This paper presents a method for three-dimensional (3D) super-resolution imaging using stochastic optical reconstruction microscopy (STORM). The technique enables nanoscale resolution in all three dimensions without requiring sample or optical beam scanning. By using optical astigmatism, the method determines the axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photo-switchable probes allows high-precision 3D localization of each probe, enabling the construction of a 3D image. The method achieves a lateral resolution of 20-30 nm and an axial resolution of 50-60 nm. This development allows the resolution of 3D morphology of nanoscopic cellular structures. STORM and PALM rely on single-molecule detection and exploit the photoswitchable nature of certain fluorophores to temporally separate the otherwise spatially overlapping images of numerous molecules, allowing high-precision localization of individual molecules. The method achieves localization accuracies as high as 1 nm in the lateral dimensions for a single fluorescent dye at ambient conditions. The shape of the image contains information about the particle's axial position. By introducing defocusing or astigmatism into the image, nanoscale localization accuracy has been previously achieved in the z dimension without significantly compromising the lateral positioning capability. In this work, the astigmatism imaging method was used to achieve 3D STORM imaging. A weak cylindrical lens was introduced into the imaging path to create two slightly different focal planes for the x and y directions. As a result, the ellipticity and orientation of a fluorophore's image varied as its position changed in z. By fitting the image with a 2D elliptical Gaussian function, the x and y coordinates of peak position as well as the peak widths were obtained, which in turn allowed the z coordinate of the fluorophore to be unambiguously determined. To experimentally generate a calibration curve of w_x and w_y as a function of z, Alexa 647-labeled streptavidin molecules or quantum dots were immobilized on a glass surface and imaged individual molecules to determine the w_x and w_y values as the sample was scanned in z. The 3D resolution of STORM is limited by the accuracy with which individual photoactivated fluorophores can be localized in all three dimensions during a switching cycle. A family of photo-switchable cyanine dyes was discovered that can be reversibly cycled between a fluorescent and a dark state by light of different wavelengths. The reactivation efficiency of these photo-switchable "reporters" depends critically on the proximity of an "activator" dye. Here, Cy3 and Alexa 647 were used as the activator and reporter pair to perform 3D STORM imaging. A red laser was used to image Alexa 647 molecules and deactivate them to the dark state, whereas a green laser was used to reactivate the fluorophThis paper presents a method for three-dimensional (3D) super-resolution imaging using stochastic optical reconstruction microscopy (STORM). The technique enables nanoscale resolution in all three dimensions without requiring sample or optical beam scanning. By using optical astigmatism, the method determines the axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photo-switchable probes allows high-precision 3D localization of each probe, enabling the construction of a 3D image. The method achieves a lateral resolution of 20-30 nm and an axial resolution of 50-60 nm. This development allows the resolution of 3D morphology of nanoscopic cellular structures. STORM and PALM rely on single-molecule detection and exploit the photoswitchable nature of certain fluorophores to temporally separate the otherwise spatially overlapping images of numerous molecules, allowing high-precision localization of individual molecules. The method achieves localization accuracies as high as 1 nm in the lateral dimensions for a single fluorescent dye at ambient conditions. The shape of the image contains information about the particle's axial position. By introducing defocusing or astigmatism into the image, nanoscale localization accuracy has been previously achieved in the z dimension without significantly compromising the lateral positioning capability. In this work, the astigmatism imaging method was used to achieve 3D STORM imaging. A weak cylindrical lens was introduced into the imaging path to create two slightly different focal planes for the x and y directions. As a result, the ellipticity and orientation of a fluorophore's image varied as its position changed in z. By fitting the image with a 2D elliptical Gaussian function, the x and y coordinates of peak position as well as the peak widths were obtained, which in turn allowed the z coordinate of the fluorophore to be unambiguously determined. To experimentally generate a calibration curve of w_x and w_y as a function of z, Alexa 647-labeled streptavidin molecules or quantum dots were immobilized on a glass surface and imaged individual molecules to determine the w_x and w_y values as the sample was scanned in z. The 3D resolution of STORM is limited by the accuracy with which individual photoactivated fluorophores can be localized in all three dimensions during a switching cycle. A family of photo-switchable cyanine dyes was discovered that can be reversibly cycled between a fluorescent and a dark state by light of different wavelengths. The reactivation efficiency of these photo-switchable "reporters" depends critically on the proximity of an "activator" dye. Here, Cy3 and Alexa 647 were used as the activator and reporter pair to perform 3D STORM imaging. A red laser was used to image Alexa 647 molecules and deactivate them to the dark state, whereas a green laser was used to reactivate the fluoroph