Many analytical applications require
fast, noninvasive characterization of
deep regions of diffusely scattering (turbid)
media, such as living tissue and
powders, with high chemical specificity.
Such information is important,
for example, in disease diagnosis,
noninvasive
probing of pharmaceutical
products for quality control, drug
authentication, and security screening.
Laser spectroscopy holds particular
promise in this area due to its ease and
speed of implementation. Presently,
fundamental optical spectroscopic
techniques that are applicable to this
class of problems include near- and
midinfrared (NIR and MIR) absorption
spectroscopy and Raman spectroscopy.
Although NIR absorption is
widely used, it often suffers from low
chemical specificity.1 In comparison,
both MIR and Raman spectroscopy
offer a substantially higher degree of
chemical specificity but have, thus far,
been confined to applications involving
mainly surface layers of turbid
media. In the case of MIR, the principal
reason for this is the excessively
high absorption of its signal by water
or sample matrix. From this perspective,
Raman spectroscopy holds great
potential since it does not suffer from
these fundamental limitations.
The Raman effect is the inelastic scattering
of photons from molecules via
interactions with the vibrational modes
of the analyte molecule.1 In this process,
a photon typically transfers a fraction
of its energy to a vibrational mode
within the molecule. Consequently,
the wavelength of the scattered photon
is spectrally red-shifted, with the degree
of the shift indicating the amount of
energy uptake by the molecule. Since
the vibrational modes are quantized
and molecule specific, the molecular
identity can be determined from the
distribution of the observed shifts in
wavelength. The shift pattern serves,
in essence, as a unique fingerprint for
the molecule, specific to its structure
and conformation.
The general applicability of this technique
is limited to samples that do not
exhibit strong fluorescence emission
in the spectral Raman region, since
this can easily swamp the relatively
weak Raman signals. However, this
problem can often be avoided by using
near-infrared excitation away from the
electronic absorption bands of most
fluorescing species, thus preventing
their excitation and consequently the
generation of fluorescence emission in
the first place.
Figure 1 - Illustration of the conventional backscattering Raman and spatially offset Raman spectroscopy (SORS) geometries.
To date, the Raman method has been
used predominantly in the backscattering
collection mode (see Figure 1, left)
capable of probing only shallow depths
of turbid media where the medium still
appears semitransparent.1 For example,
in living tissue, depths of up to several
hundred micrometers are accessible.
However, the deeper layers remain inaccessible
due to the surface Raman or
interfering fluorescence signals masking
much weaker Raman spectra of the
deeper components. The penetration
depth is somewhat higher with pharmaceutical
products such as tablets,
typically 1 mm or so, but this is still
insufficient for probing the overall bulk
content of the tablet or its composition
through thicker packaging.
A substantial extension of the
Raman technique penetration
depth was recently accomplished
using the diffuse component of light
in a manner similar to NIR absorption
tomography. The developed
methods fall into two categories:
temporal and spatial. The temporal approach relies on the impulsive
excitation of sample and fast, time-resolved
detection of the Raman signal.2–4 The spatial method (SORS,5
see Figure 1, right), which is instrumentally
much simpler since it does
not require a pulsed laser and time-gated
detection, is based on the collection
of Raman signal from spatially
offset regions away from the
point of illumination on the sample
surface. Since its development, the
technique has been used in numerous
applications, including the
noninvasive Raman spectroscopy
of bones,6,7 and in pharmaceutical8
and security applications.9
In such measurements, the laterally
offset spectra contain different relative
contributions from sample layers located
at different depths. This difference is a
result of the wider spread of the deep-layer
Raman photons emerging from
the surface in comparison with surface
Raman contributions. This difference is
brought about by the lateral diffusion of
photons; photons penetrating to deeper
layers are influenced to a greater degree
than photons interacting with the surface
layers alone. It is also beneficial that
the SORS approach is not only capable
of effectively suppressing Raman signals
from the surface layers, but also the
interfering fluorescence originating from
surface layers. This is in contrast to conventional
Raman spectroscopy, where
a Raman spectrum is collected directly
from the laser deposition area. Consequently,
its signal is dominated by the
intense surface layer components.
SORS technique application areas
encompass pharmaceutical, chemical,
and food analysis applications; security
screening for harmful or illegal
substances; and disease recognition of
subsurface tissue components such as
bones and breast cancer tissue. Below
are two examples from the pharmaceutical
and security screening areas.
