Super-Resolution Nonlinear Photothermal Microscopy

Super-resolution fluorescence microscopy enables imaging of fluorescent structures beyond the diffraction limit. However, this technique cannot be applied to weakly fluorescent cellular components or labels. As an alternative, photothermal microscopy based on nonradiative transformation of absorbed energy into heat has demonstrated imaging of nonfluorescent structures including single molecules and similar to 1-nm gold nanoparticles. However, previously photothermal imaging has been performed with a diffraction-limited resolution only. Herein, super-resolution, far-field photothermal microscopy based on nonlinear signal dependence on the laser energy is introduced. Among various nonlinear phenomena, including absorption saturation, multiphoton absorption, and signal temperature dependence, signal amplification by laser-induced nanobubbles around overheated nano-objects is explored. A Gaussian laser beam profile is used to demonstrate the image spatial sharpening for calibrated 260-nm metal strips, resolving of a plasmonic nanoassembly, visualization of 10-nm gold nanoparticles in graphene, and hemoglobin nanoclusters in live erythrocytes with resolution down to 50 nm. These nonlinear phenomena can be used for 3D imaging with improved lateral and axial resolution in most photothermal methods, including photoacoustic microscopy.