excitation beam. Microwave pulses were made by a PIN diode switch, amplified by a traveling wave tube amplifier ( MPD LAB 1 1IO-10) and fed into a split-ring cavity with Qx: 100 [ 13,141. The variable delay of the probe pulse was supplied by a BNC 7075 digital delay generator. The change in absorbance induced by the microwave pulses was observed by a Si photodiode (RCA C-30842), amplified by a Tektronix AM 502 wide-band lownoise amplifier and averaged by a PAR 160 boxcar integrator with the gate set at the maximum of the transient change in absorbance [ 61.
3. Results 3.1. Two-pulse Hahn echo
E z
1-
Or
t
,“,
b
1
t (pz.1
,echo
w
180 i 0x1.
y 0
I
1
:
RC of Rb sphaer.R-26 467 MHz 1.2K : : : :: :4 2 3 t Ips)
Fig. 1. Optical absorbance-detected electron spin echo traces monitored at the 1D I- 1E I, 467 MHz transition for various delays T between the first 75 ns (n/2) and the second 120 ns (a ) microwave pulse, by a third 75 ns (x/2) microwave probe pulse running from 0 to t ( < 5) ps. The upper part of the figure illustrates the experiment schematically. The pulse sequence occurs in the first few microseconds of the change in the intensity of the transmitted light that reflects the pulse-induced depopulation and subsequent repopulation of the ‘P sublevels. An increase in corresponds to a decrease in the absorbance AA (ApUts-AnO& concentration of the triplet state of the primary donor, [3P]. This response was 100 x ac-amplified and monitored by a boxcar with a time constant of 30 ms and an aperture time (gate) of 300 ps. The boxcar gate was moving simultaneously with the probe pulse. The same traces were obtained when the gate was kept fixed at t = 0 ps. The symbols A to G of the trace for r = 1 ps are explained in the text. The repetition rate RR was 188 Hz resulting in an effective time constant ~,~=0.5 s. The temperature was 1.2 K. The scan rates were 5 min over 2 or 5 ps.
Fig. 1 shows the response of the absorbance to a series of x/2, rr, xl2 microwave pulses at a frequency 621
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of 467 MHz, corresponding to the IDI - (El transition. The delay between the first n/2 pulse and the x pulse (called the driving pulses) is fixed at r for each trace. The delay t between the first n/2 pulse and the second n/2 pulse (the probe pulse) is varied, and the optically detected echo signal is clearly visible. Let us first consider the situation for probing the Hahn echo at t = 22 +L&, where d,,, is the sum of the effective widths deff of the driving pulses. The first n/2 driving pulse creates a coherent superposition of the T,, and 1; triplet sublevels (the ordering of the levels is TX>TY>Tz) and simult~~usly equalizes their pop~ation. This results in a decrease in transmittance of the sample (fig. I, upper part) because the triplet now decays with the average of the fast T, and the slow T, decay rates, which leads to a temporary decrease in the triplet population, hence to an increase in singlet ground states. The time evolution of the transmittance response signal is governed by the individual triplet sublevel decay rates and the light flux [ $61; the change in transmittance is monitored by a boxcar whose 300 PS wide gate covers a large part of the transient. The II:pulse refocuses the electron spins, and after a delay 7 the Hahn echo is produced, which is converted into a population difference by the 1~12probe pulse. Without irreversible loss of phase, and for infinitely narrow pulses of sufficient intensity to cover the whole ODMR linewidth, the x pulse would have no effect on the transmittance and the transmittance at the time of the echo should then be the same as if no pulses were applied. In practice, the pulses have a finite width, the cavity does not admit the pulsed microwave field immediately, and loss of phase occurs with a characteristic time T2. Therefore, the measured echo intensity corresponds to a decrease in t~nsmission of at most 25% ( fg 1, z = 300 ns) compared to the level for t x-=22 + Atot,where coherence is lost and the response of the transmittance corresponds to that for incoherent pulsed excitation of the T,,-T, transition. For r > 300 ns the echo intensity diminishes because of the loss of phase (see below). The response of the system when the 3~12probe pulse is set at times between 0 and 2t+d,, (O
23 October 1987
(mV)
RC of
Rb. qitwtr.
R-26
60
1
:
:
0 length
:
:
i
:
:
500 of microwave puke
:
:
I
:
:
1000 A
(nsl
Fig. 2. Change in the absorbance induced by a fen~h-va~ed microwave pulse resonant at 660 MHz. Other conditions as in fig. 1. Inset: schematic representation of the experiment.
