Abstract

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 photo damage 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.

Highlights

  • In recent years, coherent Raman scattering microscopy has found many applications in biomedical imaging

  • If the energy difference between them matches a molecular vibration of a molecule, energy can be transferred from one beam to the other

  • Because signal levels in Stimulated Raman Scattering (SRS) microscopy are measured as a small change on a high background intensity, care must be taken to optimize the imaging parameters to achieve the best signal-to-noise ratio (SNR) for the applied laser power

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Summary

INTRODUCTION

Coherent Raman scattering microscopy has found many applications in biomedical imaging. Because signal levels in SRS microscopy are measured as a small change on a high background intensity, care must be taken to optimize the imaging parameters to achieve the best signal-to-noise ratio (SNR) for the applied laser power. (1) and (2) and evaluating the derivative as a function of Ipump, it is found that the maximum signal is obtained when the time-averaged intensities of pump and Stokes beam are equal This is the optimal experimental choice when the noise is independent of the laser powers in non-shot noise limited implementations and for related coherent Raman methods where the signal is measured against a dark background, such as in Coherent AntiStokes Raman Scattering (CARS) and Raman Induced Kerr Effect Spectroscopy (RIKES). This 1:2 power ratio would result in a theoretical improvement of the SNR of 9 % compared to a 1:1 power

EXPERIMENTAL SET-UP
RESULTS AND DISCUSSION
CONCLUSIONS
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