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Fluorescence suppression in time-resolved Raman spectra

This content has been downloaded from IOPscience. Please scroll down to see the full text. 1977 J. Phys. E: Sci. Instrum. 10 617 (http://iopscience.iop.org/0022-3735/10/6/015) View the table of contents for this issue, or go to the journal homepage for more

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-.

FI uorescence su pp ress ion in time-resolved 'Raman

spectra

S Burgess and I W Shepherd Department of Physics, University of Manchester, Manchester M13 9PL, UK

Received 29 October 1976, in final form 27 January 1977 Abstract A pulsed Raman spectrometer is described, based on mode-locked Ar+ laser excitation and gated detection using time-amplitude conversion techniques. The equipment has been used to suppress unwanted fluorescence relative to Raman signal, and improvements by a factor of 6 for Rhodamine 6G ( T = 3.9 ns) and acridine orange ( T = 9.5 ns) have been obtained with negligible change in signal-to-noise ratio. These figures are better than those from previous attempts because electronic jitter in timing has been reduced so that the limit is set by the transit time spread in the photomultiplier tube. Large random fluctuations in fluorescence are also significantly reduced, which is of great importance when accurate Raman data are required in fluorescing solutions.

1 Introduction Fluorescence background has always been a major problem in Raman spectroscopy (Koenig and Frushour 1975, Shepherd 1975). One of the most promising techniques for decreasing this unwanted background in the spectra is to use a mode-locked laser as the radiative source and to gate the detection. Because the characteristic time of a fluorescence event (21 ns) is much

greater than the uncertainty-limited time of the Raman process it is possible to discriminate against the fluorescence by collecting only those photons emitted during the laser pulse. In this paper we review some of the ideas behind this technique and describe the equipment we have constructed. We further show how previous work (Van Duyne et a1 1974) failed to develop the full potential of the method. 2 Experimental detail 2.1 Equipment The apparatus is outlined in figure 1 . The light source was a mode-locked Spectra Physics laser (164) operating at 514.5 nm. The pulse train, consisting of approximately triangular pulses of approximately 0.5 ns duration and separated by 7 ns, was focused on to the sample cell by a lens of 10 cm focal length. The photons emitted from the sample were collected and focused on to the entrance slit of a Spex 1402 double monochromator. A photomultiplier (Mullard 56 AVP) fixed at the exit slit of the monochromator detected the emerging photons. These photons were tightly focused on to the photocathode by a lens of 2 cm focal length to reduce transit time differences in the tube. The output from the photomultiplier was fed through one section (A) of a dual discriminator (Ortec T105:N) to the START channel of a time-to-amplitude converter (TAC). The TAC is combined with a single-channel analyser (SCA) into one module (TAC/SCA, Ortec 467). Beam splitters placed immediately after the laser, taking about 15 % of the laser intensity, were used to supply two photodiodes. Photodiode A (Spectra Physics 403 high-speed light detector) was used in conjunction with a 140A Hewlett-Packard sampling scope to monitor the mode-locked laser pulse shape while the output of photodiode B was fed through a DELAY unit (SEN FE 290) and the second section, B, of the dual discriminator to the STOP channel of the Taclsc.4. With this arrangement the train of pulses to the STOP channel of the TAC is synchronized with the laser pulse rate of 143 MHz. The TAC will not respond to these pulses until a START pulse, originating from a Raman or fluorescence photon, has arrived at the TAC. This photon emission rate is usually well below the speed at which the TAC will reset (1 MHz) so that START channel pile-up does not occur. The distribution of START to STOP times is then an exact replica of the time spectrum of photons emitted by the sample. A picture of this spectrum can be built up in the multichannel analyser (Ireland MCA 500) and is displayed on a screen with the channel number linearly related to START-STOP time. The SCA gates that section of the

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Figure 1 The experimental arrangement of the spectrometer and gated detection