2.2. Transient nutation Fig. 2 shows the response of the absorbance to a single microwave pulse of width d, resonant at the IDI + IE( , 660 MHz transition. The initial sigmoidal rise represents the building up of the ffl field in the cavity. The transient nutation or Torrey oscillations are rapidly damped because of the random distribution of the chromophores and consequent distribution in effective HI fields [ 151. The Rabi period is 130 ns, corresponding to an average effective field H, NN 2.7 G. Note that yH, is smaller than the width A# of the inhomogeneous ADMR line (Ao/21~ = I2 MHz [ 11]). Note also that the nominal width d of a pulse of effective width A,@corresponding to a 7~12or 7[:pulse depends on the time db needed to build up the Ei, field in the cavity. For instance, the first maximum in fig. 2 corresponds to a rc pulse of width A= 105 ns, yielding A,=105 - 13012= 40 ns. This corresponds well with the calculated cavity response time of 50 ns. The duration of a single n/2 pulse was found to be 75 ns under the same conditions of incident power, cavity matching, etc. 3.3. Two pulses Fig. 3, upper trace, shows the response of the absorbance to two n/2 pulses of width A ( = 75 ns) and variable interval r, again resonant at 660 MHz, For r =O the pulses overlap completely; the inte-
CHEMICAL PHYSICS LETTERS
Volume 140, number 6
RC of Rb. sphaer.R-26
0
500
r
1500
1000 (nsl
0
A
1000 nr
pulse LengthA (ns)
Fig. 3. Upper: Response to two 75 ns (x/2) microwave pulses resonant at 660 MHz for varying delays. Conditions as in fig. 1, scan rate: 5 min over 2 IS. Lower: Response to a pulse of variable width at 1000 ns delay after a 75 ns (x12) pulse. RR, 366 Hz (5,,,.=0.25 s) and scan rate, 5 min over 1 ps. Further conditions asinfig. 1.
grated change in transmission during the boxcar gate then corresponds to level A. For increasing z the two pulses form first one pulse of width ‘5+ A; the change in transmission, corresponding to the first peak (100 ns pulse) in fig. 2, is represented by level B. At B the pulse width is 7 + A= 24 which now effectively corresponds to a IC pulse. At C, the pulses separate (compare the level at 150 ns pulse duration in fig. 2). The subsequent modulations D are analogous to the Rabi oscillations in fig. 2, and have the same frequency. Their particular shape is caused by the combined effects of the inhomogeneous linewidth ( yH, c AU), the distribution in the H, field seen by the molecules, and the spin dephasing during the internal 7 -A. The lower trace of fig. 3 shows the response to a x/2 pulse and a pulse of variable width A, spaced by 1000 ns and resonant at 468 MHz. The first maxi-
23 October 1987
mum, which is a minimum in 3P concentration, corresponds to partial population inversion to the quickly decaying TYsublevel. Half a Rabi period later, a minimum is reached because then the original population is partially restored. With increasing A this pattern is repeated with some damping. The Rabi period (190 ns) is somewhat larger than in fig. 2 due to the fact that the microwave transition used is different from the previous one. 3.4. Hahn echo sequence With the aid of figs. 2 and 3 we can now understand the complicated response in absorbance of fig. 1 following a three-pulse sequence resonant at 467 MHz. Let us consider the echo trace with the interval 7 between the two driving pulses, ~12 (A= 75 ns) and R (4=120ns) equalto lOOOns.Asecondn12 (A=75 ns) pulse is scanned with delay time t, starting at the position of the first n/2 pulse (t= 0). For zero delay, the n/2 pulse overlaps with the n/2 driving pulse and is therefore not effective. The combined effect of the two driving pulses is a temporary decrease in triplet concentration, and consequent increase in singlet ground-state absorbance, just as in the upper trace of fig. 3, level A. The absolute level for t = 0 agrees with level A (at A= 120 ns) in the lower trace of fig. 3. When the probe pulse immediately follows the n/2 pulse they combine to a 7cpulse that inverts the population difference, which for an ideal system (infinitely narrow pulses, homogeneous H, field distribution and yHI SAW) is brought back to the original value by the subsequent R pulse. The change in absorbance AA would then be nulled. In actual practice, the decrease amounts to only about 8% (compare level B with level A). When the two 7c/2 pulses separate, free induction decay sets in, and the population inversion becomes ineffective. When dephasing is complete, the combined effect of the three pulses is the same as for an incoherent experiment with a decrease in triplet population as a result of the transfer of population from a slow T, to a fast decaying TY sublevel (C). At D the probe pulse is precisely midway between the n/2 and the n pulse (delay t’). An echo is generated at time 2t’ and (partly) converted to a population difference by the x pulse. The peculiar shape of this echo, which manifests itself by the effect on 623
CHEMICAL PHYSICS LETTERS
Volume 140, number 6
23 October 1987
the 1D I+ 1E 1 and I D I - I E I transitions is identical. Over a temperature range 1.2-2.1 K we found that within the accuracy of the measurement, T2 is independent of temperature.
