Picosecond fluorescence depolarisation measured by frequency conversion

Chemical Physics 61 (1981) 17-23 North-Holland Publishing Company

PZCOSECONQ FTXJORESCENCE DEPOCARI[SATiiON BY FREQUENCY CONVEXWON Godfrey S. BEDDARD, Tom DOUST and George PORTER Davy Faraday Research Laboramy, The Ropl Institution, London WX4BS.

MEASURED

UK

Received 21 April 1981

A method of using sum frequency generation in a LiI$ crystal has been used with picosecond pulses from a dye laser to measure fluorescence decays in solution. Fluorescence can be detected at wavelengths greater than 1 p. Some examples of the method to measure the rotational relaxation of dyes in solution are presented. In the dye aesyi violet two sites for solvent-solute interaction are proposed; one affects mainly the elenronic properties of the molecule while the other hinders molecular rotation.

1. Introduction The non-linear phenomenon of sum frequency generation in anisotropic crystals [l] has been used with a mode-locked synchronously pumped dye laser as an optical gate with picosecond time resolution [2-4]. When used to measure fluorescence decays it offers several advantages over time correlated single photon counting [S]. As well 2s greatly improved time resolution it is possible to detect fluorescence in the far red and infrared without using special red-sensitive photomultipliers; for example, fluorescence at 900 nm can be mixed with laser light at 600 nm to generate a sum frequency at 360 nm. Furthermore, the polarisation of the fluorescence can be analysed and hence the decay of the fluorescence anisotropy can be measured directly. This offers an alternative method to anisotropic absorption [6] and transient dichroism for the measurement of rotational diffusion on a picosecond time scale C7]. Thus, frequency conversion has almost as much importance in areas covered by existing techniques as it does in extending detection ranges in both the wavelength and time domains. Two methods of collecting the fluorescence are described and compared. The rotational 0301-0104/81/0000-0000/$02.50

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diffusion times of the dyes cresyl violet* and 3,3,3’,3’-tetramethyI-l,l’-dimethyIindotricarbocyanine perch!orate (HITC) have been measured in various solvents. Both behave in a fashion consistent with soIvent attachment and have slower rotation times than predicted by “stick” behaviour. Comparison of the rotational and spectral properties of cresyl violet in various solvents with those of the dye oxazine-1 indicates the existence of two possible sites for soIvent attachment. These sites are identified as the N-O ring and the 2,2’-amino substituents.

2. Theory and demipeiiqn of the technique The two optica! configurations used are shown in figs. la and lb. In both cases a synchronously pumped mode-locked rhodamine 6G dye laser producing pulses of about 4 ps (fwhm) and 80 mW average power was used. The pulse train is split on a 50% beam splitter and half of it passed down a variable optical delay line, the length of which is controIIed by a translation stage driven by a stepping motor. The steppiug * Since this was completed similar results were reported by van Jena and Using [8] using a transient dichroism method.

glass cell mounted perpendicularly to the exciting beam and spun about an axis parallel to the beam; the fluorescence is collected off the front face of the cell along the axis of the exciting beam. In method 2 (fig. lb) the sample is flowed through a cell or pumped through a nozzle to form a jet. The Ruorescence is collected at 180” to the exciting beam and the transmitted excitation light removed by filters. The instantaneous power of the sum frequency in a crystal of length L is given approximately by [Q]

P,(L) = kL’?lPz

Fig. 1. Experimenta arrangements used for time resolved fluorescence upconversion. F are Rters, P are polarisers and C a sectored disc chopper connected to the lock-in amp. or photon counter. a+ is the laser beam and o2 the fluorescence. ‘The insert shows optical arrangement 1, the main figure arrangement 2.

motor controller also indexes the memory of a multichannel analyser so that the signal at each delay is collected in consecutive channeIs of the analyser. The complete decay can thus be collected in one scan of the delay line. If the fluorescence is weak several scans can be averaged. The undelayed portion of the pulse is used to excite the sample. The fluorescence is collected and focused into a LiIO3 &ystal (1 mm path length) along with the delayed laser pulse. This short path length, although it allows less frequency conversion is necessary to avoid group dispersion effects. In method 1 (fig. la), which is similar to that used by Hirsch et al. [4], the sample is contained in a 1 mm path length

03/w.