617

S Burgess and Z W Shepherd

time spectrum containing the Raman photons; this is accomplished internally in the TAC~SCA467 system. It is easy to set the upper and lower limits of the SCA because the SCA output, which occurs before the TAC output is generated, can be used to inhibit a TAC output unless the analogue signal is within the SCA window, and to thus limit the range of a time spectrum as it is stored in the MCA. There is no need for an auxiliary pulse generator with this arrangement. Once the window is adjusted until only the required part of the time spectrum is being stored in the h c A , it remains fixed while the output to the ratemeter is then taken from the SCA output. The ratemeter was constructed in our laboratory and counts in the range 0-3 x lo4 counts s-l. This adjustment procedure is most easily performed with a non-fluorescent sample, e.g. pure benzene, with the monochromator tuned to a Raman line. Since the lifetime of a Raman process is much less than the duration of a laser pulse the resulting time spectrum stored in the MCA is that of the laser pulse convoluted with any timing jitter in the electronics. The upper and lower limits of the SCA are set to gate the full width at half-maximum (FWHM)of this pulse. A direct measure of fluorescence suppression can be obtained by comparison of the gated signal with the output of discriminator section A, with the laser in either continuous or mode-locked operation. We believe this provides a more reliable comparison than previously reported data (Van Duyne et a1 1974) because the effects of START channel pile-up are avoided. Other differences between our arrangement and that of Van Duyne et a1 can now be pointed out. The photomultiplier was specially selected to have both low transit time spread, in order to reduce timing jitter, and a high gain (about 108). The high gain results in output pulses much greater than 50 mV so that we were able to use a threshold discriminator without any preamplification, thus reducing timing jitter even further. In our arrangement the STOP channel is triggered directly from the laser pulse, eliminating any electronic jitter or drift caused by using the mode-locked driver as trigger. Tight focusing on to the detecting diode, which was held at the relatively large voltage of - 24 V to cope with the high repetition rate, gave pulses greater than 100 mV which were sufficient to trigger the threshold discriminator. In order to ensure that the structure of the time spectra was genuine, the specimen was replaced by a random source of photons (light-emitting diode) in a test for a flat background. The photon time spectrum obtained with this source was found to have negligible structure only when (i) short leads (a few centimetres) were used between the discriminator and the TAC to minimize reflections: (ii) the pulses from photodiode B and the photomultiplier were much higher than the 50 mV discriminator threshold. Finally we were able to calibrate the multichannel analyser against the START-STOP time by triggering both the START and STOP channels of the TAC from identical outputs of the same discriminator (A). For this purpose the DELAY was inserted before the STOP channel and all leads were kept to a length of less than 10 cm, introducing a delay of about 4 ns which was subsequently corrected, The DELAY and hence the START-STOP time were increased in units of 0.5 ns from zero to 24 ns and the corresponding channel number in which pulses were stored was recorded. The calibration of the TAC START-STOP time against MCA channel number is shown in figure 2. The graph shows that the TAC is incapable of dealing with short START-STOP times and does not become linear until START-STOP times exceed 14 ns. Thus under normal circumstances the TAC cannot cope with the STOP pulse repetition time of 7 ns determined by the laser repetition rate, 143 MHz, since all START-STOP times would be 618

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Figure 2 Calibration of the multichannel analyser. Channel number is plotted against TAC START-STOP time

less than 7 ns. To solve this problem the pulse-widening facility in the discriminator was used. As the output pulses of the discriminator are widened more and more, the discriminator becomes unable to cope with the input pulse rate and ignores alternate pulses. In our arrangement the output pulses were widened to approximately 14 ns so that only every third pulse supplied by the photodiode reached the STOP channel. In this way, with pulses arriving every 21 ns at the STOP channel while laser pulses arrive every 7 ns at the sample, the required photon tirne spectrum can be stored in the MCA in those channels representing the 14-21 ns region. The TAC attempts unsuccessfully to store two identical spectra in the 0-7 and 7-14 ns regions. This 0-14 ns dead time is a general feature of any Ortec TAC including that used by Van Duyne et al. The effect of the above procedure is that two-thirds of the signal are wasted. This reduces the signal-to-noise factor in the frequency spectra to be discussed later. The aim of the above modifications was to eliminate all possible electronic jitter and to bring the time spectrum of a pure Raman process, as stored in the MCA, down to a narrow peak whose width is determined only by the finite width of the laser pulses (about +ns) and the transit time spread in the photomultiplier (about ns).

+

2.2 Samples The solvent used as a source of Raman photons was benzene (AnalaR, BDH) with the monochromator tuned to 542.2 nm corresponding to the 992cm-1 band. In order to study the fluorescence rzjection facility, benzene was doped with (a) the fluorescent dye Rhodamine 6G (BDH) with a natural lifetime of 3.9 ns; (b) the dye acridine orange (Hopkin and Williams) with a natural lifetime of 9.5 ns. 3 Results The photon time spectra for the Rhodamine 6G system in benzene are shown in figure 3. The time spectra were recorded with the monochromator tuned to the 992 cm-I (542.2 nm) Raman line of benzene and again with the monochromator tuned away from the line. The corresponding traces for the acridine orange system were much the same. The time spectrum for pure benzene is also shown, The shape and position of the benzene peak, whose FWHM is roughly 1.3 ns, were found to be highly stable once the laser was mode-locked. Slight retuning of the laser had an almost negligible effect. The position of the peak could be altered significantly (20.1 ns) only if the photomultiplier supply voltage was changed by more than +%, thus affecting the transit time, or if the