4. Discussion
I
0
:
I
:
I
:
2
2-r
I
:
I
4
:
I
:
:-
6
Ips)
Fig. 4. Decay of the spin-spin relaxation T2 at 1.2K for the two microwave transitions as monitored by a Hahn echo sequence with 75 (n/2), 120 (n) and 75 (x12) ns pulse lengths. The traces shown are the difference between a trace taken with the third pulse set at the echo (A7 =O) and one with the third pulse set at a delay A7 =800 ns after the echo. Note that AAIr= ns> MbrsOnv The response for 27 Q 700 ns results from the effect of overlapping microwave pulses. The boxcar gate was kept fixed at t=O. Further conditions as in fig. I, except RR: 400 Hz ( ‘T,~= 0.25 s) and scan rate: 2.5 min over 3 us. The traces are averages of four scans. Inset: semi-logarithmic plot of the echo intensity versus 25.
the absorbance of the position of the probe x/2 pulse, may be explained in a similar way as in ref. [ 161. At E the probe pulse starts to fuse with the n: pulse. The effect of this 3x/2 pulse is a slight decrease of total triplet population (fig. 3, lower trace). At F the probe and the A pulse overlap completely and the situation is the same as at A. When the probe pulse passes the x pulse the absorbance changes to a level C’ = C. At G, t=2r +A,,,, the spins are refocused by the x driving pulse and a Hahn echo is formed, which is converted to a population difference by the 7~12 probe pulse. 3.5. Measurement In fig. 4 the echo intensity is plotted as a function of delay time 22. The resulting curve is a single exponential with characteristic time T, = 1.16 k 0.05 ps (see inset), on which modulations are superimposed that are due to hypertine interactions. The modulations for 22 < 700 ns are due to partly overlapping microwave pulses, the effect of which is somewhat different for the two microwave frequencies because of cavity matching, etc. For 22 > 700 ns, the decay of 624
We have shown that ADMR can be used with advantage to detect spin coherence effects, such as electron spin echoes and transient nutation signals, in photo-excited triplet states. For samples with low fluorescence yield, the sensitivity is more than an order of magnitude better than the earlier employed FDMR technique. In the present work we have studied a sample in which transition dipole moments of the triplet state are randomly oriented, and in which the resonance linewidth Aw is significantly broader than YH,. Because the transition probability is proportional to H: cos%, with 8 the angle between the Hi field and the transition moment, the “tipping” angle of pulses is xl2 or ‘I[:only on the average. Since many spins experience a lower or higher tipping angle, the resulting picture of the spin dynamics in the rotating frame, as defined by the two-level description of Feynman, Vernon and Hellwarth [ 171 (reviewed in ref. [ 18 I), is very complicated. This is especially true for the shape of the response when the probe pulse fuses with the n: pulse and for the “midway” echo (fig. 1). Nevertheless, the fast damping and the low amplitude of the transient nutations can be qualitatively explained by the randomness of the sample and our experimental condition of a polarized microwave field [ 14,151. A quantitative description of the trace of fig. 1 must await a numerical simulation. For the first time, the phase memory time T2 of the triplet state of the primary electron donor in bao terial photosynthesis could be accurately determined. The observed time T2 = 1.16 + 0.05 ps at 1.2 K, is somewhat longer than that previously estimated for whole cells of Rb. sphaeroides wild type employing the less sensitive fluorescence-detected spin echo spectroscopy (0.5 ps [ 61). The temperature dependence of T2 is of special interest, since the primary donor P of bacterial reaction centers is a dimeric BChl molecule [&lo]. The coupling between the BChls of 3P can in principle be
Volume 140, number 6
CHEMICAL PHYSICS LETTERS
determined from the temperature dependence of T,, when the so-called mini-exciton model [2] may be employed. The contribution to T2 of transitions between the exciton components is then given by [ 1]
where ~~is the lifetime of the triplet excitation in the higher exciton state and AQ the difference in zerofield resonance frequencies of the two exciton states whose energy difference is equal to the coupling energy 2 1UI . t is the shift in experimental resonance frequency induced by higher temperatures. Eqs. (1) and (2) are valid when the majority of the triplet spins is in the lower exciton state, i.e. Wr,= exp( - 2 I UI lkT) CK 1, where W is the transition probability from the lower to the upper exciton state. When WB 2 ) UI a stochastic hopping of the triplet excitation occurs between the molecules of the dimer, while for W-x 2 I UI a “mini-exciton” description has to be applied [ 2 1. In principle, a temperature study of T2 and t should yield 2 ( UI, provided eq. (1) is valid over the temperature range studied. Within the accuracy of our measurement (about 0.05 us), however, no change in T2 was observed in the range 1.2-2.1 K, while up to 40 K the change in E was less than 2 MHz. This could be a result of (a) AQr,> 1, l/T2 x W and 2]U]=akT(T=2.1 K)%l.5cm-‘or(b)A.Qr,cl, l/T; a0, and the dephasing is still dominated by temperature-independent dipole-dipole interactions between the electronic spin and the nuclear spin system [ 19,201. To discriminate between these two possibilities, we plan to extend our measurements to higher temperatures, taking advantage of the high sensitivity of the present technique.
We are grateful to the members of the Center for the Study of the Excited States of Molecules for their interest and hospitality, to Dr. J. Schmidt for critically reading the manuscript, to Dr. M. Glasbeek of
23 October 1987
the University of Amsterdam for the loan of the MPD amplifier, to Ms. H.M. Nan for preparing the reaction centers and to Messrs. L. van As and J. van Egmond for their technical support. This work was supported by the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Organization for the Advancement of Pure Research (ZWO).
