Optimized signal-to-noise ratio with shot noise limited detection in Stimulated Raman Scattering microscopy

We describe our set-up for Stimulated Raman Scattering (SRS) microscopy with shot noise limited detection for a broad window of biologically relevant laser powers. This set-up is used to demonstrate that the highest signal-to-noise ratio (SNR) in SRS with shot noise limited detection is achieved with a time-averaged laser power ratio of 1:2 of the unmodulated and modulated beam. In SRS, two different coloured laser beams are incident on a sample. If the energy difference between them matches a molecular vibration of a molecule, energy can be transferred from one beam to the other. By applying amplitude modulation to one of the beams, the modulation transfer to the other beam can be measured. The efficiency of this process is a direct measure for the number of molecules of interest in the focal volume. Combined with laser scanning microscopy, this technique allows for fast and sensitive imaging with sub-micrometre resolution. Recent technological advances have resulted in an improvement of the sensitivity of SRS applications, but few show shot noise limited detection.The dominant noise source in this SRS microscope is the shot noise of the unmodulated, detected beam. Under the assumption that photodamage is linear with the total laser power, the optimal SNR shifts away from equal beam powers, where the most signal is generated, to a 1:2 power ratio. Under these conditions the SNR is maximized and the total laser power that could induce photodamage is minimized. Compared to using a 1:1 laser power ratio, we show improved image quality and a signal-to-noise ratio improvement of 8 % in polystyrene beads and C. Elegans worms. Including a non-linear damage mechanism in the analysis, we find that the optimal power ratio converges to a 1:1 ratio with increasing order of the non-linear damage mechanism