Raman micro-spectroscopy in optically stacked
micro-objects
P.R.T. Jess1, V. Garcés-Chávez1*, A.C. Riches2, S. Herrington2 and K. Dholakia1
1SUPA,
School of Physics and Astronomy, University of St. Andrews, North Haugh, St. Andrews, Fife, KY16 9SS,
United Kingdom
2Bute Medical School, Bute Medical Buildings, University of St Andrews, St Andrews, Fife, KY16 9TS,
United Kingdom
*gv3@st-and.ac.uk
Abstract: Evolution of Raman signal with an axial increment of mass of
the substance of interest inside a specific Raman excitation volume is
investigated. A vertical aligned of micro-objects (stack) of different size in
an inverted optical tweezers is performed by using a single laser beam
which also serves for the Raman excitation. Raman spectra were collected
for different number and size of stacked polystyrene microspheres. The
Raman signal to noise ratio rises with the number of micro-objects in the
stack. Furthermore, Raman spectra of a stack of live cells were obtained. A
significant enhancement of the characteristic Raman shift peaks was
observed when a stack up to three cells was achieved.
2004 Optical Society of America
OCIS codes: (000.0000) General.
References and links
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1. Introduction
In recent years Raman microspectroscopy has become popular and powerful technique
for content and structural examination of many different microscopic particles. In particular, it
is becoming especially important within the biological arena for applications from cancer
diagnosis [7] to examining the drug distribution in cellular structures [8]. One of the major
challenges of Raman spectroscopy is that the signal is very weak. The undesirable collected
light from the surrounding can completely obscure the Raman scattering. Enormous efforts
have been done for raising the ratio from signal to background noise. The Raman signal can
be enhanced by increasing the time of acquisition (~minutes). However in the case of live
cells, long acquisition time can cause damage. During all this time, the sample should be
immobilised. Frequently, the sample is adhered to a glass surface (coverslip), which gives an
extra strong scattered light (noise) component. Be able to hold a particle of interest away from
any surface without damage it is desired. Optical tweezers (OT) technique [1] is very well
known for being able to grab and separate a single microparticle of interest from the rest.
Optically trapped microparticles with power lower than 1mW can be obtained. Recently, C.
Xie et al [6] have combined optical tweezers and Raman spectroscopy (Raman Micro
Spectroscopy). The combination of both techniques lend themselves easily, as the highly
focussed trapping beam provides the necessary power for Raman excitation and trapping of a
single microparticle. The backscattered Raman signal can then be conveniently collected by
the same high numerical aperture (NA) objective used for trapping. The trapping objective is
also used to collect any backscattered Raman light for examination. The combination of these
techniques allows long time observations without need for the sample to be adhered to the
coverslip. However, for small particles, the Raman signal is very weak and it's also affected
by light scattered from the surrounding medium. More recently, a collective confocal
arrangement has been proposed. This confocal system is frequently used for simulate a thin
film situation where just a part of the excited mass volume is collected. In this way, any
backscattered light from the surroundings is suppressed. A confocal system can be
complicated and difficult to align as well as causing a loss in valuable Raman intensity.
A laser Raman beam focused at the diffraction limit (1 or 2m), by a high NA objective
describes a conical volume of excitation. In order to obtain a characteristic finger print of any
desired microparticle, the excitation volume must be optimised and the collection of the
Raman scattering must be highly efficient. The intensity distribution produced by the laser
focus determines the volume of light which can be used for Raman excitation. The collection
efficiency of a Raman microspectroscopy system depends on the ratio between the amounts of
mass present in the collection volume compared with the amount of unoccupied space in that
collection volume from where only noise is collected [5]. Although it has been demonstrated
that a microparticle trapped at or near the excitation beam focus optimises the excitation and
collection of Raman signal, for a microparticle trapped near the focal plane, with a diameter
bigger than few microns, part of this mass lies out with the region excited by the laser beam.
In this case the excitation mass is not optimised.
Here we report a very simple technique which increases significantly the ratio of Raman
signal to noise without imposing any confocal system. We have made use of optical tweezing
techniques to create a stack of several microparticles which increase the number of
microparticles in the volume of excitation. We made use of a standard inverted optical
tweezers. Stacking of microparticles is achieved by focusing the beam at the top of a sample
chamber. The trapping of several microparticles is obtained by moving the sample chamber
perpendicular to the incident beam [2]. Using just 10mW of laser power, a stack of up to five
microparticles has been achieved. We are also able to move the whole stack by moving the
laser beam. We have studied the evolution of the Raman signal as a function of mass stacked
in the excitation volume. A clear enhancement of the signal is obtained as a function of the
number of microparticles in the stack. However, our results show that this increment cans not
always being taken as a linear dependence of the mass in the volume.