References [ 1 ] C.A. van’t Hof and J. Schmidt, Chem. Phys. Letters 36 (1975) 457,460;42 (1976) 73. [ 21 B.J. Botter, C.J. Nonhof, J. Schmidt and J.H. van der Waals, Chem. Phys. Letters 43 (1976) 210; B.J. Botter, A.J. van Strien and J. Schmidt, Chem. Phys. Letters 49 (1977) 39. [ 31 H.T. Lewellyn, A.H. Zewail and C.B. Harris, J. Chem. Phys. 63 (1975) 3687. [4] W.G. Breiland, H.C. Brenner and C.B. Harris, J. Chem. Phys. 62 (1975) 3458. [ 51 W.G. van Dorp, T.J. Schaafsma, M. Soma and J.H. van der Waals, Chem. Phys. Letters 21 (1973) 47. [6] N. Nishi, J. Schmidt, A.J. Hoff and J.H. van der Waals, Chem. Phys. Letters 56 (1978) 205. [7] H.C. Brenner, J. Chem. Phys. 67 (1977) 4719. [8] G. Feher, A.J. Hoff, J.C. McElroy and R.A. Isaacson, Biophys. J. 13 (1973) 61a. [9] J.R. Norris, M.E. Druyan and J.J. Katz, J. Am. Chem. Sot. 95 (1973) 1680. [ lo] J. Deisenhofer, 0. Epp, K. Miki, R. Huber and H. Michel, J. Mol. Biol. 180 (1984) 385. [ 111H.J. den Blanken, G.P. van der Zwet and A.J. Hoff, Chem. Phys. Letters 85 (1982) 335. [ 121 H.J. den Blanken and A.J. Hoff, B&him. Biophys. Acta 681 (1982) 365. [ 13] W.N. Hardy and L.A. Whitehead, Rev. Sci. Instr. 57 ( 198 1) 213. [ 141 H.J. den Blanken, R.F. Meiburg and A.J. Hoff, Chem. Phys. Letters 105 (1984) 336. [ 15] CA. van’t Hof, Dissertation, University of Leyden (1977). [ 161 R. Vreeker, M. Glasbeek, E.T. Sleva and A.H. Zewail, Chem. Phys. Letters 129 (1986) 117. [ 171 R.P. Feynman, F.L. Vernon and R.W. Hellwarth, J. Appl. Phys. 28 (1957) 49. [ 181 J. Schmidt and J.H. van der Waals, in: Time domain electron spin resonance, eds. L. Kevan and R.N. Schwartz (Wiley, New York, 1979) p. 343. [ 191 C.A. van? Hofand J. Schmidt, Mol. Phys. 38 (1979) 309. [20] J.F.C. van Kooten and J. Schmidt, Mol. Phys. 55 (1985) 351.
625
CHEMICAL PHYSICS LETTERS
23 October 1987
ABSORBANCE-DETECTED ELECTRON SPIN ECHO SPECTROSCOPY OF NON-RADIATIVE TRIPLET STATES IN ZERO FIELD. AN APPLICATION TO THE PRIMARY DONOR OF THE PHOTOSYNTHETIC BACTERIUM RHODOBACTER SPHAEROIDES R-26 E.J. LOUS and A.J. HOFF Departmentof Biophysics,Huygens Laboratoryof the State University,P.O. Box 9504,230O RA Leyden, The Netherlands Received 30 July 1987
Electron spin echoes of triplet states in zero field were optically detected using the singlet ground-state absorbance. The technique has been applied to reaction centers of the photosynthetic bacterium RhodobactersphaeroidesR-26. The spin-spin relazation time T, of the triplet state of the primary electron donor P (a bacteriochlorophyll dimer) was 1.16 k 0.05 us, independent of temperature in the range I .2-2.1 K.
