JOURNAL OF RAMAN SPECTROSCOPY J. Raman Spectrosc. 2002; 33: 238–242 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jrs.840 Fluorescence background suppression in Raman spectroscopy using combined Kerr gated and shifted excitation Raman difference techniques P. Matousek,∗ M. Towrie and A. W. Parker Central Laser Facility, CLRC Rutherford Appleton Laboratory, Didcot, Oxfordshire OX11 0QX, UK Received 12 October 2001; Accepted 3 December 2001 An exceptionally high level of fluorescence rejection from resonance Raman spectra was achieved using a combination of two techniques, namely Kerr gated temporal rejection with shifted excitation Raman difference spectroscopy. The method was able to recover the resonance Raman spectrum from the intense fluorescence background with a signal-to-noise ratio at least 10 times higher than that achievable with either of the two approaches used individually. To demonstrate the effectiveness of the technique we obtained the resonance Raman spectrum of the laser dye rhodamine 6G (1 × 10−3 mol dm−3 ) in methanol by excitation at 532 nm and measuring under the maximum of fluorescence emission at 560–590 nm. The method reached the photon shot noise limit of the residual fluorescence providing a detection limit for Raman spectra 106 times lower than the original fluorescence intensity in an accumulation time of 800 s. A unique feature of the experiment was the way in which the optical parametric amplifier light source was configured to alternate automatically between the two excitation wavelengths using an optogalvanic mirror arrangement. The ultra-high sensitivity of the combined approach holds great promise for selective probing of complex biological systems using resonance Raman spectroscopy. Copyright 2002 John Wiley & Sons, Ltd. INTRODUCTION Resonance Raman spectra obtained using a probe beam wavelength matched to an electronic absorption band have exceptional sensitivity and selectivity, which is particularly useful when studying complex biological samples. Unfortunately, fluorescence is often strong and limits observations in ground-state studies to only strong Raman bands. In many cases, the probe beam may also induce fluorescence in contaminants or the solvent, and in biological or industrial cases particularly, the concentration of the molecule under study may be low. The normal method to deal with these problems is to make background subtractions, but Raman signals are typically very weak and easily swamped by noise induced by fluorescence, which may be 106 –108 times stronger than the signal.1 A conventional approach is to avoid the fluorescence by probing in the near-infrared (NIR) region in the absence of electronic absorption and emission transitions.2 – 4 This method does not allow the resonance effect to be utilized for most compounds and also suffers from low sensitivity owing to the inverse fourth-power law dependence of non-resonant Ł Correspondence to: P. Matousek, Central Laser Facility, CLRC Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11 0QX, UK. E-mail: p.matousek@rl.ac.uk Raman scattering on wavelength. The problem can also be circumvented by probing well below the fluorescence emission in the ultraviolet (UV) region,5 – 7 an approach that offers the benefit of much higher cross-sections for Raman scattering than in the NIR region. However, this method often leads to resonance enhancement of several sample constituents simultaneously and this may be undesirable when detecting species at low concentrations or where constituent selectivity is desired. To achieve selectivity through the resonance enhancement, one usually has to resort to probing in the near-UV–visible region of the spectrum, i.e. in the region where fluorescence is frequently present. This then requires the use of some suppression technique to eliminate the detrimental effects of the fluorescence background on the Raman signal. There are now several linear Raman techniques that have been demonstrated to reduce the fluorescence problem. These include shifted excitation Raman difference spectroscopy8 (SERDS), polarization modulation,9 shifted spectra,1,10 Fourier transform filtering1 and temporal gating.11 – 16 Recently, we have developed an effective rejection device based on a 4 ps optical Kerr shutter17,18 for separating Raman light from fluorescence in the time domain. The fluorescence suppression ratio achievable by the technique depends on the fluorescence lifetime and can be up to ¾105 for long-lived Copyright 2002 