Method of analyzing a mixed fluorescence response of a plurality of fluorophores, fluorescence analyzer, fluorescence microscope and computer program

US12339229B2 · kind B2 · utility

Assignee

• LEICA MICROSYSTEMS CMS GMBH · DE

Inventors

• Benjamin Deissler · Rockenberg, DE
• Christian Schumann · Gießen, DE
• Kai RITSCHEL · Wetzlar, DE
• Mate BELJAN · Wetzlar, DE
• Wernher Fouquet · Mannheim, DE
• Gheorghe Cojoc · Leonberg, DE

Key dates

• Filing date: Nov 28, 2022
• Grant date: Jun 24, 2025
• Expiry date: Jul 7, 2043

Classification

• Technology area (CPC G): Physics
• CPC primary: G02B21/367
• WIPO field: Measurement
• WIPO sector: Instruments

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.