A femtosecond stimulated Raman loss (fSRL) microscope for highly sensitive bond-selective imaging

Abstract

ABSTRACT We demonstrate nonlinear vibrational imaging of isolated Raman bands by detecting femtosecond pulse stimulated Raman loss. Femtosecond pulse excitation produces a stimulated Raman loss signal that is 12 times larger than what picosecond pulse excitation produces. The strong signal allowe d real-time, bond-selective im aging of deuterated palmitic acid-d 31 inside live cells, and 3D sectioning of fat storage in live C. elegans. With the high peak power provided by femtosecond pulses, this system is highly compatible with other nonlinear optical modalities such as two-photon excited fluorescence. With most of the excitation power contributed by the Stokes beam in the 1.0 - 1.2 µm wavelength range, photodamage of biological samples was not observed. Keywords: stimulated Raman scattering (SRS), nonlinear microscopy, multimodal imaging, femtosecond laser, coherent Raman scattering, lipid INTRODUCTION Label-free chemically selective imaging based on inherent molecular vibrations is an attractive alternative to fluorescence imaging. Vibrational microscopes based on spontaneous Raman scattering and infrared (IR) absorption have been routinely used for chemical imaging of unstained samples. Nevertheless, the application of IR microscopy to live cell imaging has been hindered by the poor spatial resolution and IR absorption of water. Raman microscopy avoids these problems because water is a weak Raman scatterer. Moreover, a higher spatial resolution can be achieved by visible light excitation and confocal detection. However, Raman scattering has an extremely small cross section (~ 10