John Wiley & Sons, Ltd. Efficient fluorescence background suppression in Raman Spectroscopy fluorescence species (>1 µs) and ¾102 –104 for lifetimes in the range ¾1–100 ns. The specifications of the gate are transmittance in the open state of up to ¾40% (excluding Fresnel losses on optical elements), a rejection ratio of 105 in the closed state and a usable spectral range from 300 to 700 nm. To enhance the performance of this device further for the case where an intense fluorescence background is still present after the Kerr gate, we have developed a new approach which combines the Kerr gating rejection technique with SERDS.8 The method provided an extremely high suppression not attainable with either of the two techniques used individually. In combination, the Kerr gate directly rejects fluorescence while the probe shifted technique accesses the photon shot noise level within the residual fluorescence which is otherwise typically not achievable.8 The SERDS technique relies on obtaining two Raman spectra using two probe excitation wavelengths separated by an amount comparable to the bandwidth of the measured Raman bands (typically 10–20 cm1 ). The subtraction of the two spectra from each other then produces a difference spectrum from which the Raman signal can be reconstructed by numerical analysis.8 This technique has been shown to be able to reach photon shot levels which otherwise are very difficult to attain with intense fluorescence backgrounds. A similar but less effective result can be obtained by shifting the spectrometer wavelength, which is often a technique of choice because of its instrumental simplicity.1,10 As the Kerr gate and SERDS techniques rely on different methods to recover the Raman signal, they can be combined to give higher fluorescence rejection. The combined approach is expected to be particularly beneficial in picosecond time-resolved resonance Raman (TR3 ) experiments.19 – 24 The conventional method for processing TR3 data is to subtract a positive time delay spectrum from that obtained at a negative time delay. However, this often leads to deeply distorted backgrounds brought about by the presence of various fluorescing intermediates from which the true Raman signal can be very difficult or even impossible to recover. These distortions can be neatly eliminated by the SERDS approach. In addition, the benefits of the combined method are enhanced in the situations where fluorescence originates only from the pump. As the pump wavelength is not shifted during the experiment, it consequently contributes with identical fluorescence background to both the shifted and unshifted excitation spectra. Note that the probe-induced fluorescence in the SERDS technique may, on the other hand, exhibit a small spectral shift due to the hot fluorescence component which can undergo a wavelength shift causing some distortion of the difference spectrum baseline. EXPERIMENTAL The basic principle of the optical Kerr gate has been described in our earlier publications.17,18 Briefly, the Kerr gate consists Copyright 2002 John Wiley & Sons, Ltd. of two crossed polarizers and a Kerr medium. In the closed state, light collected from the sample and collimated in an optical train is effectively blocked by the crossed polarizers. Coincident in time with the Raman scattered light from the sample, a short gating pulse of 800 nm wavelength that bypasses the polarizers creates a transient anisotropy within the Kerr medium by the optical Kerr effect. The gating beam is polarized at 45° and its intensity is adjusted to create, in effect, a half-waveplate that rotates the polarization of the light from the sample allowing it to be transmitted through the crossed polarizer for the duration of the induced anisotropy created by the picosecond gating pulse, the temporal opening of the gate being dependent on the solvent relaxation. The Kerr gate setup consists of a 2 mm long cell filled with CS2 and two 41 ð 41 mm Glan Taylor polarizers.18 The Kerr gate is followed by a spectrometer (Triplemate). A laboratory-built filter stage by-pass option was used in the experiments described here to optimize transmission. The probe laser line was blocked using a glass edge absorbing filter. A concentrated solution of copper sulphate in water in a 1 cm thick, 2 in diameter optical cell was placed in front of the spectrometer slit to block the residual 800 nm gating beam. The spectrometer throughput was determined to be by ¾25% higher for horizontal polarization (corresponding