Single-particle orientation microscopy with quantum dots for bioimaging.

Abstract

Imaging the orientation of fluorescent probes is an important multi-dimensional tool for studying a variety of biological features, such as revealing the alignment of scaffolding and structural components in cells, or observing rotational dynamics of individual biomolecules. Traditionally, orientation microscopy has been performed in a confocal configuration, limiting its applications to small volumes. However, wide-field camera-based orientation imaging can expand the techniques to image large volumes, such as entire cells. These methods measure the polarization state of probe fluorescence to determine orientation. Single-particle measurements can additionally yield localization information with resolutions of tens of nanometers in biological environments. However, probe selection, instrument design, calibration, and analysis of the various polarization images require attention to detail to generate accurate measurements. We report on a microscopy method that super-resolves probe orientation and wavelength, and demonstrate the method with quantum dot (QD) nanoparticles in small clusters. Although QD probes are examined in detail, other probes such as fluorescent dyes and proteins can use the same framework and instrumentation for non-toxic application of orientation microscopy.