Label free super-resolution by nonlinear photo-modulated reflectivity

Far-field super-resolution (SR) microscopy has developed to be an important tool in life sciences. However, it relies on, and therefore is limited by the ability to control the fluorescence of label molecules or nanoparticles. While in many cases, label-free far-field microscopy is required, an equivalent SR technique is not well established yet. Several demonstrations of label-free far-field microscopy, that were introduced recently, include transmission microscopy based on ground state depletion of charge carriers, nonlinear photo-thermal microscopy in a fluid medium, and photo-acoustics SR microscopy. Additionally, CARS based SR vibrational microscopy has been demonstrated.We introduce a new far field label-free SR methodology that is based on the nonlinear response of the reflectance to photo-modulation [1]–[3]. It relies on the ability to photo-excite a temperature and/or charge-carriers spatial distribution inside the diffraction limited spot by an ultra-short pump pulse. In our approach, Nonlinear Photo-Modulated Reflectivity (NPMR), an overlapping delayed probe pulse monitors the nonlinear reflectance changes. By scanning over the sample and measuring the NPMR, spatial resolution is enhanced. NPMR is measured by recording the high harmonics induced by the pump sine modulation in the probe laser reflectance. The improvement in resolution scales like n 1/2 , where n is the nonlinearity order. Similarly, the reduction of PSF was shown in saturation absorption fluorescence microscopy (SAX), and in saturated excitation microscopy of plasmonic nanoparticles.In order to achieve a reliable response in high harmonics, pure sine modulation is required. By using an acousto-optic modulator coupled to an arbitrary wave-form generator, we have succeeded in modulating our pump source with harmonic impurity down to 10 -4 , at high modulation frequency of up-to 1 MHz.Examples of resolution enhancement due to nonlinearities are presented: The change of thermo-reflectance of the heating process of a nanostructured gold on ITO surfaces, together with high modulation rate, to achieve scanning speed of up to 20 μm/s. Moreover, it is possible to couple the methodology of NPMR to other resolution enhancement approaches to further reduce the point spread function. Such methods include the spatial modulation of the pump beam and apodization of the pump beam. We will also show that under specific conditions, the method can be simplified to use only a single laser pulse, to serve both as the pump and as the probe. Using these approaches, super-resolution down to 85nm is demonstrated with a wavelength of 400 nm.We also show that adjustment of the probe beam optical frequency to correlate to plasmonic resonances can dramatically improve the sensitivity of detection. Our methodology for detection of nonlinearities can be applied to other imaging modalities.Finally, NPMR is general and is suitable to characterize semiconductors and metals in vacuum, ambient, and liquid, semi-transparent and opaque systems in reflection.