to measurements with the polarizers oriented in parallel, i.e. ’without the Kerr gate’) compared with the vertical orientation (polarizers crossed, ’with the Kerr gate’) in the spectral region where Raman signal was detected. No correction was made to the detected spectra to account for this difference in throughput. The grating density was 600 lines mm1 . Raman scattered light was collected at 90° to the probe beam direction. A liquid nitrogen-cooled, back-illuminated CCD camera with an array 2000 ð 800 pixels (Jobin Yvon) was used to record Raman spectra. The CCD was binned vertically across 400 pixels and horizontally across 5 pixels. One count corresponds to one photoelectron. The spectral resolution was ¾25 cm1 and was limited by the spectrometer slit width of 200 µm (the linewidth limit of our TR3 system is 10 cm1 ). The sample solution was re-circulated in an open liquid jet of 0.5 mm diameter. Unless stated otherwise, the overall acquisition time for Raman spectra at each wavelength was 400 s in the SERDS measurements, i.e. 800 s for the difference spectrum, and the spectra were collected by alternating every 10 s between the two shifted probe wavelengths. The probe wavelength and pulse duration were 532 nm and 1 ps, respectively, and the pulse energy was 5 µJ, corresponding to an average power of 5 mW. The beam was focused to ¾200 µm. Spectra were calibrated using acetonitrile Raman bands and in absolute terms were accurate to ¾10 cm1 . Owing to the huge fluorescence background without the Kerr gate, these spectra were obtained with an overall acquisition time of 66 s (6600 acquisitions each 0.01 s long) and with a neutral density (ND) filter of optical density 1. The spectrum in J. Raman Spectrosc. 2002; 33: 238–242 239 240 P. Matousek, M. Towrie and A. W. Parker this measurement was obtained by rotating the polarizers in parallel and blocking the gating pulse. The quoted signal-to-noise ratio (S/N) values are related to the Raman signal level present in a given measurement (taking into account the effect of the ND filter and the 40% Kerr gate throughput). The rhodamine 6G (Rh6G) resonance Raman bands were measured to have intensities of 30–300 counts s1 using the combined approach. The noise level is taken as the distribution width in counts s1 of the data channel where ¾90–95% of data points lie. Where photon shot noise is discussed the corresponding width of š2 range on the ’data channel’ is used, i.e. 4, where is the statistical standard deviation defined in the usual sense. A schematic diagram of the overall system is shown in Fig. 1. Our ps-TR3 apparatus25 uses a regenerative amplifier system followed by a two-pass linear amplifier (SuperSpitfire, Spectra Physics/Positive Light) providing 800 nm, 1 ps, 2–3 mJ fundamental pulses at a 1 kHz repetition rate. The fundamental laser output is split in two: 500 µJ is used for the gating pulse to drive the Kerr gate and the remainder is frequency doubled to pump two laboratory-built optical parametric amplifiers that generate the synchronized, independently tunable pump and probe pulses for various time-resolved spectroscopic experiments. In the experiments reported in this paper only one amplifier was used, modified as detailed below. probe (λ, λ+δ) Raman Raman/fluorescence a) CCD Kerr gate shutter / 4 ps sample spectrometer 800 nm, 0.5 mJ Kerr gate 2-3 mJ/1 kHz/800 nm/1 ps (Ti:sapphire) SHG 400 nm 2-pass OPG grating λ+δ λ OPA probe 532 nm, 5 µJ b) optogalvanic mirror aperture Figure 1. (a) Principle of the combined Kerr gated and SERDS techniques. (b) Schematic diagram of the laser/OPA setup for the generation of the dual wavelength for the Raman excitation source. Copyright 2002 John Wiley & Sons, Ltd. The versatile optical parametric generator/amplifier (OPG/OPA) technology adopted in our laser system allows us to generate the two excitation wavelengths in a very simple way. The OPG generated a broadband seed beam which was spectrally dispersed using a grating and the selected wavelength was steered into the OPA stage using an optogalvanic mirror [see Fig. 1(b)]. The moving galvanometer mirror permits automatic alternation between two spectrally shifted probe wavelengths every 10 s, each cycle being synchronized with the CCD readout. The spectral shift was set to 28 cm1 , which was well within the OPA acceptance bandwidth and no other adjustment to the OPG/OPA system was needed. The setup suffered from a slight pointing change but this would be eliminated if the optical path was