Multi-color fluorescence analysis with single wavelength excitation

A method for determining one or more characteristics of particles using multiple fluorescence analysis comprising:

• directing an incident light beam at particles under analysis, the particles including at least three fluorescent markers each having different emission spectra, but each marker being excitable by light at the same wavelength, said incident light beam causing the excitation of said markers such that spectrally different wavelengths of fluorescence are emitted from said particles without substantial spectral overlap;
• spectrally separating with optical elements the different wavelengths of emitted fluorescence to simultaneously refine the emitted fluorescence to enable the separate detection of the different wavelengths;
• simultaneously detecting the three or more different fluorescence emissions associated with the particles under analysis;
• and
• associating said detected fluorescence with one or more characteristics of said particles.

A method for determining one or more immunofluorescent characteristics of cells or the like flowing in a liquid stream using multiple fluorescence analysis comprising:

• moving cells, substantially one at time, in a liquid flow stream, said cells including at least three cell-surface fluorescent markers thereon suitable for immunofluorescence analysis, each marker having different emission spectra in which the emission maxima are sufficiently spectrally separated so that there is no substantial spectral overlap;
• directing a light beam of a single wavelength at the cells in said flow stream to excite all of the fluorescent markers such that different wavelengths of fluorescence are emitted from said cells;
• spectrally separating with optical elements the different wavelengths of emitted fluorescence to enable the separate detection of the different wavelengths;
• simultaneously detecting the three or more different fluorescence emissions associated with the cells moving in the flow streams;
• and
• using said detected fluorescence to determine one or more characteristics of said cells.

An apparatus for determining one or more characteristics of cells or the like flowing in a liquid stream using multiple fluorescence analysis comprising:

• means for moving cells, substantially one at a time, in a liquid flow stream;
• means for directing a light beam at the cells in said flow stream in order to excite multiple fluorescence markers on said cells by a single wavelength of light;
• at least three fluorescence detection means for detecting spectrally separated fluorescence without substantial spectral overlap emitted by the cells moving the flow stream, each detection means including a select combination of optical light filter means which permit the simultaneous collection of spectrally separate fluorescence emission of at least three spectrally wavelengths;
• and
• means for using said detected fluorescence to determine one or more characteristics of said cells.

An apparatus for determining one or more characteristics of particles using multiple flourescence analysis comprising:

• means for directing a light beam at particles under analysis in order to excite multiple flourescence markers on said cells by a single wavelength of light;
• at least three fluorescence detection means for detecting spectrally separated fluorescence without substantial spectral overlap emitted by the particles, each detection means including a select combination of optical light filter means which permit the simultaneous collection of spectrally separate fluorescence emission of at least three spectrally separated wavelengths;
• and
• means for associating said detected fluorescence with one or more characteristics of said particles.