Method for simultaneous detection of multiple fluorophores for in situ

Abstract

A fluorescent in situ hybridization method comprising the steps of (a) providing a cell nuclei having chromosomes hybridized with at least one nucleic acid probe including at least one nucleic acid molecule labeled with at least one fluorophore; (b) viewing the cell nuclei through a fluorescence microscope optically connected to an imaging spectrometer for obtaining a spectrum of each pixel of the cell nuclei by (i) collecting incident collimated light simultaneously from all pixels of the cell nuclei; (ii) passing the incident collimated light through an interferometer system so that the light is first split into two coherent beams and then recombine to interfere and form an exiting light beam; (iii) focusing the exiting light beam on a detector having an array of detector elements, so that at each instant each of the elements is the image of one and always the same pixel for the entire duration of the measurement and so that each of the elements produces a signal which is a particular linear combination of light intensity emitted by the pixel at different wavelengths; (iv) rotating or translating one or more of the elements of the interferometer, so that the optical path difference is scanned simultaneously for all the pixels of the cell nuclei; and (v) recording signals of each of the detector elements as function of time using a recording device to form a first spectral cube of data; and (c) interpreting the first spectral cube of data using a mathematical algorithm.