Light sheet-excited spontaneous Raman imaging of a living fish by optical sectioning in a wide field Raman microscope

Abstract

Spontaneous Raman microscopy is a potentially useful technique for imaging living cells, tissue and small animals without any probe or dye labeling. We have developed a spontaneous Raman imaging system in wide-field view, which we term ‘light sheet-excited direct Raman spectroscopy’ (LSDRS). This system, which we reported previously, consists of a background-free electrically tunable Ti:Sapphire laser (BF-ETL), a cylindrical lens, a CCD camera, and a narrow bandpass filter. Here, we have adapted the LSDRS system for microscopy systems, such as single-plane illumination microscopy (SPIM) for biomedical applications, and demonstrated spontaneous Raman imaging of a living fish. The results suggest that our Raman microscopy system enables investigation of the differentiation process and mechanism of iridocytes during development. This is the first report in which Raman imaging of a living animal was successfully demonstrated by spontaneous Raman scattering signals, but not nonlinear Raman effects such as CARS and SRS.