reconfigured to enable the grating itself to be rotated between the two positions as opposed to the galvanometer mirror (see Fig. 1). RESULTS To test the performance of the combined Kerr gated and SERDS approach, we chose one of the most difficult samples and extreme conditions that a Raman spectroscopist can expect to be confronted with, the laser dye rhodamine 6G (Rh6G) at 1 ð 103 mol dm3 concentration in methanol (the quantum yield of fluorescence26 is ¾0.3 at this concentration), probing at 532 nm to provide a very efficient means of fluorescence excitation. Under these conditions the Raman spectrum between 900 and 1800 cm1 was detected in the fingerprint region, i.e. 560–590 nm, which is under the fluorescence maximum of Rh6G. First, each of the suppression techniques was tested on their own, demonstrating that if used individually they could not recover the Raman signal (see Fig. 2). The fluorescence background without applying any rejection method was measured to be up to 2 ð 107 counts s1 strong. As stated earlier, owing to excessive intensity of the fluorescence, this measurement was carried out using a neutral density filter with optical density 1 and over a shorter total acquisition time, 66 s. The displayed fluorescence intensity is corrected for the attenuation by the neutral density filter to show the true level present within the experiment. Two excitation wavelength-shifted spectra were obtained in this way to obtain the shifted excitation Raman difference spectrum [see Fig. 2(a)]. The difference spectrum is shown as measured, i.e. it is not scaled up to account for the ND filter as above. The resultant difference spectrum exhibits a very poor S/N, 1 : 27–1 : 270. Here the expected photon shot noise limit is about two times lower. Hence for direct comparison with Kerr gate results, where no ND filter and 400 s acquisition were used, it is necessary to scale up the signal 60-fold. This corresponds to a factor of about eighttimes higher S/N, i.e. ¾1 : 3.4–1 : 34 assuming the statistical square root improvement of S/N with counts. Even if the photon shot noise was reached the S/N would still be only J. Raman Spectrosc. 2002; 33: 238–242 intensity (counts/s) 25,000,000 a) 20,000,000 i) 15,000,000 10,000,000 ii) 5,000,000 0 800 10,000 5,000 0 −5,000 −10,000 −15,000 −20,000 −25,000 intensity (counts/s) Efficient fluorescence background suppression in Raman Spectroscopy 1000 1200 1400 1600 1800 16,000 14,000 12,000 10,000 8,000 6,000 4,000 2,000 0 800 400 b) i) 300 200 100 ii) 0 −100 1300 1800 intensity (counts/s) intensity (counts/s) wavenumbers (cm−1) −200 wavenumbers (cm−1) Figure 2. Attempts to recover the Raman spectrum of Rh6G in methanol using the two fluorescence suppression techniques individually: (a) with shifted excitation Raman difference spectroscopy only, where trace (i) is the fluorescence spectrum obtained with no suppression and (ii) is the difference spectrum; (b) with the Kerr gated technique only, where trace (i) is the remaining fluorescence after the Kerr gate and (ii) is the residual of fifth-order polynomial fit to the background (i). 1 : 1.7–1 : 17 for 400 s accumulation without the ND filter, i.e. still insufficient for observing the Raman spectrum. The Kerr gated approach alone resulted in an S/N of 1 : 1–1 : 10 and clearly reveals some of the main features of the Rh6G Raman signal. However, the noise is not photon shot noise limited but rather of the form of a heavily deformed ‘sloping’ background. This distorted the Raman spectrum to a degree that it was not possible to separate the Raman bands from this noise. The combined Kerr gate and SERDS approach cleanly distinguishes the Raman signal of Rh6G [see Fig. 3(a)] as well as a prominent Raman band of methanol at ¾1034 cm1 with a 10 times better S/N of 1 : 0.1–1 : 1. This S/N level matches the calculated photon shot noise limit estimated from the remaining fluorescence after the Kerr gate. This therefore represents an improvement by a factor of more than 10 in S/N over what each technique could provide individually under the most favourable conditions this corresponds to at least a 100-fold improvement in acquisition time. The observed 17-fold improvement in the S/N of the combined approach compared with SERDS comes from the fact that in both cases the photon shot noise limit is reached. Therefore, the improvement in S/N can be viewed as being due to the direct suppression of fluorescence by the Kerr gate reduced by the Kerr gate throughput, D 0.4, and can be