Center for Relativistic Laser Science

The stimulated Raman scattering (SRS) utilizing inherent chemical contrast of sample is applicable to the label-free imaging with enhanced Raman signal intensity and chemical selectivity. It has been widely employed to investigate biomolecules and its dynamics in living organisms without perturbation of exogenous labels¹. However, to monitor biomolecular dynamics covering whole fingerprint region in biological targets, conventional SRS microscopy has limitation since it targets a single vibration mode of specific chemical structure based on a two-pulse scheme of pump and Stokes pulses. There are various approaches to achieve a broad- or multi-spectral investigation with SRS microscopy by changing the pulse characterization as well as time delay between two pulses.

Here, we present a complementarily balanced real-time two-color SRS microscopy using two pairs (pump and Stokes) of chirped laser pulses. Both pulses are linearly chirped with glass rods (N-SF57) and changing time delay of two pulses enables to target different Raman shift within about 130 cm^-1 of spectral window². To achieve two-color SRS imaging, resonating with different vibration modes simultaneously without any additional nonlinear process, both pulse-pairs are tailored to 45° polarization and temporally (~10 ps) divided into orthogonal polarization to each other³. Since both polarization of pump and Stokes experience the same environmental condition at the sample position, a simple balancing procedure between SRS processes of two different vibrational modes can decrease background noise level without signal loss⁴. A mixture of melamine and polystyrene beads is investigated targeting on the NH and CH stretching mode at 2964 and 3055 cm^-1, respectively. Two distinctive Raman images are detected at the same time and works as off-resonance signals to each other. With this technique, we expect to explore cellular dynamics with simultaneously monitoring lipid and DNA compositions in bio-assays.

References

[1] C. Krafft, I. W. Schie, T. Meyer, M. Schmitt and J. Popp, “Developments in spontaneous and coherent Raman scattering microscopic imaging for biomedical appications”, Chem. Soc. Rev., 2016, 45, 1819.

[2] Dan Fu, Gary Holtom, Christian Freudiger, Xu Zhang, and Xiaoliang Sunney Xie, “Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers”, J. Phys. Chem. B 2013, 117, 4634-4640.

[3] Ruoyu He, Yongkui Xu, Lili Zhang, Shenglong Ma, Xu Wang, Dan Ye, and Minbiao Ji, “Dual-phase stimulated Raman scattering microscopy for real-time two-color imaging”, Optica 2017, vol. 4, No. 1, 44.

[4] Francesco Crisafi, Vikas Kumar, Tullio Scopigno, Marco Marangoni, Giulio Cerullo, and Dario Pollo, “In-line balanced detection stimulated Raman scattering microscopy”, Scientific Reoprts 2017, 7, 10745.