1. Introduction The measurement of electron spin coherence of photoexcited triplet states is a valuable tool to gain information on the dynamics of spin relaxation processes [ l-41. A special attraction of photoexcited systems is that the detection of coherence can be carried out by optical means. Usually this is done via the phosphorescence of the excited triplet state. Following the observation of zero-field transitions of triplet states in non-phosphorescent molecules by monitoring the fluorescence [ 51, we demonstrated several years ago that spin coherence may also be observed via the fluorescence [6]. In these experiments spin coherence was converted into a population difference (the observable in optical detection methods) by an additional x/2 probe pulse. Using this technique, which was independently suggested by Brenner [ 71, we studied the T2relaxation of triplet states of porphin free base in n-octane and of reaction centers of a photosynthetic bacterium. The phase memory time T2of a system is a sensitive probe of its structure and environment. This is especially of interest for the photoactive reaction centers in photosynthesis. The current notion is that at least in photosynthetic bacteria, the reaction center triplet state is located on a dimeric (bacterio) chlorophyll molecule [ 8-101. Hence thermally 620
induced transitions between the exciton components of the dimer state are expected to influence the phase memory time [2]. From this temperature dependence one might derive the interaction between the dimer components. Optically detected electron spin echo (ESE) spectroscopy of triplet states in zero field has, compared to high-field ESE, many advantages for measuring T2 with high accuracy, since (a) microwave excitation and optical detection are decoupled, leading to a better time resolution, (b) hyperfme interactions are quenched to first order, leading to a less modulated decay trace, (c) the sensitivity is enhanced because in zero field the resonance line of a randomly oriented system is not broadened by the anisotropic electron spin dipolar interaction. Up to now, however, efforts to measure T2in the non-phosphorescent bacterial reaction centers by optically detected spin echoes have been frustrated by the low sensitivity of the fluorescence-detection method employed [ 61. Recently, we have shown that for non-phosphorescent systems with low quantum yield of fluorescence, zero-field resonance of the triplet state may be observed via the singlet ground-state absorbance with a sensitivity that is one to two orders greater than when fluorescence is used [ 11,121. In this communication we report on the application of absorbance-detected magnetic resonance ( ADMR) to the
0 009-2614/87/$ 03.50 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
Volume 140, number 6
CHEMICAL PHYSICS LETTERS
study of spin coherence. We show that electron spin echoes can be observed with good sensitivity at temperatures where helium is superfluid. The phase memory time Tz of the triplet state of bacterial reaction centers was found to be virtually independent of temperature in the range 1.2-2.1 K, suggesting that T, is still determined by interactions of the triplet spin with the nuclear system rather than by thermally activated transitions between two exciton states.
23 October 1987
ot+_--_-,_~~j -enrors&
---i
5 300
trme lpsl
ImV) 210
z.150 "S
I-
2. Experimental Reaction centers of Rb. sphaeroides R-26 were prepared as described [ 121. Reduction of the primary electron acceptor was carried out by adding 10 mM ascorbate to a buffer-66% glycerol suspension and freezing in the light. Illumination at low temperature then produces the triplet state on the dimeric primary electron donor P by a radical recombination reaction. The concentration of the triplet state 3P is monitored by measuring the absorbance of the primary donor at 890 nm, at which wavelength the triplet state has little absorption, The absorbance of a sample (OD = 0.15 at 890 nm) was monitored with a single-beam apparatus as in refs. [ 11,121, in which the optical probe beam also serves as the excitation beam. Microwave pulses were made by a PIN diode switch, amplified by a traveling wave tube amplifier ( MPD LAB 1 1IO-10) and fed into a split-ring cavity with Qx: 100 [ 13,141. The variable delay of the probe pulse was supplied by a BNC 7075 digital delay generator. The change in absorbance induced by the microwave pulses was observed by a Si photodiode (RCA C-30842), amplified by a Tektronix AM 502 wide-band lownoise amplifier and averaged by a PAR 160 boxcar integrator with the gate set at the maximum of the transient change in absorbance [ 61.
3. Results 3.1. Two-pulse Hahn echo
E z
1-
Or
t
,“,
b
1
t (pz.1
,echo
w
180 i 0x1.
y 0
I
1
:
RC of Rb sphaer.R-26 467 MHz 1.2K : : : :: :4 2 3 t Ips)
Fig. 1. Optical absorbance-detected electron spin echo traces monitored at the 1D I- 1E I, 467 MHz transition for various delays T between the first 75 ns (n/2) and the second 120 ns (a ) microwave pulse, by a third 75 ns (x/2) microwave probe pulse running from 0 to t ( < 5) ps. The upper part of the figure illustrates the experiment schematically. The pulse sequence occurs in the first few microseconds of the change in the intensity of the transmitted light that reflects the pulse-induced depopulation and subsequent repopulation of the ‘P sublevels. An increase in corresponds to a decrease in the absorbance AA (ApUts-AnO& concentration of the triplet state of the primary donor, [3P]. This response was 100 x ac-amplified and monitored by a boxcar with a time constant of 30 ms and an aperture time (gate) of 300 ps. The boxcar gate was moving simultaneously with the probe pulse. The same traces were obtained when the gate was kept fixed at t = 0 ps. The symbols A to G of the trace for r = 1 ps are explained in the text. The repetition rate RR was 188 Hz resulting in an effective time constant ~,~=0.5 s. The temperature was 1.2 K. The scan rates were 5 min over 2 or 5 ps.
