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. Today 1997; 33: 353.
7. Sands HS, Hayward IP, Kirkbride TE, Bennett R, Lacey RJ,
Batchelder DN. J. Forensic Sci. 1998; 43: 509.
8. Shreve AP, Cherepy NJ, Mathies RA. Appl. Spectrosc. 1992; 46:
707.
9. Angel SM, DeArmond MK, Hanck KW, Wertz DW. Anal. Chem.
1984; 56: 3000.
10. Bell SEJ, Bourguignon ESO, Dennis A. Analyst 1998; 123: 1729.
11. Harris JM, Chrisman RW, Lytle FE, Tobias RS. Anal. Chem. 1976;
48: 1937.
12. Tahara T, Hamaguchi H. Appl. Spectrosc. 1993; 47: 391.
13. Yaney PP. J. Opt. Soc. Am. 1972; 62: 1297.
14. Howard J, Everall NJ, Jackson RW, Hutchinson K. J. Phys. E: Sci.
Instrum. 1986; 19: 934.
15. Fujii T, Kamogawa K, Kitagawa T. Chem. Phys. Lett. 1988; 148: 17.
Copyright  2002 John Wiley & Sons, Ltd.
16. Deffontaine A, Delhaye M, Bridoux M. In Time-resolved Vibrational Spectroscopy, Laubereau A, Stockburger M (eds). Springer:
Berlin, 1985; 20–24.
17. Matousek P, Towrie M, Stanley A, Parker AW. Appl. Spectrosc.
1999; 53: 1485.
18. Matousek P, Towrie M, Ma C, Kwok WM, Phillips D, Toner WT,
Parker AW. J. Raman Spectrosc. 2001; 32: 983.
19. Kwok WM, Ma C, Matousek P, Parker AW, Phillips D,
Toner WT, Towrie M, Umapathy S. J. Phys. Chem. A 2001; 105:
984.
20. Benniston AC, Matousek P, Parker AW. J. Raman Spectrosc. 2000;
31: 503.
21. Coates CG, Olofsson J, Coletti M, McGarvey JJ, Onfelt B,
Lincoln P, Norden B, Tuite E, Matousek P, Parker AW. J. Phys.
Chem. B 2001; 105: 12 653.
22. Schneider S, Kurzawa J, Stockmann A, Engl R, Daub J,
Matousek P, Towrie M. Chem. Phys. Lett. 2001; 348: 277.
23. Everall N, Hahn T, Matousek P, Parker AW, Towrie M. Appl.
Spectrosc. 2001; 55: 1701.
24. Vlček Jr. A, Farrell IR, Liard DJ, Matousek P, Towrie M,
Parker AW, Grills DC, George MW. J. Chem. Soc., Dalton Trans.
in press.
25. Towrie M, Parker AW, Shaikh W, Matousek P. Meas. Sci. Technol.
1998; 9: 816.
26. Bindhu CV, Harilal SS, Nampoori VPN, Vallabhan CPG. Mod.
Phys. Lett. B 1999; 13: 563.
27. Li W-H, Li X-Y, Yu N-T. Chem. Phys. Lett. 1999; 312: 28.
28. Majoube M, Henry M. Spectrochim. Acta, Part A 1991; 47: 1459.
29. Hildebrandt P, Stockburger M. J. Phys. Chem. 1984; 88: 5935.
30. Kagan MR, McCreery RL. Anal. Chem. 1994; 66: 4159.
31. Gumy J-C, Vauthey E. J. Phys. Chem. 1996; 100: 8628.
32. Zander C, Sauer M, Drexhage KH, Ko DS, Schulz A, Wolfrum J,
Brand L, Eggeling C, Seidel CAM. Appl. Phys. B: Lasers Opt. 1996;
63: 517.
J. Raman Spectrosc. 2002; 33: 238–242