Multi-dimensional fluorescence apparatus and method for rapid and highly sensitive quantitative analysis of mixtures

An apparatus that provides rapid and sensitive quantitative analysis of a sample by fluorescence, the apparatus comprising:

• a single-mode pulsed laser that is directed to the sample to generate pulsed fluorescence in the sample, the pulsed laser having a shot-to-shot fluctuation no greater than one percent and a pulse energy greater than 1 micro-Joule:
• a fluorescence wavelength-selector that receives as an input a portion of the pulsed fluorescence from the sample and that outputs a fraction of the input fluorescence that lies within a specified wavelength range;
• a photodetector that receives fluorescence photons within the specified wavelength range as an input from the fluorescence wavelength-selector and outputs a time-dependent electrical signal;
• a digitizer coupled to the photodetector that receives the time-dependent electrical signal as an input and that converts the time-dependent electrical signal into a digitized signal;
• a recorder that receives the digitized signal from the digitizer and outputs a wavelength-time matrix that includes fluorescence decay curves for at least two emission wavelengths;
• and
• an analyzer that receives the wavelength-time matrix from the recorder and outputs a numerical value for the contribution of at least one fluorescent component to the data contained within the wavelength-time matrix.

An apparatus that provides rapid and sensitive quantitative analysis of a sample by fluorescence, the apparatus comprising:

• a repetitively pulsed excitation light source that is directed to the sample to generate pulsed fluorescence in the sample, the light source adapted to selectively output light pulses at various excitation wavelengths, the light source having a shot-to-shot fluctuation no greater than three percent at any of the excitation wavelengths;
• a fluorescence wavelength-selector that receives as an input a portion of the pulsed fluorescence from the sample and that outputs a fraction of the input fluorescence that lies within a specified wavelength range;
• a photodetector that receives fluorescence photons within the specified wavelength range as an input from the sample and outputs a time-dependent electrical signal;
• and
• a signal processor coupled to the photodetector that receives the time-dependent electrical signal as an input and determines a numerical value for the contribution of at least one component of the sample based on the time-dependent electrical signal.

An apparatus that provides rapid and sensitive quantitative analysis of a sample by fluorescence, the apparatus comprising:

• a single-mode input pulsed laser;
• an excitation wavelength-converter that receives as an input light pulses from the single-mode input pulsed laser, that is directed to the sample to generate pulsed fluorescence in the sample, and that selectively outputs light pulses at various excitation wavelengths, the light pulses having a shot-to-shot fluctuation no greater than one percent at any of the excitation wavelengths;
• a fluorescence wavelength-selector that receives as an input a portion of the pulsed fluorescence from the sample and that outputs a fraction of the input fluorescence that lies within a specified wavelength range;
• a photodetector that receives fluorescence photons within the specified wavelength range as an input from the sample and outputs a time-dependent electrical signal;
• a digitizer coupled to the photodetector that receives the time-dependent electrical signal as an input and that converts the time-dependent electrical signal into a digitized signal;
• a recorder that receives the digitized signal from the digitizer and outputs a wavelength-time matrix that includes fluorescence decay curves for at least two excitation wavelengths;
• and
• an analyzer that receives the wavelength-time matrix from the recorder and outputs a numerical value for the contribution of at least one fluorescent component to the data contained within the wavelength-time matrix.

A fluorometric method comprising:

• irradiating a sample with a plurality of light pulses having a shot-to-shot fluctuation no greater than three percent to generate pulsed fluorescence in the sample;
• selecting a portion of the pulsed fluorescence from the sample within a specified wavelength range;
• generating a time-dependent electrical signal based on the selected portion of the pulsed fluorescence;
• and
• determining a numerical value for the contribution of at least one component of the sample based on the time-dependent electrical signal.

