Double-helix Point-spread-function

The phase mask is placed in the microscope’s emission path to reshape light from each fluorescent point source into a characteristic two-lobed pattern. Its optical action creates an image whose geometry changes with molecular depth, allowing three-dimensional information to be encoded directly in the detected fluorescence rather than treated as a purely lateral signal.

The molecule’s lateral position is obtained from the center of the two-lobed pattern, while its axial position is inferred from the pattern’s rotation angle. These measurements separate the two spatial components within a single image feature, enabling localization of fluorescent molecules in three dimensions and supporting nanometer-scale positional analysis.

A rotating double-helix pattern converts changes in molecular depth into changes in image orientation. Because the orientation is characteristic of the molecule’s axial position, researchers can follow depth-dependent localization across an extended imaging range. This is especially valuable when molecular motion or organization must be examined beyond a single focal plane.

Fluorescently labeled proteins, nucleic acids, and other molecules are suitable targets identified in the provided context. The method can therefore address molecular organization, transport, and interactions rather than being limited to one biochemical class. Its value comes from assigning three-dimensional positions to labeled components within complex biological samples.

Fluorescence is collected from a labeled molecule through the microscope’s emission path, where the phase mask reshapes the point-source image into a double-helix pattern. The recorded pattern is then evaluated through its center and rotation angle to determine lateral and axial position. This workflow converts one optical signal into three-dimensional localization information.

The approach provides three-dimensional positions for fluorescently labeled molecules, including their lateral location and depth. Repeated localization can support tracking over extended depths, making it possible to investigate how proteins, nucleic acids, or other molecules are organized, transported, or positioned relative to one another in complex biological samples.

Biochemical systems often contain labeled molecules whose organization and interactions vary across depth. By encoding depth through image rotation while retaining lateral information from the pattern center, the method supports nanometer-scale localization and tracking throughout an extended three-dimensional region. This helps connect molecular position with transport, organization, and interactions in biological samples.