Fluorescence suppression in time-vesolued Raman spectra I

these systems were recorded both in a fluorescence-suppressed mode from the output of the SCA and in a conventional way from the output of discriminator section A. The result is shown for the Rhodamine 6 G system in figure 4. Again the traces obtained with the acridine orange system were much the same. It can be seen that the fluorescence background in the time-resolved spectrum has been reduced from that in the conventional spectrum by an amount such that the Raman-tofluorescence ratio, R/F (density of Raman counts/density of fluorescence counts at 542.2nm) has been increased by a factor of 6. The slope of the background and the erratic steps in this background, which can occasionally obscure the Raman peak in the conventional spectra, are much less in evidence. Apart from these large fluctuations the signal-to-noise ratio is substantially unchanged.

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4 Discussion In figure 3 it can be seen that the FWHM of the photon time spectrum for pure benzene is less than 15 ns. The absence of

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Figure 3 Photon time distributions for benzene and benzene/Rhodamine 6G. 1, distribution for the pure benzene sample with the monochromator tuned to the Raman line 542.2 nm (992 cm-l). 2, distribution for the benzeneiRhodamine 6G sample with the monochromator tuned to 542.2 nm. 3, distribution for the benzene1Rhodamine 6G sample with the monochromator detuned from 542.2 nm. The area B is the total area under curve 3 while A is the total area bounded by curves 2 and 3. Areas B' and A' are those sections of B and A respectively which lie between the limits of the SCA

discriminator threshold voltage was changed by 50 mV so that the discriminator triggered at a different part of the input pulse. With the upper and lower levels of the SCA set on the FWHM of this pure benzene trace, the Raman frequency spectra of

subsidiary peaks shows that echo and memory effects are negligible. In view of the laser pulse width (&+ns) and the transit time spread in the photomultiplier (about 5 ns) we can conclude that there is little or no timing jitter in the rest of our detection system. The corresponding width measured by Van Duyne et a1 was 3-3.5 ns. A very rough estimate can be made of the expected increase in RIF from the time spectra in figure 3. This is done from a measure of the areas A , B, A' and B' from which the expected RIFimprovement is given by A'BiAB'. This estimate is consistent with the observed increase by 6. The signal-to-noise ratio S", in these experiments has been discussed by Van Duyne et a1 (1974), based on the work of Tobin (1970). They obtained fairly good agreement between calculations and experiment although they made no mention of the expected increase in RIF. Due to the pulse overlapping in the time spectra and other complications introduced by the laser pulse shape function convoluted with the transit time spread, any calculations of S!iV or RIF must contain many approximations. Assuming that the convoluted pulse shape is an isosceles triangle of base T, that the counting intenal is identical to this, that the repetition time is tr and that the fluorescence can be characterized by a single time T , it is possible to calculate the factor I by which the ratio R I F increases. The full expression is a complicated function, but under the condition ~ < t rwe obtain exp (- T I T ) - 2 exp (Tl2.7))

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and for r % t r

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10-

I = (T/tr)-'.

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The values appropriate to the present study are T = 1.3 ns, tr=7 ns and T = 3.9 ns for Rhodamine 6G and 9.5 ns for acridine orange. Equation (1) is clearly not valid for these values of 7 but we obtain values of I = 5+ from equation (2) in both cases. This is in good agreement with observation considering the approximations made. A full treatment of SjN is complicated and we rely on the following close estimates. A Raman frequency spectrum with an overwhelmingly large background, caused in this case by a fluorescence count of nF counts s-l, will have an S / N ratio of nR/nF112, nR being the Raman count rate. That is to say the noise is entirely associated with the high fluorescence count rate. In our experiments n~ has been reduced by a factor of 6 with respect to H R , causing Sih'to increase by a factor of 116. However, because the power available from a mode-locked laser is limited to one-third of the continuous power available

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Figure 4 Raman frequency spectra of the benzene/ Rhodamine 6G sample obtained with a mode-locked laser. A, conventional spectrum; B, fluorescence-suppressed spectrum taken with the SCA operational

