Multicolour localization microscopy by point-spread-function engineering.

Super-resolution microscopy has revolutionized cellular imaging in recent years1-4. Methods relying on sequential localization of single point emitters enable spatial tracking at ~10-40 nm resolution. Moreover, tracking and imaging in three dimensions is made possible by various techniques, including point-spread-function (PSF) engineering5-9 -namely, encoding the axial (z) position of a point source in the shape that it creates in the image plane. However, a remaining challenge for localization-microscopy is efficient multicolour imaging - a task of the utmost importance for contextualizing biological data. Normally, multicolour imaging requires sequential imaging10, 11, multiple cameras12, or segmented dedicated fields of view13, 14. Here, we demonstrate an alternate strategy, the encoding of spectral information (colour), in addition to 3D position, directly in the image. By exploiting chromatic dispersion, we design a new class of optical phase masks that simultaneously yield controllably different PSFs for different wavelengths, enabling simultaneous multicolour tracking or super-resolution imaging in a single optical path.