Background-free stimulated Raman scattering microscopy with acousto-optic tunable filter

Stimulated Raman Scattering (SRS) microscopy is a Coherent Raman technique that emerged in recent years as a powerful tool for biomedical imaging due to its high specificity, high speed, and label-free capability. Despite its advantages, SRS microscopy can be affected by nonlinear competing phenomena, namely two-photon absorption (TPA), cross-phase modulation (XPM), and thermal lensing (TL), which generate a background signal that reduces the achievable specificity and sensitivity. These competing processes are quasi-instantaneous and spatially non-uniform in heterogeneous samples and require customized setups to be canceled in SRS acquisitions. A robust approach for background-free SRS measurements is the frequency-modulation (FM) SRS, which is based on the broad spectral dependence of the parasitic effects (typically tens of nanometers) compared to the narrower band of the SRS effect (~ 1 nm). Performing a differential measurement at two different wavenumbers, respectively on- and off- Raman resonance, it is possible to selectively detect the SRS process. Different solutions for background cancellation via FM have been reported in the literature, but they present various drawbacks, such as the limited applicability over certain ranges of the vibrational spectrum or the necessity to modify the optical setup when performing measurements at different Raman shifts. We propose an FM-SRS configuration realized for the first time with an acousto-optic tunable filter, able to perform measurements from the fingerprint to the CH-stretch region of the spectrum without any modification of the optical setup. We determined its efficiency in canceling the background signal due to different types of competing effects on various samples: polymer beads, human hair, and human cells. These results underline the importance of an effective cancellation of background signals of diverse nature when collecting SRS images. Our FM-SRS setup demonstrated critical advantages compared to other FM configurations.