Abstract: A method of in situ point spread function (PSF) retrieval is disclosed which includes encoding 3D location of molecules into PSFs, receiving molecule-generated images containing PSFs, segmenting the images into sub-PSFs, initializing template PSFs from a pupil function, determining a maximum normalized cross correlation (NCC) coefficient (NCCmax) between the sub- and template PSFs, associating each of the sub-PSFs with a template PSF based on the NCCmax and storing the sub-PSFs in associated bins, aligning and averaging the binned sub-PSFs, applying a phase retrieval algorithm to the averaged sub-PSFs to update the pupil function, regenerating the template PSFs, repeating until a difference between a new and a prior generation pupil function is below a predetermined threshold, generating in situ PSFs from the last pupil function, and applying a maximum likelihood estimation algorithm based on the in situ PSFs and the sub-PSFs to thereby generate lateral and axial locations of molecules.

Abstract: A method of in situ point spread function (PSF) retrieval is disclosed which includes encoding 3D location of molecules into PSFs, receiving molecule-generated images containing PSFs, segmenting the images into sub-PSFs, initializing template PSFs from a pupil function, determining a maximum normalized cross correlation (NCC) coefficient (NCCmax) between the sub- and template PSFs, associating each of the sub-PSFs with a template PSF based on the NCCmax and storing the sub-PSFs in associated bins, aligning and averaging the binned sub-PSFs, applying a phase retrieval algorithm to the averaged sub-PSFs to update the pupil function, regenerating the template PSFs, repeating until a difference between a new and a prior generation pupil function is below a predetermined threshold, generating in situ PSFs from the last pupil function, and applying a maximum likelihood estimation algorithm based on the in situ PSFs and the sub-PSFs to thereby generate lateral and axial locations of molecules.