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Traceable Measurements with Stimulated Raman Scattering MicroscopyAli Rae and Debdulal Roy

National Physical Laboratory, Hampton Road, Teddington, Middlesex, TW11 0LW, UKali.rae@npl.co.uk

AcknowledgementsThe authors gratefully acknowledge NEW02 Raman project of the European Metrology Research Programme (EMRP) for funding this work.

References1. 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 NeedStimulated 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 quanti� cation of the SRS signal are essential. Traceable quanti� cation of lipids within cell and tissue systems can be an invaluable probe of disease. However, it is not only biomedical imaging that would bene� t, for example, traceable and non-destructive quanti� cation 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 CharacterisationBefore 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 quanti� cation of the point spread function and therefore the focal volume of both SRS and CARS microscopes.

Traceable Quantitative MeasurementsWith 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 ca� eine solutions. Using this calibration data, the concentration of a 4th unknown ca� eine solution was determined. The solution concentration determined via SRS was validated using UV/Vis spectroscopy.

Conclusion In this work we have demonstrated traceable quanti� cation 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 quanti� cation 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 quanti� cation of resolution in SRS and CARS microscopes. The reference sample consists of 100 nm polystyrene beads dispersed on a 100 nm polyacrylonitrile thin � lm. Inset spectra show the individual Raman spectra of a) the polystyrene and b) the polyacrylonitrile thin � lm. 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) 3D representation of the point spread function/focal volume of the microscope.

Figure 4 – Traceable ca� eine solution concentrations measured by SRS microscopy.

a) b) c)