Noninvasive authentication
of pharmaceutical drugs
The first example is the authentication
of drugs in the supply chain.
The need for such authentication is driven by the increasing infiltration of
the market by counterfeit drugs.8,10,11
For example, the occurrence of fake
antimalarial drugs presents a serious
threat to health in eastern Asia. This
problem is exacerbated by the rapid
spread of Internet vendors, which significantly
increases the complexity of
the supply chain.
The existing monitoring tools
include NIR absorption spectroscopy,
which has limited chemical specificity
restricting its effectiveness, and
Raman spectroscopy, which is used
in its conventional backscattering
collection mode, limiting its use
to surface layers of turbid samples.
Although the Raman technique is
effective with many pharmaceutical
blister packs, darkly colored tablet
coatings or capsules or thick packaging
yield excessively intense Raman
and/or fluorescence signals. This
reduces the technique’s sensitivity
and, in some cases, prevents its
deployment entirely.
Recently, it was demonstrated8 that
SORS can substantially enhance the
detection sensitivity and even permit
the probing of pharmaceutical products
through opaque plastic bottles,
which often present a major insurmountable
challenge to the existing
Raman approach. In this study, SORS
outperformed, in all investigated
cases, the conventional Raman technique
by effectively suppressing the
contributions from the blister packs
and capsule shells, and permitted
unobstructed probing of the content
of white plastic bottles.
Figure 2 - Noninvasive Raman spectra of paracetamol tablets measured through a white, diffusely scattering 1.7-mm-thick plastic container in drug authentication. Conventional Raman and SORS raw data are shown together with the tablet’s reference Raman spectrum. The conventional Raman spectrum is overwhelmed with the Raman signal of the container, masking the Raman signal of paracetamol held within.
Figure 2 shows the performance of
SORS and conventional backscattering
Raman geometry in probing noninvasively
white plastic bottles containing
pharmaceutical tablets. In this
application, the conventional Raman
approach was ineffective due to the
presence of very intense interfering
Raman signals from the container.
Even under such challenging conditions,
the SORS approach, after the
scaled subtraction of two SORS spectra
measured at different spatial offsets
(0 and 3 mm), yielded a clean Raman
spectrum of the pharmaceutical tablets
within the bottle. The experiments were performed at 830 nm, with an
acquisition time of 10 sec and a laser
power of 50 mW.
Security screening
The recent heightened terrorist threat
underlines the importance of the
availability of robust security screening
techniques with high chemical specificity.
Examples include the screening
of passengers and their luggage at airports
or the screening of envelopes at
mail sorting centers.
Figure 3 - Noninvasive measurement of powders in envelopes in security screening applications. The measurement was performed using an envelope containing sugar representing a hidden illegal or harmful substance. a) Raw conventional and SORS spectra, b) background-subtracted SORS spectrum (eliminating fluorescence contribution) and sugar reference spectrum. The conventional Raman spectrum is dominated with interfering fluorescence background originating from the envelope material masking the Raman signal of the substance within.
The use of SORS was reported9 for
probing powders held in envelopes,
and the results were compared with
those attained using conventional
backscattering Raman spectroscopy.
In these measurements, a standard
brown envelope containing white
sugar, representing a harmful substance,
was used. Comparative conventional
backscattering Raman
measurement yielded a massive fluorescence
background originating from
the envelope material, entirely masking
the Raman signal of the substance
contained within (see Figure 3). In
contrast, the SORS spectrum clearly
shows the Raman components of sugar
held within the envelope. The experiments
were performed using 830 nm,
and the acquisition time was 10 sec
with a laser power of 55 mW.
Conclusion
The SORS method has stimulated the
development of numerous new analytical
methods for probing tissue and powders
at previously inaccessible depths.
Potential application areas include
disease diagnosis, such as osteoporosis,
brittle bone disease, and breast cancer;
quality control and authentication of
pharmaceutical products; and security
screening applications.
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The authors are with the Central Laser Facility,
Science and Technology Facilities Council,
Rutherford Appleton Laboratory, Didcot,
Oxfordshire OX11 0QX, U.K.; tel.:
+44 1235 445377; fax: +44 1235 445693;
e-mail: [email protected].