1. 3D Localization Microscopy With Ground State Depletion (GSD 3D)

2. Basic Principles of the GSD Super-Resolution Technology for Displaying Highly Resolved Cellular Structures in Three Dimensions
The exact position of a fluorochrome is based on the principle of temporal separation;
This requires switching of fluorochromes between on and off states followed by a sequential readout;

3. Super-resolution imaging
The crucial step of dSTORM/GSD is to temporarily switch off the majority of fluorophores to allow the precise localization of single fluorophores.

4. Super-resolution as reconstructed z-stack
Fluorophore in the focal plane: the image appears round;
Below the focus: elongated in the vertical direction;
Higher than the focus position: elongated in the horizontal direction.

5. Section localization scheme

6. Super-resolution as reconstructed z-stack
Coordinates in z are assigned to a specific layer in a z-stack based on a calibration of the deformed PSF by the cylinder lens.
Resolution around 70 nm can be achieved.

7. Evolution of resolution
AlexaFluor 488
AlexaFluor 532
Rhodamine 6G
AlexaFluor 647
Others.