Stimulated Raman Scattering; Life, in the fast lane

Ali Rae and Debdulal Roy

National Physical Laboratory, Teddington TW11 0LW, UK

Introduction
Raman microscopy is a powerful tool for the chemical and structural analysis of biological systems. However, spectral acquisition times in spontaneous Raman scattering are long and spectral quality su ers greatly in the presence of strong uorescent background. Stimulated Raman scattering, a coherent, non-linear, technique overcomes these limitations and allows for robust, label free, high speed imaging of biological samples with vibrational contrast.

Theory
Virtual State

Experimental
GLP APE Picoemerald 1064 nm and OPO Reference Clock PD Lock in ampli er BBM DAC + PC p s OBJ SS OBJ SPF BBM

p s
V1 V0
SRG

GLP Glan-Laser polariser, BBM Broadband mirror, OBJ Objective, SS Sample Stage, SPF Shortpass lter, PD Photodiode.

SRL

SRL 80 MHz Modulated Energy Transfer

Figure 2. Schematic diagram of the experimental set up and modulated energy transfer detection scheme for SRS microscopy.

s
20 MHz

Figure 1. Schematic diagram of the stimulated Raman scattering process. Coherent excitation at the beat frequency between the pump (p) and stokes (s) pulses results in a transfer of energy between the two elds.

Stimulated Raman scattering microscopy utilises two, picosecond pulsed elds, the pump and stokes elds, to coherently excite the sample. The frequency di erence between these elds, p s, is tuned on resonance with a vibrational resonance of the species of interest within the sample. When these two spatially and temporally overlapped elds come into resonance with this species, the molecule is excited from the ground (v0) to the rst vibration state (v1) via a virtual state (---). The resulting annihilation of a pump photon and the creation of a stokes photon alters the incident eld intensities. These intensity changes can be probed as a contrast mechanism for microscopy1. The stimulated Raman scattering process is illustrated in Figure 1.

SRS microscopy utilises a detection scheme based on the high frequency modulated transfer of energy between pump and stokes elds. At biologically compatible laser powers, I/I is on the order of 104 and is buried within the laser noise2. By modulating one of the input elds at 20 MHz, a lock-in ampli er can be used to achieve an almost shot-noise limited sensitivity in the detection of the SRS signal. Pump and stokes beams are provided by an APE Picoemerald laser, with an OPO and SHG providing tunings in the range of 375 nm to 2100 nm. These beams pass into a spontaneous Raman microscope (Witec CRM 200) and the pump beam is detected by a fast photodiode (Thorlabs DET10A). Demodulation of the SRS signal is carried out by a RF lock-in ampli er (Stanford Research Systems SR844).

Results
To show the potential of SRS microscopy for the analysis of pharmaceuticals and narcotics, SRS imaging of trace cocaine concentrations on the surface of 3 m polystyrene beads has been demonstrated. Figure 3a shows an SRS map of the spiked beads, tuned on resonance with the 1720 cm1 mode of cocaine. By tuning the di erence frequency, p s, across the CH region, it is possible to reconstruct the spontaneous Raman spectra of the sample. This demonstrates SRS provides spectroscopic vibrational information identical to that obtained via spontaneous Raman spectroscopy.

a)

b)
i

Conclusions
Stimulated Raman scattering microscopy is a powerful tool for the high speed, label free, in vivo imaging of biological systems with contrast based on vibrational spectroscopy. This work demonstrates the ability of SRS to detect trace concentrations of narcotics and lays the foundations for the in vivo imaging of pharmaceuticals and narcotics in biological samples normally inaccessible by spontaneous Raman microscopy.

ii

Figure 3. a) SRL image of cocaine coated polystyrene beads spread on a glass coverslip and reconstruction of the spontaneous Raman spectrum via SRS. b) Polystyrene (i) and cocaine (ii) molecules.

References
1. P. Nandakumar, A. Kovalev, A. Volkmer, New, J. Phys. 11, 033026 (2009). 2. T. Ye, D. Fu, W.S. Warren, Photochem. Photobiol. 85, 631-645 (2009).

www.npl.co.uk/nanoanalysis http://projects.npl.co.uk/NEW02-Raman/

Acknowledgements
Ali Rae and Debdulal Roy gratefully acknowledge funding from the European Metrology Research Programme

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