Copyright 2002 John Wiley & Sons, Ltd. p estimated from the formula I0 /IKG , where I0 and IKG are the fluorescence intensities recorded without and with the Kerr gate, respectively. The resulting difference spectrum was fitted to Lorentzian bands with purpose-written software using a published method.8 The reconstructed spectrum is shown in Fig. 3 and is broadly similar to those obtained using other Raman techniques.27 – 30 The spectrum should be viewed as an example of the S/N that can be obtained with the combined approach. We wish to point out that, in general, the exact spectral profile of a Raman spectrum reconstructed from the difference spectrum is subject to some uncertainty in the regions where multiple bands overlap. A more rigorous method was proposed by Bell et al.10 involving the measurement and data processing of three shifted spectra (0, υ and 2υ). We are currently developing an algorithm which enables us to reconstruct the Raman spectrum directly from the raw difference spectrum using only linear data manipulation and eliminating the ambiguous fitting step or deconvolution process. The experiment was repeated with Rh6G dissolved in water at a slightly lower concentration (saturated solution) yielding a similar spectrum [see Fig. 3(b)]. Here the reconstructed spectrum correctly lacks the prominent methanol band. The fluorescence lifetime of Rh6G in methanol and water is ¾4 ns.31,32 Overall, the combined approach provides a powerful method for the suppression of fluorescence with the detection limit for Raman bands being 106 times lower than the original fluorescence level after an 800 s acquisition time. Even higher suppression could be obtained for samples with longer lived fluorescence. We believe that this technique holds great promise for studying complex fluorescent biological and chemical samples which so far have defied the attempts of spectroscopists to measure their resonance Raman spectra. CONCLUSIONS By combining the Kerr gating fluorescence suppression method with the conventional probe wavelength-shifted technique we have been able to obtain an exceptionally high level of fluorescence suppression not attainable using either of the two approaches individually. In a test experiment, the resonance Raman spectrum of Rh6G, one of the most difficult samples, was detected under its fluorescence maximum. The method allowed the photon shot noise level to be reached providing a detection limit for Raman spectra ¾106 times lower than the level of fluorescence with an 800 s acquisition time. To date, we believe, the most powerful method for fluorescence rejection has been achieved using the Kerr gate alone where we have reported up to a 105 fluorescence suppression capability. The combined approach provides still an order of magnitude better S/N than that achievable with the Kerr J. Raman Spectrosc. 2002; 33: 238–242 241 P. Matousek, M. Towrie and A. W. Parker 450 a) 650 i) 350 550 300 intensity (counts/s) 450 ii) 350 250 150 b) 400 i) intensity (counts/s) 242 ii) 250 200 150 100 iii) * 50 50 −50 800 iii) 0 1000 1200 1400 1600 1800 wavenumber (cm−1) −50 800 1000 1200 1400 1600 1800 wavenumber (cm−1) Figure 3. Kerr gated shifted excitation Raman difference spectra (i) of Rh6G in (a) methanol (1 ð 103 mol dm3 ) and (b) water (<1 ð 103 mol dm3 , saturated solution) probed at ¾532 nm. The figure shows both the experimental data ( ) and fit (solid line). The difference spectrum traces of pure solvents are also shown (ii). Raman spectra numerically reconstructed using the fitted (solid line) and experimental data () are shown at the bottom (iii). The band marked with asterisks denotes a prominent methanol band which is absent in the spectrum reconstructed for Rh6G in water. The accumulation time for each shifted spectrum was 400 s. ° gated approach alone. Its ultra-high sensitivity holds great promise for selective probing of complex biological systems using resonance Raman spectroscopy in the near-UV or visible region, in cases where selective resonance Raman enhancement of sample subcomponents or chromophores is desired. REFERENCES 1. Mosier-Boss PA, Lieberman SH, Newbery R. Appl. Spectrosc. 1995; 49: 630. 2. Schrader B, Hoffmann A, Keller S. Spectrochim. Acta, Part A 1991; 47: 1135. 3. Fujiwara M, Hamaguchi H, Tasumi M. Appl. Spectrosc. 1986; 40: 137. 4. Chase DB. J. Am. Chem. Soc. 1986; 108: 7485. 5. Bowman WD, Spiro TG. J. Raman Spectrosc. 1980; 9: 369. 6. Li C, Stair PC. Catal. 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