Femtosecond Stimulated Raman microscopy: home-built realization and a case study of biological imaging

Abstract

The progresses of femtosecond laser physics have had a significant impact on research and development of nonlinear microscopy techniques, such as Stimulated Raman scattering microscopy. The main advantages of this technique, based on vibrational spectroscopy, is its abilities to perform label-free imaging with high sensitivity, high spatial and spectral resolution, 3D sectioning capability and fast time of image acquisition (typically a few seconds). The main aim of this paper is to demonstrates the home-built realization of an imaging system based on Stimulated Raman Scattering (SRS) powered by femtosecond laser sources, using a National Instrument data acquisition (NI-DAQ) card. The SRS microscope is obtained by the integration of an SRS experimental set-up with a commercial laser scanning microscope. The successful implementation of the imaging system is demonstrated reporting label free images of polystyrene beads with diameter of 3 microns and evaluating their signal to noise ratio. Finally, the reliability of our system in a biological environment is tested. Taking advantage of SRS microscopy, subcellular localizations of lipid droplets inside adipocyte cells are demonstrated.