Fig. 1
Principle and design of CRS microscopy. Modified from Freudiger et al. [33]. CRS microscopy mainly includes two sub-types, coherent anti-Stokes Raman scattering (CARS) microscopy and stimulated Raman scattering (SRS) microscopy, both of which can be performed by setting the wavelength of the pump beam in a single setup. a The principle of SRS microscopy. Two input beams (Stokes and pump) are focused on the sample; when the difference in energy between the two beams (Ω) matches that of a specific chemical bond in the sample, then an additional signal is produced. Input and output spectra of SRS and CARS is shown. SRS leads to an intensity increase in the Stokes beam (SRG) and an intensity decrease in the pump beam (SRL). Also shown (not to scale) is the CARS signal generated at the anti-Stokes frequency ωAS when the energy difference between the pump beam photon and the Stokes beam photon matches the vibrational frequency (Ωvib) of a specific chemical bond. b Agreement of the SRL spectrum (red circles) with the spontaneous Raman spectrum (black line) of the Raman peak (1595 cm−1) of 10 mM retinol in ethanol. The distorted CARS spectrum (blue squares) exhibits a typical peak shift, a dispersive shape, and non-resonant background. c Plant cell wall imaging of the chemical composition of stem and root tissues. A CRS microscope with forward and epi detection is illustrated. The Stokes beam is modulated by an electro-optic modulator. The transmitted or reflected pump beam is filtered and detected by a large-area photodiode. The SRL is measured by a lock-in amplifier to provide a pixel of the image. The CARS signal is detected by the NDD. d Schematic of the process of CRS imaging of plant cell wall composition