Interferometric Scattering Microscopy to Characterize Nanometric Objects and Subcellular Structures: Towards Fast 3D Imaging at Nanoscale

Abstract

It is of great interest to visualize nano-scale objects in bioscience. Several ingenious methods such as super-resolution microscopic techniques have been developed to meet the need. These techniques, however, bear evident limitations in measurement time, time resolution, and requirement for fluorescent proteins or probes. In order to circumvent such problems, a new scattering-based method named iSCAT (interferometric scattering microscopy) has been recently introduced. Since this technique relies on scattering signal from a target particle and detects the interference of the signal with a constant reference, it is, in principle, not subject to the aforementioned limitations: theoretically unlimited signal-to-noise ratio to improve time resolution, practically unlimited measurement time, and lack of photophysical and photochemical artefacts.In this presentation, first, we show our recent application of this technique to image biological cells.Interestingly, the iSCAT technique enables us to visualize subcellular structures with remarkable spatial, temporal resolutions and contrast without any labeling.Second, to characterize the position and orientation of nanorods, we utilized scattering of polarized light from anisotropic scatterers, the strength of which is a function of relative orientation of the scatterers to the direction of polarization. From this scheme named psiSCAT (polarization selective iSCAT), we successfully demonstrate that one can capture the orientation of anisotropic nanorods without sacrificing the bright-field image of the entire view-field. Third, we can track the position of nanoparticles along the axial dimension and to image the sample at different depth with minimal image distortion by adopting the remote-focusing technique. We believe that the adoption of the remote-focusing approach significantly expands the strength and utility of iSCAT. This scattering-based microscopy technique would be an indispensable tool in visualizing the nanoscopic world and shed new light on phenomena occurring in the nanospace.