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Stimulated emission depletion (STED) microscopy has been established as an important technique for imaging below the diffraction limit facilitating new discoveries in an array of biological systems. In STED microscopy a “donut-shaped” laser focus is super-imposed onto the diffraction-limited focus of an excitation laser. The dounut-shaped beam suppresses fluorescence in the periphery of the excitation spot, reducing the effective point spread function to a sub-diffraction size. However, the application of multicolor STED microscopy in living cells poses a number of challenges. Here we detail a novel STED system specifically designed for two-color STED applications. Our system employs FPGA-based gated detection and fast beam scanning to reduce pixel dwell time and photobleaching. We demonstrate the instrument’s capability with two-color continuous imaging of intracellular targets below the diffraction limit allowing observation of rare events within live-cells.

Localization-based superresolution imaging is dependent on finding the positions of individual fluorophores in a sample by fitting the observed single-molecule intensity pattern to the microscope point spread function (PSF). For three-dimensional imaging, system-specific aberrations of the optical system can lead to inaccurate localizations when the PSF model does not account for these aberrations. Here we describe the use of phase-retrieved pupil functions to generate a more accurate PSF and therefore more accurate 3D localizations. The complex-valued pupil function contains information about the system-specific aberrations and can thus be used to generate the PSF for arbitrary defocus. Further, it can be modified to include depth dependent aberrations. We describe the phase retrieval process, the method for including depth dependent aberrations, and a fast fitting algorithm using graphics processing units. The superior localization accuracy of the pupil function generated PSF is demonstrated with dual focal plane 3D superresolution imaging of biological structures.

Stimulated emission depletion (STED) microscopy provides diffraction-unlimited resolution in fluorescence microscopy. Imaging at the nanoscale, however, requires precise alignment of the depletion and excitation laser foci of the STED microscope. We demonstrate here that adaptive optics can be implemented to automatically align STED and confocal images with a precision of 4.3 ± 2.3 nm.

When near the focus, the image of a point emitter has little information about the emitter's position along the optical axis. To improve the 3D localization precision, several methods such as astigmatic imaging, double helix point spread functions, and multiple focal plane strategies have been developed to encode the axial position in one or more 2D images of the same emitter. In each of these strategies, a proper model for the 3D microscope point spread function (PSF) is essential to avoid inaccuracies in localization. Generating PSF models from phase retrieved (PR) pupil functions has the advantage that the model images are noise free, can be calculated for any focal plane, and include aberrations specific to the optical system. This representation can also be extremely compact by representing the pupil function as a set of Zernike polynomials. Here we demonstrate the use of PSFs generated from PR pupil functions for 3D single molecule localization based super-resolution imaging using a dual focal plane setup. We show the phase retrieval process, as well as the details of fast PSF calculation by implementation on graphics processing unit hardware. We show a comparison between fitting with ideal, aberration free PSF models and that generated from PR pupil functions using a variety of cellular structures including the ER, microtubules and the membrane proteins STIM1 and ORAI1.

The resolution attainable with stimulated emission depletion (STED) microscopy greatly depends on the quality of the STED laser focus. So far, visual inspection of a measured STED focus has been the only convenient means of gauging the source of aberrations. Here we describe a method, requiring no instrument modifications, for obtaining an equivalent to the complex pupil function at the back aperture of the objective and show that it provides quantitative information about aberration sources (including aberrations induced by the objective or sample). We show the accuracy of this field representation to be sufficient for reconstructing the STED focus in three dimensions and determining corrective steps.