Invited Article: Spectral focusing with asymmetric pulses for high-contrast pump–probe stimulated Raman scattering microscopy

Abstract

Purely label-free imaging to directly monitor small molecules in a biological organism is still challenging despite recent technical advancements. Time-resolved pump–probe coherent Raman scattering microscopy is a promising label-free approach to increase chemical specificity. However, conventional time-resolved methods involve a compromise between three conflicting requirements: high spectral resolution, low background levels, and high sensitivity. Here, we present an advanced spectral-focusing technique using asymmetric pulses produced by nonlinear chirping and demonstrate its performance in pump–probe phase-modulated stimulated Raman scattering microscopy. In addition, we report for the first time a novel frequency-scanning spectral-focusing system using tunable bandpass filters. Our concept uses the filters not only as a frequency allocation tool for the probe pulses but also as a pulse-shaping tool that provides a strong nonlinear chirp. The spectral resolution and signal-to-noise ratio are greatly improved by highly efficient time-resolved detection using asymmetric spectrally focused probe pulses. We achieve a spectral resolution of ∼25 cm−1, a reduced nonresonant background level on the order of 10−8, and a detectable concentration limit of 0.01% dimethyl sulfoxide/water solution (1.5 mM). Using this method, we demonstrate high-contrast imaging of a small-molecule drug in a tissue. These advancements will allow time-resolved coherent Raman microscopy to be used as a practical drug-imaging tool for biomedical sciences.