Visualizing the precise localization of molecules and structures is crucial for a better understanding of cellular processes. The Leica SR GSD 3D widefield fluorescence microscope utilizes the unique GSDIM (Ground State Depletion followed by Individual Molecule Return) technology complementing the super-resolution methodology developed by Nobel Prize winner Stefan Hell. The Leica SR GSD 3D offers not only 2D, but also 3D super-resolution imaging – with the highest precision, reproducibility and maximum possible resolution in widefield microscopy so far. The Leica SR GSD 3D is based on a fully automated TIRF system. As a multi-functional system, it gives researchers the freedom to tailor the system exactly to their applications in live cell or advanced fluorescence imaging.
With stochastical optical reconstruction microscopy (GSD or dSTORM) it is possible to achieve super-resolution images with a lateral resolution of up to 20 nm. However the z-resolution of a widefield microscope was still limited around several hundred nanometers. As a pioneer in super-resolution microscopy Leica Microsystems has developed the Leica SR GSD 3D to overcome this limit down to a resolution of 50 nm in axial direction and to set new standards in 3D localization precision.
The Leica SR GSD 3D scores with its unique precision for localizing single molecules, its system stability, optical performance and its easy operation. These attributes are essential for reproducible, high-quality results in the shortest possible time. All the optical components are apochromatically corrected to high Leica Microsystems standards. The software enables precise calibration and besides the standard calibration with gold beads, color-specific calibrations with fluorescence beads or dye molecules of the specimen are available.
The Leica SR GSD 3D is equipped with a 160x high-performance objective specifically developed for super-resolution microscopy. Its design is optimized for high-power laser emissions. The extremely low autofluorescence ensures a high signal-to-noise ratio, which is ideal for single molecule detection applications. The outstanding apochromatic correction further improves the image results.
For localization of single molecules in GDSIM or dSTORM fluorescence microscopy the point spread function (PSF) of each molecule is recorded and a fit is performed to determine the position of the fluorophore. With a regular fluorescence microscope the lateral position of the fluorophore is defined with the regular PSF (left). If a cylindrical lens is added to this system, a different PSF (astigmatism) is obtained (right). Corresponding images allow determination of the z position of a detected molecule, thereby enabling 3D reconstruction.
To ensure reproducibility of results, the cylindrical lens is precisely positioned into the emission beam path as soon as the system is switched from 2D to 3D recording – not by hand, but automatically by a single mouse click through software control.
With the SuMo stage Leica Microsystems has introduced a new technology in drift compensation which puts the maximum system drift below the resolution during acquisition. This makes it possible to observe the super-resolution image as it is being acquired. The special SuMo technology (SUpressed MOtion) ensures not only minimum drift but also maximum stability during detection.