0)

where the subscripts on the frequency o and the power/unit area P refer to the gating pulse, the fluorescence and the sum frequency respectively. The frequencies are related by 03 = oz*tol. It can be seen that by appropriate phase matching the difference frequency can aIs0 be generated. This can be used to measure fluorescence in the UV ClOf where the sum frequency would be at very short wavelengths where detection becomes difficult or where cays&Is transparent at such short wavelengths are not available. This may be of use in measuring fluorescence excited by the second harmonic of the laser or by multi-photon absorption. Since the power of the laser pulses is constant to within a few percent, the sum frequency signal is proportional to the fluorescence intensity at a particular time delay. The time resolution in a thin crystal is only limited by the width of the laser pulse as the optical delay can be adjusted by increments corresponding to far smaller times than the duration of the Iaser pulse. By angle tuning the crystal the phase veIocities of the laser pulse and the fluorescence can be matched for di5erent fluorescence wavelengths. This allows decays to be measured at diEerent wavelengths in the emission spectrum. In these experiments, type 1 phase matching was used with the two input beams on the ordinary ray and the sum freqnency on the extraordinary ray. Tlnis phase matching condition also acts as a frequency selecting element: in the crystal used a bandwidth of about 3 .& was upconverted at a particular crystal orienta-

G.S. Beddard et al. 1 Picosecond fluorescence depohsation

tion. Time gated spectra can be measured at a given time after excitation either by tuning the laser frequency [11] or at a fixed laser frequency by turning the crystal to select the fluorescence wavelength. In our experiments the fundamental and second harmonic of the gating pulse are removed by filters and a monochromator is set to select the sum frequency. The beam exciting the fluorescence is modulated at about 700 Hz by a sectored disc chopper and the signal from the photomultiplier recovered by a lock-in amplifier. Aitematively the photomultiplier can be made to operate in a photon counting mode and the signal collected in an up-down counter triggered by the chopper. In both cases the signal can be normalised to the dye laser intensity which can be measured separately (not shown in fig. 1). To measure the autocorrelation function of the laser pulse the lens and sample cell can be replaced by a mirror in method 1 or the sample removed in method 2. The fluorescence intensity r5 generated by the exciting pulse has a time profile given by

J I

Qt) =

-m

I#‘) L(t - I’) dt’,

where t’ is the time delay, t(t) is the laser pulse shape and I;(t) the molecular fluorescence intensity at time t. The signal after the crystal S(f) is given by s(r) =

Jm I,Wt(t- t’)di’, -.-m

which combined with eq. (2) gives f S(r) = I&‘) A(t-r’) dt’, I -5a

19

Fig. 2. Fluorescence from HITC in methanol detected at 770 nm and excited at 590nm. The laser autocorrelation function has been displaced slightly for clarity. The solid line through the fluorescence is the convolution of the laser autocorrelation firnction wiih the sum of two exponential

temn. 3. ResuIts Fig. 2 shows the fluorescence decay of HITC in methanol at 770 nm together with the laser autocorrelation function and the computed fit (sum of two exponentials) to the data. By angle tuning the crystal the fluorescence decay could be observed as far to the red as 980 nm, close to the limit of HITC emission. Fig. 3 shows fluorescence collected with polarisation parahel

(3)

(4

where A(t) is the measured autocorrelation function of the laser pulse. As the autocorreiation function is readily measured the signal can be fitted to the desired decay function at the shortest times with no a priori knowledge of the laser pulse shape. This offers a significant advantage over transient absorption methods [6,7] where it is necessary to make assumptions about the laser pulse shape to facilitate curve fitting.

Fig. 3. Ruorescence profile of cresyl violet detected parallel top curve and perpendiculviy to the excitation pnlarisation. The detection wavelength was 770 nm.

20

G.S. Beddard et al. / Picosecond fluorescence depolarisarion

where Ij~=[1+2r(t)Je-“~~, I, = [l -r(t)]

and perpendicular to the polarisation of the exciting light for cresyl violet in iso-propanol measured using method 2. The fluorescence anisotropy r(t), fig. 4, was cakulated from the equation [12, 131

r(t) = [I&)G -I,(t)ll[lub)G

Table 1 Exp=rimentill excited state Froperties Solvent

Viscosity (cP!

acetone methanol water ethanol

propanoi

0.32 0.55 1.03 I.2 2.2

(5)

+=~(t)l,

of cresyl vi&t,

@a)

e-““.

(6b)

I, and IL are the fluorescence intensities polarised parallel and perpendicular to the excitation and rf is the fluorescence lifetime. The normalisation factor G was determined in two ways. Firstly, in very mobile liquids the signal at long times is free of the effects of rotational motion and the I, and 1, signals should have the same magnitudes so G can be determined by tail matching at long times. Secondly, if the laser pulse is short compared with the molecular events convolution does not significantly distort the signals at short times, thus the condition Ii! = 31, at t = 0 can be used to calibrate the cmves. The anisotropy r(t) can then be related to the rotational relaxation lifetime depending upon its shape [12,13]. The rotational relaxation lifetime can also be calculated from either the 41 or I, gignals and if, as is the case, the fluorescence decay is single exponential the Ii1 signal is described by eq. (6a) and the I, signal by eq.

HITC and oxanine (all lifetimes given in pi;oseconds)

Cresyl violet 7R

‘;-f

78i4 134*4 13Or5

X70 X50 2390 3510 3870

350514 696515

Oxazine”’

HITC -R

71

22654

1080

377*11 436zi7

1300 1500

TR

rr

57 84 141 136 237

1122 813 552 1024 1122

caiculated stick lines for: cresyl violet (vol = 228 A’) 84 ps/cP HITC iv01 = 473 A”) 91 ps[cP oxtine (vol= 317 A3) 120 ps/cP measured r0 values cresyl violet = 0.355% 0.02 HITC = 0.38 f 0.02 a) Taken from ref. 1141.

