Stimulated Raman scattering simulation for imaging optimization

Abstract

Two simulation programs of a stimulated Raman scattering microscopy (SRS) imaging system with lock-in amplifier (LIA) detection were developed. SRS is an imaging technique based on the vibrational Raman cross-section as the contrast mechanism and enables fast, label-free imaging. Most SRS implementations are based on LIA detection of a modulated signal. However, building and operating such SRS set-ups still poses a challenge when selecting the LIA parameter settings for optimized acquisition speed or image quality. Moreover, the type of sample, e.g. a sparse sample vs. a densely packed sample, the required resolution as well as the Raman cross-section and the laser powers affect the parameter choice.A simulation program was used to find these optimal parameters. The focal spot diameters of the individual lasers (pump and Stokes) were used to estimate the effective SRS signal focal spot and the (optical) spatial resolution. By calibrating the signal and noise propagation through an SRS system for a known molecule, we estimated the signal and noise input to the LIA. We used a low pass filter model to simulate the LIA behavior in order to find the optimal parameters (i.e. filter order and time constant).Optimization was done for either image quality (expressed as contrast to noise ratio) or acquisition time. The targeted object size was first determined as a measure for the required resolution. The simulation output consisted of the LIA parameters, pixel dwell time and contrast to noise ratio.In a second simulation we evaluated SRS imaging based on the same principles as the optimal setting simulation, i.e. the signals were propagated through an imaging system and LIA detection. The simulated images were compared to experimental SRS images of polystyrene beads.Finally, the same software was used to simulate multiplexed SRS imaging. In this study we modeled a six-channel frequency-encoded multiplexed SRS system demodulated with six LIA channels. We evaluated the inter-channel crosstalk as a function of chosen LIA parameters, which in multiplex SRS imaging also needs to be considered.These programs to optimize the contrast to noise ratio, acquisition speed, resolution and crosstalk will be useful for operating stimulated Raman scattering imaging setup, as well as for designing novel setups.

Multi-Parameter Analysis of Nanoplastics in Flow: Taking Advantage of High Sensitivity and Time Resolution Enabled by Stimulated Raman Scattering

“…Each SRS measurement was conducted over the whole injection time of 20 min with a 60.5 μs acquisition time. For the lock-in amplifier (LIA) settings, a time constant of 13 μs and a filter order of 8 were chosen to support this time resolution . For the detection in epi-mode, the laser power at the sample was set to 33 mW for the 1064 nm beam (Stokes) and 17 mW for the OPO output beam (pump), whereas for detection in transmission mode, the laser powers were set to 66 and 34 mW, respectively.…”

“…The LIA mixes the detected signal with a reference frequency used for the Stokes beam modulation and applies a low pass filter. The result is a DC signal proportional to the amplitude of the detected signal (pump beam) modulation, i.e., the SRS signal . Although SRS has been applied successfully for the analysis of microplastics on a filter, in a silicone tissue phantom, and in suspension, , the detection of plastic particles below 1 μm (NPLs) with SRS has not been demonstrated so far.…”

Practical considerations for quantitative and reproducible measurements with stimulated Raman scattering microscopy

“…More detailed information can be found in a publication by Zada et al , who describe the effects of various lock-in amplifier settings on SRS signal and image formation, and their freely available simulation programme for SRS imaging. 35…”

Noises investigations and image denoising in femtosecond stimulated Raman scattering microscopy

“…In the literature of SRS, great attention is given to technological and application advances, while the investigations of noise are often overlooked, although SNR is a crucial issue not only for biological imaging, but also for spectroscopic applications [25,26]. The hardware characterizations, usually, are carried out to assess the shot noise condition [27][28][29][30][31][32]. A systematic study of SRS noise was reported in ref.…”