Stimulated Raman scattering (SRS) microscopy offers label-free, high-speed measurements of small molecules in living cells and tissues, holding promise for potential biomedical applications such as drug screening and therapeutic drug monitoring. Nevertheless, with conventional SRS microscopy, fluctuations in the background Raman signal from cells and tissues limit quantitative measurements at drug concentrations below a few mM. Here we present novel SRS microscopy using temporal filtering combined with a phase-locking technique to break the detection limit due to the background Raman signals. We demonstrate that the new phase-locked system enables drug measurements at sub millimolar concentrations with linear calibration curves.
Stimulated Raman scattering (SRS) offers high sensitivity to monitor low-concentration drugs in real time. However, the "needle" of a drug signal at millimolar concentrations is hidden in the "haystack" of tissue background Raman signals, which practically limits the specificity of the drug measurements. Here, we compare two promising solutions, namely frequency-modulated SRS (FM-SRS) and time-resolved phase-modulated SRS (PM-SRS). We show that the temporal response of FM-SRS is a simple sinusoidal function and its specificity must be compromised. However, the time-resolved PM-SRS with shaped probe pulses provides temporal high-pass filtering with steep cutoff characteristics, enabling higher specificity in the drug measurement.
Quantitative measurements of a small amount of chemicals in label-free tissue imaging by conventional SRS microscopy remain challenging because of background signals. We present a time-resolved, phase-modulated (PM) SRS microscopy method with increased signal contrast. In addition to removing background signals generated via amplitude modulation, PM-SRS can reduce intrinsic tissue background signals by temporally separating the excitation and detection processes. Furthermore, polarization artifacts in tissues can also be removed by rapidly modulating the relative delay between the pump and probe pulses. This new technology enables robust quantitative measurements in tissue and extends the potential of SRS imaging in biomedical applications.
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