Three-dimensional information from two-dimensional scans: a scanning microscope with postacquisition refocusing capability

Laser scanning microscopes are helpful tools for the visualization of 3D structures at the submicron scale. By pointwise raster scanning the sample with a tightly focused laser spot, they collect sample information in a sequential manner. We present a scanning microscope that has the ability to record 3D sample information from a single 2D scan. By combining camera detection with double-helix phase engineering in the emission path, the microscope allows for postacquisition refocusing within an axial range of roughly 400 nm with a high-NA lens (NA 1.35). Refocused images are extracted from a single 2D data set by applying different synthetic pinholes, i.e., by integrating over a small predefined area in the image acquired for every sampling point. 3D cross-sectional images of a stained microtubule network within fixed African green monkey kidney cells are presented, demonstrating the capability of the system. The imaging paradigm enables faster data acquisition with potentially lower phototoxic side effects.