Single-pulse Stimulated Raman Photothermal Microscopy and Direct Visualization of Cholesterol-rich Membrane Domains

By measuring the thermal effects resulting from stimulated Raman excitations, stimulated Raman photothermal (SRP) microscope offers a new access to spatially and temporally resolved Raman signatures across various sample types. Unlike stimulated Raman scattering (SRS) microscopy, SRP is highly compatible with noisy ultrafast laser sources, allowing the use of high peak power, low repetition rate optical parametric amplifier (OPA) lasers to boost sensitivity and imaging speed. Here, we report a single pulse SRP (spSRP) microscope system in which SRP signals are induced by individual pairs of laser pulses generated by an OPA laser. Extensive pulse chirping is used to maximize SRS excitation rate and to minimize photodamage. The single-pixel limit of detection (LOD) of spSRP on dimethyl sulfoxide (DMSO) reaches 890 μM, which is a ~44-fold improvement over SRS microscope. Versatile applications to fungi, cells, and tissues are demonstrated. Live cell spSRP imaging was carried out at speed of 10 frames per second. Particularly, the spSRP system enables direct visualization of cholesterol-rich domains, co-localized with caveolin immunofluorescence, in the plasma membrane of HeLa cells.