Multicolour localization microscopy by point-spread-function engineering

Super-resolution microscopy has revolutionized cellular imaging in recent years(1-4). Methods that rely on sequential localization of single point emitters enable spatial tracking at a resolution of similar to 10-40 nm. Moreover, tracking and imaging in three dimensions is made possible by various techniques, including point-spread function (PSF) engineering(5-9) namely, encoding the axial (z) position of a point source in the shape that it creates in the image plane. However, efficient multicolour imaging remains a challenge for localization microscopy a task of the utmost importance for contextualizing biological data. Normally, multicolour imaging requires sequential imaging(10,11), multiple cameras(12) or segmented dedicated fields of view(13,14). Here, we demonstrate an alternate strategy: directly encoding the spectral information (colour), in addition to three-dimensional position, 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.