Furthermore, a stack of RBC was also created. We observed how the cells align along the
beam propagation by rotating 90 degrees [10]. In this way the stack filled up the axial
direction of the excitation volume. The shape of a RBC is like a disc of 7m of diameter and a
thickness of 3m. Due to the alignment of the cells, the whole cell can be contained into the
excitation volume. Our results show that by stacking up to three cells, the Raman signal is
enhanced significantly in comparison to a single tweezed cell. No further computational
analysis of the taken data was necessary. This can be useful for increasing the very weak
signal obtained from traditionally challenging samples. In order to test further the usefulness
of this technique the same experiment was performed on yeast cells. Stacks of up to three cells
were formed and a large increase in signal to noise ratio was also observed.
2. Experiment
A block diagram of our kit built Raman tweezers can be seen in figure 1. The trapping laser is
an 80mW@785nm circularised Laser Diode (Sanyo DL-7140-201s) with a stable temperature
controller. The undergoes an initial expansion and is then introduced to the system via a
holographic notch filter, HNF (Tydex notch-4) into what is recognisable as an inverted optical
tweezers utilising a x100 1.25NA (Nikon) oil immersion objective. The backscattered Raman
light is collected by the objective and passed through the HNF to the spectrograph (Triax 550
Jobin Yvon) that transfers the light onto a CCD camera (Symphony OE STE Jobin Yvon).
Fig. 1. Block diagram of the experimental setup. HNF: Holographic Notch Filter; BEF: Band edge filter.
The sample chamber used in the experiments was formed using an 80m deep vinyl
spacer between two standard thickness No.1 coverslips. As a sample, we used dilute solutions
of polymer microspheres of various sizes (5 to 25m) in distilled water. Stacking in inverted
tweezers is easily achieved by focussing the beam at the top of the sample chamber where the
first particle is trapped; subsequent particles are moved into the stack by moving the beam
over them whereupon they are guided toward the focus via the gradient force and are stored
beneath the already trapped particles. Using different sized particles and stacks of varying
numbers of spheres we were able to form structures of various depths. The number of spheres
in the stack depends on the power of the laser beam and the depth of the chamber. A stack of
up to five microspheres was achieved by using 20mW of laser beam power. By slightly
moving the position of the laser, the whole stack of microspheres could be demonstrating the
ability of the Raman microspectroscopy system in the stacking of microspheres.
The maximum Raman signal was recorded from each polystyrene stack formed. The
1
maximum signal was monitored using the 1000cm Raman band, being the most suitable as
it is the most prominent in the spectra, which can be seen in figure 2. The Raman signal was
integrated for two seconds and found to be highly repeatable.
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Fig. 2. Raman spectra of a 5m polymer microsphere.
The evolution of the Raman signal was investigated in the following situations. Initially the
Raman signal was obtained as a function of the number of trapped microspheres in a stack
(increasing the axial amount of mass). Secondly, the size of a single trapped sphere was
changed (axial and lateral increment of mass). Comparison of Raman signal intensity of two
10m and four 5m polystyrene spheres is analysed. Finally, we have studied the influence of
the Raman signal to changes the excitation intensity. In this case, the power of the laser was
varied from 10mW to 30mW measured at the back aperture of the objective. Moreover, we
report the first experimental result of the evolution of the Raman signal for a single, two and
three live cells held in a stack. The most prominent peaks were increased and peaks not
normally visible at short integration times became visible when three cells were stacked.
3. Results and discussion
3.1 Influence of the Raman signal with the number of trapped spheres in a stack (increase of
the mass of substance along the axial direction)
The peak intensity of the 1000cm-1 Raman signal of polymer was recorded for stacks of
m spheres. The optical power, reaching the objective back aperture,
was kept constant at 30mW. The resultant plots of signal intensity with number of stacked
spheres can be seen in figure 3(a-b).
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Intensity vs the number of
stacked 10 micron spheres
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Intensity vs the number of
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Fig. 3 (a-b) Intensity of the 1000cm
Raman band versus the number of spheres trapped in a stack.
A clear increase in intensity with number of particles in a stack is observed. Interestingly the
intensity value for a single 10m sphere is very similar than two 5m spheres. Also, we can
see the same behaviour in the case of two 10m spheres related with the intensity obtained for
four 5m spheres. These results suggest that the axial increase of mass in the excitation
volume gives a major contribution to the Raman signal to noise ratio. This also suggests that
increasing the mass axially provides a much greater contribution to collected signal than
increases in mass in the lateral direction. In order to test this issue further, a plot of the Raman
intensity versus depth of the sample is shown in the figure 4. The values obtained for single
20 and 2 m particles are included.