Fig. 1 shows the response of the absorbance to a series of x/2, rr, xl2 microwave pulses at a frequency 621
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of 467 MHz, corresponding to the IDI - (El transition. The delay between the first n/2 pulse and the x pulse (called the driving pulses) is fixed at r for each trace. The delay t between the first n/2 pulse and the second n/2 pulse (the probe pulse) is varied, and the optically detected echo signal is clearly visible. Let us first consider the situation for probing the Hahn echo at t = 22 +L&, where d,,, is the sum of the effective widths deff of the driving pulses. The first n/2 driving pulse creates a coherent superposition of the T,, and 1; triplet sublevels (the ordering of the levels is TX>TY>Tz) and simult~~usly equalizes their pop~ation. This results in a decrease in transmittance of the sample (fig. I, upper part) because the triplet now decays with the average of the fast T, and the slow T, decay rates, which leads to a temporary decrease in the triplet population, hence to an increase in singlet ground states. The time evolution of the transmittance response signal is governed by the individual triplet sublevel decay rates and the light flux [ $61; the change in transmittance is monitored by a boxcar whose 300 PS wide gate covers a large part of the transient. The II:pulse refocuses the electron spins, and after a delay 7 the Hahn echo is produced, which is converted into a population difference by the 1~12probe pulse. Without irreversible loss of phase, and for infinitely narrow pulses of sufficient intensity to cover the whole ODMR linewidth, the x pulse would have no effect on the transmittance and the transmittance at the time of the echo should then be the same as if no pulses were applied. In practice, the pulses have a finite width, the cavity does not admit the pulsed microwave field immediately, and loss of phase occurs with a characteristic time T2. Therefore, the measured echo intensity corresponds to a decrease in t~nsmission of at most 25% ( fg 1, z = 300 ns) compared to the level for t x-=22 + Atot,where coherence is lost and the response of the transmittance corresponds to that for incoherent pulsed excitation of the T,,-T, transition. For r > 300 ns the echo intensity diminishes because of the loss of phase (see below). The response of the system when the 3~12probe pulse is set at times between 0 and 2t+d,, (O
23 October 1987
(mV)
RC of
Rb. qitwtr.
R-26
60
1
:
:
0 length
:
:
i
:
:
500 of microwave puke
:
:
I
:
:
1000 A
(nsl
Fig. 2. Change in the absorbance induced by a fen~h-va~ed microwave pulse resonant at 660 MHz. Other conditions as in fig. 1. Inset: schematic representation of the experiment.
2.2. Transient nutation Fig. 2 shows the response of the absorbance to a single microwave pulse of width d, resonant at the IDI + IE( , 660 MHz transition. The initial sigmoidal rise represents the building up of the ffl field in the cavity. The transient nutation or Torrey oscillations are rapidly damped because of the random distribution of the chromophores and consequent distribution in effective HI fields [ 151. The Rabi period is 130 ns, corresponding to an average effective field H, NN 2.7 G. Note that yH, is smaller than the width A# of the inhomogeneous ADMR line (Ao/21~ = I2 MHz [ 11]). Note also that the nominal width d of a pulse of effective width A,@corresponding to a 7~12or 7[:pulse depends on the time db needed to build up the Ei, field in the cavity. For instance, the first maximum in fig. 2 corresponds to a rc pulse of width A= 105 ns, yielding A,=105 - 13012= 40 ns. This corresponds well with the calculated cavity response time of 50 ns. The duration of a single n/2 pulse was found to be 75 ns under the same conditions of incident power, cavity matching, etc. 3.3. Two pulses Fig. 3, upper trace, shows the response of the absorbance to two n/2 pulses of width A ( = 75 ns) and variable interval r, again resonant at 660 MHz, For r =O the pulses overlap completely; the inte-
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RC of Rb. sphaer.R-26
0
500
r
1500
1000 (nsl
0
A
1000 nr
pulse LengthA (ns)
Fig. 3. Upper: Response to two 75 ns (x/2) microwave pulses resonant at 660 MHz for varying delays. Conditions as in fig. 1, scan rate: 5 min over 2 IS. Lower: Response to a pulse of variable width at 1000 ns delay after a 75 ns (x12) pulse. RR, 366 Hz (5,,,.=0.25 s) and scan rate, 5 min over 1 ps. Further conditions asinfig. 1.
