Stimulated Raman scattering (SRS) imaging is a fast, label‐free, and sensitive technique to map the distribution of a vibrational species in a microscopy setting. It has great potential for applications in many fields, such as lipid imaging in biomedicine. However, depth penetration of the light into the sample is an issue with any light‐based technique, especially with multiphoton techniques such as SRS. Using longer wavelengths allows deeper penetration into densely scattering materials, but applying wavelengths above 1,500 nm is challenging technically. We have built a flexible SRS microscope system capable of imaging with a combination of 1,064 nm and wavelengths over 1,500 nm, using the idler output of an optical parametric oscillator (OPO). For comparison, the same system was also operated in the more common configuration, using 1,064 nm in combination with the OPO signal output around 800 nm. With the long‐wavelength settings, we show improved depth penetration in polyethylene plastic material and in a silicone phantom with embedded polymer microbeads, and we report images of lipid structure in biological tissue. These results demonstrate the technical feasibility of using these long wavelengths for SRS imaging. Disadvantages such as poorer spatial resolution and lower signal strength are also discussed. The application of this new approach to SRS microscopy can allow greater insight into deep‐lying structures in a non‐invasive way.

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.

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 a 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.

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.