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Fig. 4 Intensity of the 1000cm
Raman band versus the sample depth.
These results show that for a small depth values, a linear dependence is observed. However as
the depth increases the Raman signal decays exponentially.
Excitation volume and collection efficiency of the Raman signal play an important role in
the data shown before. Close to the beam waist the microscope is able to collect the Raman
signal more efficiently, however, for the high numerical aperture objective this falls off
rapidly [12]. Interestingly, our results show that in the case of micro-objects held close to the
focus of a laser beam, increasing the number of microparticles stacked in the excited volume
increases also the intensity of the Raman signal.
Figure 5 shows how the excitation volume, defined by focusing a laser beam with x100
objective, can be filled up by stacking several micro-objects. In this figure, the laser beam is
sent in an upward direction. A single 5, 10, 20 and 25m sphere was placed in the excitation
volume. We can see clearly how four 5m spheres do not fill the same amount of volume than
one single 20m sphere. However a similar intensity Raman signal was obtained.
Fig. 5. Filling up the excitation volume with 1 (20m), 2 (10m) and 4 (5m) polysterene spheres in a stack.
Raman excitation volume by using a x100@1.25NA objective. Upward laser beam propagation.
So with increasing depths, such as those that may be encountered in in-vivo tissue
measurements, our results suggest that we may not automatically assume that the Raman
signal is directly proportional to the amount of sample present.
3.2 Influence of the Raman signal with size of a single trapped sphere.
We have also measured the Raman signal generated from a single microsphere from 5 to
25m of diameter (see fig. 6).
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A Chart display intensity of the 1000cm-1 Raman Peak for
Polymer vs Sample depth
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Fig. 6. Chart displaying Raman intensity vs. sample depth for single micro spheres.
In this case the mass in the excitation volume has been incremented in the axial and lateral
direction. This graph shows that as the size of the microspheres increase we observe decay in
collected Raman intensity. A linear profile (until approximately 10m) and a subsequent
exponential decay of the signal are observed. The ability of the system to collect the Raman
signal depends on the position on the trapped sphere related to the focus position of the laser
beam. Our results confirm that the collection efficiency is higher at the focus beam position.
These data are in agreement with the results shown by T.E. Bridges et al [5].
3.3. Influence of the Raman signal with changes in the excitation intensity.
Finally we observed how the power reaching the back aperture of the objective affected the
intensity profile of the collected Raman scatter. In this experiment up to five 5m spheres
were stacked.
Intensity/ AU
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A chart to show the intensity of the 1000cm-1 peak in polymer
vs no. of 5 micron spheres stacked for different excitation powers
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Fig. 7 A chart showing how the Raman profile varies with power reaching the objective back aperture.
This data shows an increase of Raman intensity collected as the laser power in increased.
However we can see a slightly dip when a stack of three spheres was performed. When three
particles are stacked, the beam was focusing down into the stack in order to keep the spheres
aligned. As a result, due to geometrical considerations, we have less material occupying the
beam hence we see a reduced signal. When we reduce the optical power to 18mW we see a
smooth curve, indicating the optimal power for the stable stacking of polymer particles. At
10mW we see a different curve again, this because 10mW is not quite enough power to stably
stack large numbers of particles.
3.4 Stack of live cells (red blood and yeast cells)
One of the major prospects for Raman spectroscopy is obtaining a bio chemical finger print of
a single cell or perhaps even more challenging, Raman signals from subcellular structures
such as the membrane or nucleus. Our data shows here that by increasing the number of
homogeneous cells (as in the case of RBCs) in the excitation volume the peaks of the Raman
signal can be enhanced. This may reduce the acquisition time and further analysis of the data.
In order to do that, we created a stack of RBCs which were suspended in phosphate buffered
saline (PBS) at room temperature. It is well understood that the human RBCs present a
flattened biconcave disk shape. The optical forces exerted in an optical tweezers align the
RBCs to the electromagnetic field [10]. We have stacked up to three RBCs by using just
10mW of laser power. Figure 8 shows a cartoon of how the stack of cells was built up in an
inverted optical tweezers geometry (upward laser beam).
Fig. 8. Stacking of red blood cells in an inverted optical tweezers geometry.
The Raman signal was collected for one (figure 9(a)), two (figure 9(b)) and three (figure 9(c))
RBC tweezed in a stack. The figure 9 shows the evolution of the spectrum obtained by using a
60 second integration time and a 1200 l/mm grating. By making used of the software for
controlling the centre position of the grating, an extended spectrum from 550 to 1750 cm -1 was
obtained. Three different positions were used here in order to obtained The column 1 (left)
shows the spectra obtained when the centre position was placed at 800 cm-1, column 2 (centre)
at 1180 cm-1 and column 3 (right) at 1560 cm-1.
