Patent ID: 11960102
Assignee: PURDUE RESEARCH FOUNDATION
Field: Measurement (Instruments)
Classification: CPC G | IPC G

Claim 0:
1. A method of point spread function (PSF) retrieval for three-dimensional nanoscopy, comprising:
A) encoding 3D location of molecules into a plurality of fluorescence emission patterns expressed as point spread functions (PSFs) detected on a camera;
B) receiving one or more images containing a plurality of PSFs generated by molecules within a specimen;
C) segmenting the received one or more images into a plurality of sub-regions containing isolated PSFs (sub-PSFs) from point sources;
D) initializing a plurality of template PSFs, each of which is generated from an initial pupil function based on a corresponding axial distance between an image plane (where the point-source locates) and a focal plane of the objective;
E) cross correlating each of the plurality of sub-PSFs with each of the plurality of template PSFs, thereby generating a corresponding plurality of normalized cross correlation coefficients;
F) determining a maximum normalized cross correlation coefficient from the plurality of normalized cross correlation coefficients;
G) associating each of the plurality of sub-PSFs with a corresponding template PSF of the plurality of template PSFs based on which template PSF produced the maximum normalized cross correlation coefficient and storing each of the plurality of sub-PSFs in an associated bin corresponding to each of the plurality of template PSFs;
H) aligning each of the bin-associated sub-PSFs;
I) averaging the aligned sub-PSFs;
J) applying a phase retrieval algorithm to the averaged sub-PSFs to thereby update the pupil function;
K) regenerating the plurality of template PSFs from the updated pupil function;
L) repeating steps E-K until a difference between a new and a prior generation pupil function is below a predetermined threshold;
M) generating in situ PSFs from the last pupil function; and
N) applying a maximum likelihood estimation algorithm based on the in situ PSFs and the plurality of sub-PSFs to thereby generate lateral and axial locations of molecules within the specimen.