Rotating Coherent Scattering microscopy enabling specificity and height profiling

• Applicant: Professor Dr. Alexander Rohrbach
• Subject Area: Optics, Quantum Optics and Physics of Atoms, Molecules and Plasmas
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 413220392

Rotating coherent scattering (ROCS) microscopy is a fluorescence-free imaging technique, which can image dynamic structures at a few ms temporal resolution and at 160nm spatial resolution. These numbers make ROCS unique among existing label-free imaging techniques. Using bluish lasers and highly oblique illumination during 2 azimuthal beam rotation at > 100 Hz it turns out that coherent speckles change into meaningful objects without the necessity of image reconstructions. In the previous DFG project we successfully investigated the correlation and compatibility of ROCS with fluorescence imaging on the one hand, and, on the other hand, demonstrated that ROCS in 2D can generate material specificity based on electronic polarizabilities of differently absorbing beads, such that different materials are displayed in different colors (“color-ROCS”). In the new two-part project we want to explore in project part A the limits of these novel imaging principles, in particular of the material identification by their complex polarizabilities at distinct spectral sample points. Instead of simple beads, we now want to use specificity based Color-ROCS to investigate biological materials or even living cells by optimised sampling in the violet-blue spectrum and even extend Color-ROCS imaging to 3D. This would represent a significant improvement to the state of art, opening many new applications dealing with small dynamic structures. However, this goal requires a series of lasers in the wavelength range from 400-500nm to reliably sample different absorption spectra, since broadband light sources reduce image contrast significantly. Project part B is also motivated by the previous project, where we showed that in brightfield ROCS a reference wave amplifies the light backscattered at the object. It has turned out that the many interference fringes from the object wave (typically from a biological cell) and the reference wave are to a good approximation invariant against the rotating illumination. These interference fringes make it difficult to identify small cellular structures, hence are detrimental. Since the removal of the fringes is not possible for the partial coherent ROCS imaging technique, the goal is to exploit the interference fringes to generate height profiles of the cells with update times of a few seconds. These height profiles will give highly valuable 3D information about the dynamic, structural changes of the cell-surface, which are relevant especially during infection mechanisms caused by viruses, bacteria or particulates.

• DFG Programme: Research Grants
• Major Instrumentation: Laser combiner box
• Instrumentation Group: 5790 Sonstige Laser und Zubehör (außer 570-578)