grated change in transmission during the boxcar gate then corresponds to level A. For increasing z the two pulses form first one pulse of width ‘5+ A; the change in transmission, corresponding to the first peak (100 ns pulse) in fig. 2, is represented by level B. At B the pulse width is 7 + A= 24 which now effectively corresponds to a IC pulse. At C, the pulses separate (compare the level at 150 ns pulse duration in fig. 2). The subsequent modulations D are analogous to the Rabi oscillations in fig. 2, and have the same frequency. Their particular shape is caused by the combined effects of the inhomogeneous linewidth ( yH, c AU), the distribution in the H, field seen by the molecules, and the spin dephasing during the internal 7 -A. The lower trace of fig. 3 shows the response to a x/2 pulse and a pulse of variable width A, spaced by 1000 ns and resonant at 468 MHz. The first maxi-
23 October 1987
mum, which is a minimum in 3P concentration, corresponds to partial population inversion to the quickly decaying TYsublevel. Half a Rabi period later, a minimum is reached because then the original population is partially restored. With increasing A this pattern is repeated with some damping. The Rabi period (190 ns) is somewhat larger than in fig. 2 due to the fact that the microwave transition used is different from the previous one. 3.4. Hahn echo sequence With the aid of figs. 2 and 3 we can now understand the complicated response in absorbance of fig. 1 following a three-pulse sequence resonant at 467 MHz. Let us consider the echo trace with the interval 7 between the two driving pulses, ~12 (A= 75 ns) and R (4=120ns) equalto lOOOns.Asecondn12 (A=75 ns) pulse is scanned with delay time t, starting at the position of the first n/2 pulse (t= 0). For zero delay, the n/2 pulse overlaps with the n/2 driving pulse and is therefore not effective. The combined effect of the two driving pulses is a temporary decrease in triplet concentration, and consequent increase in singlet ground-state absorbance, just as in the upper trace of fig. 3, level A. The absolute level for t = 0 agrees with level A (at A= 120 ns) in the lower trace of fig. 3. When the probe pulse immediately follows the n/2 pulse they combine to a 7cpulse that inverts the population difference, which for an ideal system (infinitely narrow pulses, homogeneous H, field distribution and yHI SAW) is brought back to the original value by the subsequent R pulse. The change in absorbance AA would then be nulled. In actual practice, the decrease amounts to only about 8% (compare level B with level A). When the two 7c/2 pulses separate, free induction decay sets in, and the population inversion becomes ineffective. When dephasing is complete, the combined effect of the three pulses is the same as for an incoherent experiment with a decrease in triplet population as a result of the transfer of population from a slow T, to a fast decaying TY sublevel (C). At D the probe pulse is precisely midway between the n/2 and the n pulse (delay t’). An echo is generated at time 2t’ and (partly) converted to a population difference by the x pulse. The peculiar shape of this echo, which manifests itself by the effect on 623
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23 October 1987
the 1D I+ 1E 1 and I D I - I E I transitions is identical. Over a temperature range 1.2-2.1 K we found that within the accuracy of the measurement, T2 is independent of temperature.
4. Discussion
I
0
:
I
:
I
:
2
2-r
I
:
I
4
:
I
:
:-
6
Ips)
Fig. 4. Decay of the spin-spin relaxation T2 at 1.2K for the two microwave transitions as monitored by a Hahn echo sequence with 75 (n/2), 120 (n) and 75 (x12) ns pulse lengths. The traces shown are the difference between a trace taken with the third pulse set at the echo (A7 =O) and one with the third pulse set at a delay A7 =800 ns after the echo. Note that AAIr= ns> MbrsOnv The response for 27 Q 700 ns results from the effect of overlapping microwave pulses. The boxcar gate was kept fixed at t=O. Further conditions as in fig. I, except RR: 400 Hz ( ‘T,~= 0.25 s) and scan rate: 2.5 min over 3 us. The traces are averages of four scans. Inset: semi-logarithmic plot of the echo intensity versus 25.
the absorbance of the position of the probe x/2 pulse, may be explained in a similar way as in ref. [ 161. At E the probe pulse starts to fuse with the n: pulse. The effect of this 3x/2 pulse is a slight decrease of total triplet population (fig. 3, lower trace). At F the probe and the A pulse overlap completely and the situation is the same as at A. When the probe pulse passes the x pulse the absorbance changes to a level C’ = C. At G, t=2r +A,,,, the spins are refocused by the x driving pulse and a Hahn echo is formed, which is converted to a population difference by the 7~12 probe pulse. 3.5. Measurement In fig. 4 the echo intensity is plotted as a function of delay time 22. The resulting curve is a single exponential with characteristic time T, = 1.16 k 0.05 ps (see inset), on which modulations are superimposed that are due to hypertine interactions. The modulations for 22 < 700 ns are due to partly overlapping microwave pulses, the effect of which is somewhat different for the two microwave frequencies because of cavity matching, etc. For 22 > 700 ns, the decay of 624
