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SO, 2014. 10989/0814
www.npl.co.uk
Traceable Measurements with Stimulated Raman Scattering Microscopy
Ali Rae and Debdulal Roy
National Physical Laboratory, Hampton Road, Teddington, Middlesex, TW11 0LW, UK
ali.rae@npl.co.uk
Acknowledgements
The authors gratefully acknowledge NEW02 Raman project of the
European Metrology Research Programme (EMRP) for funding this work.
References
1. Freudiger, C.W., et al., Label-Free Biomedical Imaging with High
Sensitivity by Stimulated Raman Scattering Microscopy. Science, 2008.
322(5909): p. 1857-1861.
The Need
Stimulated Raman scattering (SRS) microscopy is a powerful
tool for the label free analysis of biological samples with sub-
micron resolution and linear concentration dependence [1]. In
recent years it has become clear that SRS has huge potential for
application across a range of industries from diagnostic medical
imaging to agriculture. However, for SRS to be widely adopted
within industry, particularly for regulatory purposes, accurate
and traceable methods for quantifcation of the SRS signal are
essential. Traceable quantifcation of lipids within cell and tissue
systems can be an invaluable probe of disease. However, it is
not only biomedical imaging that would beneft, for example,
traceable and non-destructive quantifcation of pesticides in
plant tissue could provide a much needed screening tool for
the agricultural industry. Clearly, measurement traceability is an
important next step forwards in the development of coherent
Raman imaging techniques.
Focal Volume Characterisation
Before accurate quantitative measurements can be made with
any confocal microscopy system, the point spread function of
the system must be evaluated. Knowledge of the point spread
function of an imaging system allows determination of the focal
volume of that system. Knowledge of the volume probed by
the system allows the direct comparison between the signal
magnitude and the number of molecules present. To this
end, we have developed a reference sample for the traceable
quantifcation of the point spread function and therefore the
focal volume of both SRS and CARS microscopes.
Traceable Quantitative Measurements
With the focal volume
measurements made
using the reference
sample detailed in Focal
Volume Characterisation,
is it possible to make
traceable concentration
measurements with SRS.
To demonstrate this, a
calibration curve was
prepared from 3 traceably
prepared aqueous cafeine
solutions. Using this calibration data, the concentration
of a 4th unknown cafeine solution was determined. The
solution concentration determined via SRS was validated
using UV/Vis spectroscopy.
Conclusion
In this work we have demonstrated traceable quantifcation
of the focal volume of an SRS microscope and presented the
traceable determination of the concentration in a simple
aqueous system. This work paves the way for traceable
quantifcation of substances in more complex media, such as
cells and tissues. The addition of traceability to SRS microscopy
measurements will be invaluable in the wide scale adoption of
SRS microscopy as an industrial tool. Figure 1 Schematic diagram of the SRS microscope.
Figure 2 Schematic diagram of the proposed reference sample for the traceable quantifcation of resolution in SRS and
CARS microscopes. The reference sample consists of 100 nm polystyrene beads dispersed on a 100 nm polyacrylonitrile thin
flm. Inset spectra showthe individual Raman spectra of a) the polystyrene and b) the polyacrylonitrile thin flm. The 1600
cm
-1
of polystyrene and the 2250 cm
-1
mode of polyacrylonitrile are used for SRS measurements.
a) b)
Figure 3 a) Lateral and b) axial resolution of our SRS microscope as determined by the proposed reference sample.
C) 3Drepresentation of the point spread function/focal volume of the microscope.
Figure 4 Traceable cafeine solution concentrations
measured by SRS microscopy.
a) b) c)