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.
Highlights
This technique requires laborious sample preparation steps to introduce a specific chemical tag
By using stretched frequency-locked pulses for narrowband excitation, the spectral resolution of time-resolved coherent anti-Stokes Raman scattering (CARS) (T-CARS) has been improved to below 30 cm−1.20,21 We have demonstrated that spectral focusing can be used for the selective probing of impulsively excited molecular vibrations in pump– probe phase-modulated stimulated Raman scattering (SRS) (PM-SRS) microscopy.[22]
We presented an advanced spectral-focusing technique using asymmetric pulses produced by nonlinear chirping and demonstrated its performance in time-resolved pump–probe SRS microscopy
Summary
This technique requires laborious sample preparation steps to introduce a specific chemical tag. Time-resolved pump–probe coherent Raman scattering microscopy[14,15,16,17,18,19] is a promising labelfree approach to increase the chemical specificity This microscopy offers the selective detection of a long-lived vibrational mode from the targeted molecules while rejecting less specific short-lived Raman background from water and abundant macromolecules such as lipids or proteins, as well as the nonresonant background caused by electronic four-wave mixing processes. Even though one can provide a longer probe delay time to reduce the background, this would reduce the Raman signal level due to the finite relaxation time of the excited molecular vibrations In this context, conventional spectral focusing based on linear chirping is not always the best strategy for the pump–probe setup. The system design presented here will be a significant addition to the pulse-shaping toolbox for the coherent Raman microscopy and other nonlinear optics communities
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