Superior contrast and resolution by image formation in rotating coherent scattering (ROCS) microscopy

Abstract

Small structures, as inside living cells, move on millisecond timescales, which is usually far beyond the imaging rate of superresolution fluorescence microscopes. In contrast, label-free imaging techniques providing high photon densities can operate at >100 Hz. For simple structures, an oblique, coherent illumination with a static laser beam increases image contrast and resolution considerably, whereas illumination of complex structures results in an image full of speckles. Remarkably, an artifact-free image is generated by subsequent oblique illumination of the structure from all azimuthal directions. This is the working principle of ROCS microscopy, which currently achieves 150 nm spatial and 10 ms temporal resolution without fluorophore bleaching, and is therefore highly beneficial for live-cell imaging. However, the complicated formation of ROCS images and image spectra during one sweep, i.e., the superposition of different speckle patterns is still unclear. Here, we investigate with experiments and computer simulations the influence of speckle-like interference patterns on the final image contrast and resolution, in darkfield mode and, by adding a reference wave, in brightfield mode. In close comparison to experimental results, we present a theoretical framework, which describes the ROCS image formation in real space and in k space by identifying different spectral components. In addition, we vary the degree of coherence by a rotating diffuser and thereby demonstrate that maximal spatial coherence and maximal speckle interference from multiple scattering provide the best image contrast and resolution. We find that the cross correlations of elementary waves emitted in a distance of several micrometers to each other positively contribute to image formation and do not, as commonly believed, distort image formation. By understanding the composition of image speckles in time and space, future coherent microscopes should provide new insights into the high-speed world of living cells.