Pump-probe stimulated Raman scattering microscopy for monitoring the transport of gaseous molecules (Conference Presentation)

The development of a technology that allows for analyzing microscopic spatial distribution and dynamics of small gaseous molecules such as inhalational anesthetics and odors would advance our understanding of its biological activities in living cells. However, direct observation of such small molecules by optical microscopy is still challenging. We propose a new pump–probe stimulated Raman scattering (SRS) microscopy method for studying the localization, transport and metabolism of gaseous molecules in a living organism in a label-free manner. A technical challenge is how to detect the Raman signal of a small amount of drug molecules that is typically overwhelmed by unwanted nonlinear background, including nonresonant background and coherent Raman scattering of surrounding cells and tissues. In particular, the latter Raman-induced background is essentially inevitable in most standard coherent anti-Stokes Raman scattering (CARS) and SRS systems. We show that these background issues can be overcome by introducing a new pump–probe, time-resolved SRS detection approach coupled with a pair of spectrally-focused, asymmetrically shaped probe pulses (T. Ito et al. APL Photonics (2018)). In the pump–probe scheme, a long-lived vibration of the targeted molecules can be efficiently probed after short-lived vibrations of other background molecules such as water and fatty acids become silent. This unique lifetime-selective signal detection provides a significantly enhanced vibrational signal contrast. As a proof-of-concept experiment, we demonstrate that the passive transport of inhalational anesthetic molecules from aqueous solution to adipose cells can be monitored by time-lapse SRS imaging.