619

S Bwgess and I W Shepherd

and because we can only gate one out of three pulses due to TAC dead time, both nx and n F are decreased by a factor of 9. The final value of S / N in the time-resolved spectra is therefore approximately 3 4 6 of the SjN which could be obtained with the laser operated in a continuous mode. It can be seen from figure 4 that the S", ratio is essentially the same in the two cases. The above estimate which predicts a marginal decrease in signal-to-noise ratio with the fluorescence suppression facility is based on the assumption that the noise is signal shot noise. Any reduction in this type of signal-to-noise can easily be regained by increasing the time of measurement. However there is a far greater problem inherent in spectra which have a large fluorescence background. In figure 4 the conventional spectrum is seen to exhibit sudden large steps in fluorescence background. Even in such low concentrations of fluorescent material as 10-9 M, typical in this work, there are still over 108 fluorescing molecules in our scattering volume (10-5 cm3) so that the effect cannot be due to thermal molecular movement. The effect must be caused by either: (a) random movement of large aggregates of molecules; or (b) a bleaching (Tobin 1970) of the fluorescing material by the laser in the scattering volume which could subsequently be disturbed by mass movement within the liquid; or ( c ) an anisotropic distribution of fluorescent material in the vicinity of the scattering volume caused by the high laser flux density (Ashkin 1970, 1971) which would have the same effect as (b). In view of these considerations the suppression of fluorescence becomes even more important. Under certain circumstances the loss of laser power on mode locking may even be desirable. If the signal-to-noise ratio were substantially reduced by the technique, the slower scan speed required to recover this loss would increase the density of these background lurches, In such a case one would need to decide whether the technique was indeed an improvement over conventional methods. 5 Conclusion The experiments reported in this paper have demonstrated that the mode-locked laser Raman technique can produce spectra with a fluorescence background suppressed by a factor of 6 relative to the Raman signal for T values of the order of several nanoseconds or more with little loss in signal-to-noise ratio. All equipment used is commercially available. Bearing in mind the erratic fluctuations in solutions and steep slope of a fluorescence background this suppression, a marked improvement over previous attempts, means that the technique is now worthwhile for extensive use where accurate Raman data are required in fluorescing systems. It has been shown that the jitter in the electronics is negligible compared with the transit time spread in the photomultiplier as specified by the manufacturers. We feel that development of faster electronics and direct time gating is superfluous with modern photomultipliers. Furthermore we feel that a visible check on the photon time spectrum and the SCA setting, made possible by the TAClSC.4 technique, facilitates the experiment and is reassuring to the experimenter. In some recent experiments Harris et a1 (1976, private communication) employed a mode-locked cavity-dumped argon ion laser with which they were able to decrease the laser pulse rate and hence remove overlapping in the time spectra referenced to adjacent pulses. Due to the fast rise time of the laser pulses they were able, by gating only the early part of the Raman photons emitted by each pulse, to reduce the background of acridine orange in benzene by a factor of about 30. We are not clear from their definition of enhancement how much corresponding Raman signal is lost. Unfortunately, 620

mainly because of their direct time gating technique which employs a 24 ps cycle time sampling-scope to monitor the photon time spectrum, their signal-to-noise ratio was reduced by three orders of magnitude. Furthermore the FWHM of their photon time spectrum, 2.711s for pure benzene, was high. Nevertheless the use of a mode-locked cavity-dumped laser could be important for a further advancement in time-resolved Raman spectroscopy. Acknowledgments We should like to thank members of the Department of Physics at Manchester University, in particular Dr Baker, Mr Waddington, Dr Hamilton and Dr Birch who have assisted with the experimental programme, and Mr Callaghan for his assistance with the drawings. We should also like to thank Dr Phillips, Mr Swords and Dr Reid of Southampton University, Mr Beddard of the Royal Institution and Mr Playford of UKAEA for their advice on various aspects of equipment. Finally we should like to acknowledge the financial support of the SRC Polymer Committee. References Ashkin A 1970 Phys. Reu. Left. 24 156-9, 25 1321-4 Ashkin A 1971 Appl. Phys. Lett. 19 283-5 Koenig J L and Frushour B G 1975 Aduaizces in Znfrared and Raman Spectroscopy vol 1 (London: Heyden) chap 2 Shepherd I W 1975 Rep. Prog. Phys. 38 565-620 Tobin M C 1970 Laser Raman Spectroscopy (New York: Wiley Interscience) Van Duyne R P, Jeanmaire D L and Shriver D F 1974 Anal. Chem. 46 213-22

Journal of Physics E: Scientific Instruments 1977 Volume 10 Printed in Great Britain 8 1977