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

TL;DR

This paper introduces a single-pulse stimulated Raman photothermal (spSRP) microscopy technique that enhances sensitivity and speed, enabling direct visualization of cholesterol-rich membrane domains in live cells with high spatial and temporal resolution.

Contribution

The study develops a single-pulse SRP system using an OPA laser, achieving significant sensitivity improvements and enabling real-time imaging of membrane domains in biological samples.

Findings

spSRP detects dimethyl sulfoxide at 890 μM, a 44-fold improvement over SRS.

Live cell imaging at 10 frames per second demonstrates high-speed visualization.

Direct imaging of cholesterol-rich domains in HeLa cell membranes was achieved.

Abstract

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 {\mu}M, 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.