Label-free chemical imaging in vivo: three-dimensional non-invasive microscopic observation of amphioxus notochord through stimulated Raman scattering (SRS)

Abstract

Notochord is a rod-shaped axial supporting structure unique only to chordates. In this study, we use cephalochordate amphioxus (Branchiostroma belcheri, a living basal chordate) and zebrafish (Danio rerio), a vertebrate, as model animals and employ stimulated Raman microscopy (SRS), a newly developed label-free technique, to investigate notochord structure and chemical composition in live animals. Coherent anti-Stokes Raman scattering (CARS) images have also been acquired for comparison. Both CARS and SRS images can construct the detailed three-dimensional structure of the notochord with resolution better than 1 μm. Label-free live imaging allows us to obtain the whole animal's intact internal morphology, which is difficult to obtain through other mechanical or optical sectioning methods. Intrinsically, chemical sensitive SRS images, with a simple data processing procedure, show that the amphioxus notochord is protein-rich. Our result agrees well with that drawn from the labeling and cryo-sectioning observation, verifying that the coherent Raman scattering techniques are useful to examine the structure and chemical composition of live animals without labels in a fast, simple and accurate way.