Measuring single molecule 3D orientational behavior is a challenge that, if solved in addition to 3D localization, would provide key elements for super resolution structural imaging. Orientation contains indeed information on local conformational properties of proteins, while orientational fluctuations are signatures of local steric, charges or viscosity constraints. Both these properties are not perceptible in pure super resolution imaging, which relies on position localization measurements. Imaging 3D orientation together with 3D localization is however not easily accessible due to the intrinsic coupling between spatial deformation of the single molecules’ point spread function (PSF) and their off-plane orientations, as well as the requirement to measure six parameters which are not directly distinguishable (two angles of orientation, aperture of angular fluctuations, and three spatial position coordinates). In this work, we report a method that is capable of resolving these six parameters in a modality that is compatible with super resolution imaging. The method is based on the use of a stress-engineered spatially-variant birefringent phase plate placed in the Fourier plane of the microscope detection path. This modifies the PSF of single emitters in a way that can be non-ambiguously decomposed onto the nine 3D-analogs of the Stokes parameters. Moreover, the use of two complementary co/counter circular polarizations projections provides a non-ambiguous determination of the 3D spatial position of single emitters with tens of nanometers precision. This method, which opens to nanoscale structural imaging of proteins organization, is presented on model nano-beads emitters and applied to single fluorophores used for cytoskeleton labelling.
Highly Inclined and Laminated Optical sheet (HILO) microscopy is an optical technique that employs a highly inclined laser beam to illuminate the sample with a thin sheet of light that can be scanned through the sample volume<sup>1</sup> . HILO is an efficient illumination technique when applied to fluorescence imaging of thick samples owing to the confined illumination volume that allows high contrast imaging while retaining deep scanning capability in a wide-field configuration. The restricted illumination volume is crucial to limit background fluorescence originating from portions of the sample far from the focal plane, especially in applications such as single molecule localization and super-resolution imaging<sup>2-4</sup>. Despite its widespread use, current literature lacks comprehensive reports of the actual advantages of HILO in these kinds of microscopies. Here, we thoroughly characterize the propagation of a highly inclined beam through fluorescently labeled samples and implement appropriate beam shaping for optimal application to single molecule and super-resolution imaging. We demonstrate that, by reducing the beam size along the refracted axis only, the excitation volume is consequently reduced while maintaining a field of view suitable for single cell imaging. We quantify the enhancement in signal-tobackground ratio with respect to the standard HILO technique and apply our illumination method to dSTORM superresolution imaging of the actin and vimentin cytoskeleton. We define the conditions to achieve localization precisions comparable to state-of-the-art reports, obtain a significant improvement in the image contrast, and enhanced plane selectivity within the sample volume due to the further confinement of the inclined beam.