公开(公告)号：US20230175963A1

公开(公告)日：2023-06-08

申请号：US17994434

申请日：2022-11-28

Applicant: LEICA MICROSYSTEMS CMS GMBH

Inventor： Benjamin DEISSLER ,  Christian SCHUMANN ,  Kai RITSCHEL ,  Mate BELJAN ,  Wernher FOUQUET ,  Gheorghe COJOC

IPC: G01N21/64

CPC classification number: G01N21/64 ,  G01N2201/127

Abstract: A method of analyzing a mixed fluorescence response of a plurality of fluorophores in a microscopic sample includes reconstructing individual fluorescence responses from a mixed fluorescence response using spectral un-mixing based on reference emission spectra for fluorophores to be reconstructed, and a procedure for determining and validating reference emission spectra including providing a plurality of image acquisition settings for a sequence of images of the sample equal to, or greater than, the plurality of fluorophores and including an illumination setting for each image, acquiring the sequence of images using the plurality of image acquisition settings and storing each image together with the corresponding illumination setting, determining candidate reference emission spectra for the fluorophores to be reconstructed from the sequence of images of the sample using one or more reference emission spectra determination algorithms, and conditionally using the candidate reference emission spectra as the reference emission spectra in the spectral un-mixing.