Fig. 9. Raman signal obtained by (a) a single, (b) a stack of two and (c) a stack of three red blood cells.
When a stack of up to three red blood cells was examined, we clearly observed stronger
Raman peaks with a much reduced noise background. The evolution of the some of the
Raman peaks (664, 774, 995, 1115, 1161, 1215, 1333, 1361, 1433 and 1609 cm -1
wavenumbers) reported previously [13] as a function of the number of cells in a stack were
analysed (see figure 10). These intensity values were obtained by substraction of the
background noise from the spectra. Notably these values enhanced when the stacking of
several cells was performed. This is a clear demonstration of the improvement of the Raman
signal by performing the spectroscopy on optically stacked cells.
Fig. 10. Intensity of the Raman signal versus number of RBCs in a stack.
In order to test further the usefulness of this technique we applied the same method
to saccharomyces cerevisiae yeast cells. These cells were selected to show the how the
technique can also be applied to non uniform biological samples when area specific
information is not required. Stacks of up to three yeast cells were also formed by using the
Raman laser (785nm) described in the fig. 1. However, sometimes the cells got stuck on the
bottom of the coverslip and is not easy to stack them. In order to trap these sticky cells, we
used a laser beam with much higher power. A 1070nm@5W Ytterbium fibre laser was
introduced to the system by using a band edge filter which strongly reflect 1070nm
wavelength. This laser provides a second optional higher power tweezers to aid the formation
of optical stacks when working with the biological samples [11]. This laser was used to form
the stacks but was the turned off as the 30mW@785nm excitation power used to excite the
Raman signal in the sample was found to be sufficient to maintain the stack of cells.
Otherwise, the light is transmitted. In this case the 300 lines/mm grating was used to examine
the sample in conjunction with a reduced integration time of only 30 seconds. This slight
change in technique represents an ongoing improvement in our experimental arrangement and
methods. Although the 300 lines/mm grating does represent a loss in resolution we find we
are still able to resolve the main peaks easily and also allows us to significantly reduce our
integration times. The results can be seen below in figure 11.
Fig. 11. Raman signal obtained by (a) a single, (b) a stack of two and (c) a stack of three yeast cells.
In this case an increment of the noise is also observed. However the graphs show a
significant enhancement of the ratio signal to noise. For instance, the Raman peaks at 891 and
1122 cm-1 shown in the bottom spectrum (Fig. 11 (c)) were impossible to resolve in the top
spectrum (Fig. 11 (a)). One of the advantages shows that it is coming clear after seeing the
spectrum in the row (c) is the increment of the ratio signal to noise. For instance, the peak at
1490 cm-1 is remarkable magnifying in relation of the background noise. These spectra were
obtained by using the same exposition time. However, it is clear that in the case of having
three cells, the exposition time can be significantly decreased.
A plot of the Raman signal versus the number of yeast cells in a stack is shown in the
figure 12. Raman peaks show in the figure are: 754, 891, 1122, 1339, 1490, 1696, 1866 cm -1
respectively. The Raman peak shifts obtained were similar than the peaks recently reported
somewhere else [14].
Fig. 12. Intensity of Raman shifts versus the number of cells hold in a stack.
4. Conclusion
We have demonstrated that an increase in mass of substance of interest in the excitation
volume enhances the collected Raman peaks. A stack of micro-objects diminishes the
background noise collected from the area surrounding the sample. The results have shown a
stronger Raman signal when the excitation volume was filled up axially. No confocal system
permitted us to increase the collection efficiency of the Raman shift peaks in the case of a
stacking of micro-objects. Our results show that the maximum contribution to the Raman
signal is obtained by an axial increase in the mass of interest. Finally the results gave us
insight into the ease and dynamics of stacking polymer spheres showing an optimal optical
power of 18mW. We believe this technique represents a real alternative to the use of, by their
nature, inefficient confocal systems for an increase in signal to noise ratio in uniform samples
or for traditionally difficult samples when non area specific information is required.
The ability to trap and manipulate an ensemble of multiple microparticles in a unison or
independently is desirable for multiple applications in several fields of research. In particular,
optical creation and control of structures is an important issue for cell culture and nucleation.
Formation of 3D trapped microparticles structures assisted by optical tweezers can be useful
for the stacking of multiple selected micro-objects. Our stacking technique can be taken
forward by making use of novel laser beams or spatial light modulators. Multiple number of
stack of cells can be performed which may be a key for cell culturing and cell engineering.