, E:.p Cresy!

vlole:‘

Oxazine

1

Me

Me HITC

21

G.S. Beddard et ai. / Picosecond fluorescence depolarisarion

(6b). In all cases so far reported [7, 131 in isotropic liquids r(r) has been described by an exponential function i.e. r(t) = r0 exp (-t/rR). Hence, knowing 7f either from up-conversion measurements with the polariser at 54.7” or from single photon COUUtiUg allows TR and ro to be calculated. These values are very similar to those calculated by generating r(t) directly from eq. (5). Table 1 shows the measured tluorescence decay times and rotational reorientation times for HITC and cresyl violet in different solvents. Fig. 5 shows a pIOt Of TR versus SOlVent viscosity for these dyes together with the lines calculated on the basis of the Debye-Einstein theory at the “stick” boundary condition [13], for an oblate ellipsoid in the case of cresyl violet and a prolate ellipsoid for HITC. In both cases the deviation from the stick boundary condition is clear, the experimentally determined rotational relaxation times being slower than those predicted by theory. This contrasts sharply with the behaviour of oxazine 1 where the measured slope 87 ps/cP [14] is closer to 800

the calculated slip boundary condition where resistance to motion arises from solvent displacement in non spherical molecules. The calculated values for oxazine 1 are 60 ps/cP for slip and 110-134 ps/cP for stick depending on the exact moiecular shape. Much stronger solvent-solute interactions are therefore present in cresyl violet than oxazine 1.

4. Discussion The changes in emission maxima in both oxazine and cresyl violet indicate an interaction at a different site on the solute molecule than that suggested by the rotational relaxation measurements_ For example, in both compounds the spectra in 1,4-dioxan (hydrogen bonding and polar) and acetonitrile (non hydrogen bonding but polar) showed shifts in the fcrmer but not in the latter. This suggests that hydrogen bonding provides a greater contribution to the spectral shii than is caused by solvent polarity.

r

b a

0

0

I .80

0.60 VISCOSITY

2.40

3.00

Ccentipoise3

Fig. 5. Experimental rotational relaxation times (picoseconds) for cresyl-violet (0). HITC (+) and oxazine (1) [la] oloucd against solvent viscosity (cP). Also shown are the calculated stick lines for the same solutes (a), (b) and (c), respectively.

22

G.S. Beddard er ai. / Picosecond fluorescence depolarisation

Cresyl violet exhibits anomalous behaviour in water; a blue shift in fluorescence maximum compared to propanol is observed. Similar effects occur in merocyanine and have been attributed to changing and opposite contributions of the dipole moment and polarisability of the solute with the strength of the reaction field of the solvent [16]. The excited states of cresyi viclet and oxazine also show similar behaviour both being quenched in water relative to the alcohols, table 1. It is probable that either intersystem crossing . is more rapid in water than alcohols as occurs with xanthene dyes [17] or that internal conversion is more rapid in aqueous solution. A deuterium isotope effect rD/rH of 1.26 for oxazine in DzO (7~ = 695 ps) and Hz0 (7~ = 552 ps) supports the suggestion of Forster [18] that direct solute-solvent interaction enhances internal conversion. These similarities in the ground and excited state properties of cresyl violet and oxazine are in contrast to their rotational diffusion behaviour. One factor causing this difference could be the change in shape between the two molecules, but HITC is more prolate than oxazine yet does not show simiIar behaviour but acts like other cyanines such as DODCI following stick behaviour. Specific hydrogen bonding to the amino group on cresyl violet although not to the diethy amino group on oxazine will explain the difference in behaviour. A strong hydrogen bond will have the effect of slowing molecular rotation and will be particularly effective if bonding occurs at the end of the long axis of the ellipsoid of revolution. Solvent attachment on the central 0, N containing ring will be near to the symmetry axis of the molecule and wili impede moIecular motion less. Rotation times slower than expected from stick behaviour have been observed in other compounds such as ft;Jorescein derivatives [13, 201. These differences and similarities between oxazine and cresy! violet can thus be explained qualitatively by two sites of solvent-soIute inter: action. The spectral and excited state properties are sensitive to interaction on the 0 and N con-

taining ring. These interactions, although large enough to change electronic properties are nevertheless sufficiently small that they do not afiect rotational relaxation. The other interaction occurs on the amino subs&rent groups and while strong enough to slow rotation does not appear to interact appreciably -with the pi-electron system. An exception to this behaviour appears to be cresyl v
Acknowledgement We acknowledge the SRC for financial support for the work; G.S.B. for an Advanced Fellowship and T.D. for a Studentship.

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G.S. Beddard

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