Conical diffraction illumination opens the way for low phototoxicity super-resolution imaging

<div><p>We present a new technology for super-resolution fluorescence imaging, based on conical diffraction. Conical diffraction is a linear, singular phenomenon, taking place when a laser beam is diffracted through a biaxial crystal. We use conical diffraction in a thin biaxial crystal to generate illumination patterns that are more compact than the classical Gaussian beam, and use them to generate a super-resolution imaging modality.</p><p>While there already exist several super-resolution modalities, our technology (biaxial super-resolution: BSR) is distinguished by the unique combination of several performance features. Using BSR super-resolution data are achieved using low light illumination significantly less than required for classical confocal imaging, which makes BSR ideal for live-cell, long-term time-lapse super-resolution imaging. Furthermore, no specific sample preparation is required, and any fluorophore can be used. Perhaps most exciting, improved resolution BSR-imaging resolution enhancement can be achieved with any type of objective no matter the magnification, numerical aperture, working distance, or the absence or presence of immersion medium.</p><p>In this article, we present the first implementation of BSR modality on a commercial confocal microscope. We acquire and analyze validation data, showing high quality super-resolved images of biological objects, and demonstrate the wide applicability of the technology. We report live-cell super-resolution imaging over a long period, and show that the light dose required for super-resolution imaging is far below the threshold likely to generate phototoxicity.</p></div>