We have shown that ADMR can be used with advantage to detect spin coherence effects, such as electron spin echoes and transient nutation signals, in photo-excited triplet states. For samples with low fluorescence yield, the sensitivity is more than an order of magnitude better than the earlier employed FDMR technique. In the present work we have studied a sample in which transition dipole moments of the triplet state are randomly oriented, and in which the resonance linewidth Aw is significantly broader than YH,. Because the transition probability is proportional to H: cos%, with 8 the angle between the Hi field and the transition moment, the “tipping” angle of pulses is xl2 or ‘I[:only on the average. Since many spins experience a lower or higher tipping angle, the resulting picture of the spin dynamics in the rotating frame, as defined by the two-level description of Feynman, Vernon and Hellwarth [ 171 (reviewed in ref. [ 18 I), is very complicated. This is especially true for the shape of the response when the probe pulse fuses with the n: pulse and for the “midway” echo (fig. 1). Nevertheless, the fast damping and the low amplitude of the transient nutations can be qualitatively explained by the randomness of the sample and our experimental condition of a polarized microwave field [ 14,151. A quantitative description of the trace of fig. 1 must await a numerical simulation. For the first time, the phase memory time T2 of the triplet state of the primary electron donor in bao terial photosynthesis could be accurately determined. The observed time T2 = 1.16 + 0.05 ps at 1.2 K, is somewhat longer than that previously estimated for whole cells of Rb. sphaeroides wild type employing the less sensitive fluorescence-detected spin echo spectroscopy (0.5 ps [ 61). The temperature dependence of T2 is of special interest, since the primary donor P of bacterial reaction centers is a dimeric BChl molecule [&lo]. The coupling between the BChls of 3P can in principle be
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determined from the temperature dependence of T,, when the so-called mini-exciton model [2] may be employed. The contribution to T2 of transitions between the exciton components is then given by [ 1]
where ~~is the lifetime of the triplet excitation in the higher exciton state and AQ the difference in zerofield resonance frequencies of the two exciton states whose energy difference is equal to the coupling energy 2 1UI . t is the shift in experimental resonance frequency induced by higher temperatures. Eqs. (1) and (2) are valid when the majority of the triplet spins is in the lower exciton state, i.e. Wr,= exp( - 2 I UI lkT) CK 1, where W is the transition probability from the lower to the upper exciton state. When WB 2 ) UI a stochastic hopping of the triplet excitation occurs between the molecules of the dimer, while for W-x 2 I UI a “mini-exciton” description has to be applied [ 2 1. In principle, a temperature study of T2 and t should yield 2 ( UI, provided eq. (1) is valid over the temperature range studied. Within the accuracy of our measurement (about 0.05 us), however, no change in T2 was observed in the range 1.2-2.1 K, while up to 40 K the change in E was less than 2 MHz. This could be a result of (a) AQr,> 1, l/T2 x W and 2]U]=akT(T=2.1 K)%l.5cm-‘or(b)A.Qr,cl, l/T; a0, and the dephasing is still dominated by temperature-independent dipole-dipole interactions between the electronic spin and the nuclear spin system [ 19,201. To discriminate between these two possibilities, we plan to extend our measurements to higher temperatures, taking advantage of the high sensitivity of the present technique.
We are grateful to the members of the Center for the Study of the Excited States of Molecules for their interest and hospitality, to Dr. J. Schmidt for critically reading the manuscript, to Dr. M. Glasbeek of
23 October 1987
the University of Amsterdam for the loan of the MPD amplifier, to Ms. H.M. Nan for preparing the reaction centers and to Messrs. L. van As and J. van Egmond for their technical support. This work was supported by the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Organization for the Advancement of Pure Research (ZWO).
References [ 1 ] C.A. van’t Hof and J. Schmidt, Chem. Phys. Letters 36 (1975) 457,460;42 (1976) 73. [ 21 B.J. Botter, C.J. Nonhof, J. Schmidt and J.H. van der Waals, Chem. Phys. Letters 43 (1976) 210; B.J. Botter, A.J. van Strien and J. Schmidt, Chem. Phys. Letters 49 (1977) 39. [ 31 H.T. Lewellyn, A.H. Zewail and C.B. Harris, J. Chem. Phys. 63 (1975) 3687. [4] W.G. Breiland, H.C. Brenner and C.B. Harris, J. Chem. Phys. 62 (1975) 3458. [ 51 W.G. van Dorp, T.J. Schaafsma, M. Soma and J.H. van der Waals, Chem. Phys. Letters 21 (1973) 47. [6] N. Nishi, J. Schmidt, A.J. Hoff and J.H. van der Waals, Chem. Phys. Letters 56 (1978) 205. [7] H.C. Brenner, J. Chem. Phys. 67 (1977) 4719. [8] G. Feher, A.J. Hoff, J.C. McElroy and R.A. Isaacson, Biophys. J. 13 (1973) 61a. [9] J.R. Norris, M.E. Druyan and J.J. Katz, J. Am. Chem. Sot. 95 (1973) 1680. [ lo] J. Deisenhofer, 0. Epp, K. Miki, R. Huber and H. Michel, J. Mol. Biol. 180 (1984) 385. [ 111H.J. den Blanken, G.P. van der Zwet and A.J. Hoff, Chem. Phys. Letters 85 (1982) 335. [ 121 H.J. den Blanken and A.J. Hoff, B&him. Biophys. Acta 681 (1982) 365. [ 13] W.N. Hardy and L.A. Whitehead, Rev. Sci. Instr. 57 ( 198 1) 213. [ 141 H.J. den Blanken, R.F. Meiburg and A.J. Hoff, Chem. Phys. Letters 105 (1984) 336. [ 15] CA. van’t Hof, Dissertation, University of Leyden (1977). [ 161 R. Vreeker, M. Glasbeek, E.T. Sleva and A.H. Zewail, Chem. Phys. Letters 129 (1986) 117. [ 171 R.P. Feynman, F.L. Vernon and R.W. Hellwarth, J. Appl. Phys. 28 (1957) 49. [ 181 J. Schmidt and J.H. van der Waals, in: Time domain electron spin resonance, eds. L. Kevan and R.N. Schwartz (Wiley, New York, 1979) p. 343. [ 191 C.A. van? Hofand J. Schmidt, Mol. Phys. 38 (1979) 309. [20] J.F.C. van Kooten and J. Schmidt, Mol. Phys. 55 (1985) 351.
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