Abstract: A system (100) and method for creating three dimensional images using probe molecules is disclosed and described. A sample is mounted on a stage (160). The sample has a plurality of probe molecules. The sample is illuminated with light, causing the probe molecules to luminesce. The probe luminescence can be split into at least four paths corresponding to at least four detection planes corresponding to object planes in the sample. The at least four detection planes are detected via a camera (155). Object planes in corresponding recorded regions of interest are recorded in the camera (155). A signal from the regions of interest is combined into a three dimensional image.

Abstract: A system (100) and method for creating three dimensional images using probe molecules is disclosed and described. A sample is mounted on a stage (160). The sample has a plurality of probe molecules. The sample is illuminated with light, causing the probe molecules to luminesce. The probe luminescence can be split into at least four paths corresponding to at least four detection planes corresponding to object planes in the sample. The at least four detection planes are detected via a camera (155). Object planes in corresponding recorded regions of interest are recorded in the camera (155). A signal from the regions of interest is combined into a three dimensional image.

Abstract: An optical microscope (101) with heightened resolution and capable of providing three dimensional images is disclosed and described. The microscope (101) can include a sample stage (160) for mounting a sample having a plurality of probe molecules. At least one non-coherent light source (127) can be provided. At least one lens (140a, 140b) can be configured to direct a beam of light from the at least one non-coherent light source (127) toward the sample causing the probe molecules to luminesce. A camera (155) can be configured to detect luminescence from the probe molecules. A light beam path modification module (132, 150) can be configured to alter a path length of the probe molecule luminescence to allow camera luminescence detection at a plurality of object planes.

Abstract: A Polarization Fluorescence Photoactivation Localization Microscopy (P-FPALM) system and method are provided to simultaneously image the localizations and fluorescence anisotropics of large numbers of single molecules within a sample. The system modifies known FPALM systems by adding a polarizing beam splitter. The beam splitter polarizes emissions perpendicular and parallel to an axis in the sample to allow spatially separate imaging of fluorescence emitted from a sample. The system includes lenses and mirrors so that the separate, polarized beams are detected simultaneously. The present invention includes methods of using the system to image localizations and fluorescence anisotropics of single molecules, and methods of using data obtained with the system to predict 3-D orientation of the molecules. The system and method achieve substantially improved lateral resolution within even dense samples over known microscopic imaging techniques, and does not compromise speed or sensitivity.