A fluorometric method comprising:

• irradiating a sample using a repetitively pulsed excitation light source having a shot-to-shot fluctuation no greater than three percent to generate pulsed fluorescence in the sample;
• receiving a portion of the pulsed fluorescence from the sample at a fluorescence wavelength-selector;
• selecting a fraction of the fluorescence received at the fluorescence wavelength-selector that lies within a specified wavelength range using the fluorescence wavelength-selector and outputting fluorescence photons within the specified wavelength range from fluorescence wavelength-selector;
• receiving the fluorescence photons within the specified wavelength range from the fluorescence wavelength-selector at a photodetector;
• converting the fluorescence photons received by the photodetector into a time-dependent electrical signal using the photodetector and outputting the time-dependent electrical signal from the photodetector;
• receiving the time-dependent electrical signal from the photodetector at a signal processor;
• and
• determining a numerical value for the contribution of at least one component of the sample based on the time-dependent electrical signal using the signal processor.

A fluorometric method comprising:

• irradiating a sample using a single-mode pulsed laser having a shot-to-shot fluctuation no greater than one percent and a pulse energy greater than 1 micro-Joule to generate pulsed fluorescence in the sample;
• receiving a portion of the pulsed fluorescence from the sample at a fluorescence wavelength-selector;
• selecting a fraction of the fluorescence received at the fluorescence wavelength-selector that lies within a specified wavelength range using the fluorescence wavelength-selector and outputting fluorescence photons within the specified wavelength range from fluorescence wavelength-selector;
• receiving the fluorescence photons within the specified wavelength range from the fluorescence wavelength-selector at a photodetector;
• converting the fluorescence photons received by the photodetector into a time-dependent electrical signal using the photodetector and outputting the time-dependent electrical signal from the photodetector;
• receiving the time-dependent electrical signal from the photodetector at a digitizer;
• digitizing the time-dependent electrical signal;
• recording the digitized time-dependent electrical signal as a wavelength-time matrix that includes fluorescence decay curves for at least two emission wavelengths;
• and
• analyzing the wavelength-time matrix to determine a numerical value, wherein the numerical value represents the contribution of at least one fluorescent component to the data contained within the wavelength-time matrix.

A fluorometric method comprising:

• irradiating a sample with a plurality of light pulses selectively at two or more excitation wavelengths to generate pulsed fluorescence in the sample, the light pulses at each excitation wavelength having a shot-to-shot fluctuation no greater than three percent;
• selecting a portion of the pulsed fluorescence from the sample within a specified wavelength range;
• generating a time-dependent electrical signal based on the selected portion of the pulsed fluorescence;
• and
• determining a numerical value for the contribution of at least one component of the sample based on the time-dependent electrical signal.

A fluorometric method comprising:

• irradiating a sample using a repetitively pulsed excitation light source that selectively outputs light pulses at various excitation wavelengths and that has a shot-to-shot fluctuation no greater than three percent at any of the excitation wavelengths to generate pulsed fluorescence in the sample;
• receiving a portion of the pulsed fluorescence from the sample at a fluorescence wavelength-selector;
• selecting a fraction of the fluorescence received at the fluorescence wavelength-selector that lies within a specified wavelength range using the fluorescence wavelength-selector and outputting fluorescence photons within the specified wavelength range from fluorescence wavelength-selector;
• receiving the fluorescence photons within the specified wavelength range from the fluorescence wavelength-selector at a photodetector;
• converting the fluorescence photons received by the photodetector into a time-dependent electrical signal using the photodetector and outputting the time-dependent electrical signal from the photodetector;
• receiving the time-dependent electrical signal from the photodetector at a signal processor;
• and
• determining a numerical value for the contribution of at least one component of the sample based on the time-dependent electrical signal using the signal processor.

A fluorometric method comprising:

• generating a series of light pulses using a single-mode input pulsed laser;
• receiving the light pulses at an excitation wavelength-converter;
• selecting light pulses at various wavelengths using the excitation wavelength-converter and outputting the light pulses at the selected excitation wavelengths from the excitation wavelength-converter, the light pulses having a shot-to-shot fluctuation no greater than one percent at any of the excitation wavelengths;
• irradiating a sample with the light pulses output from the excitation wavelength-converter to generate pulsed fluorescence in the sample;
• receiving a portion of the pulsed fluorescence from the sample at a fluorescence wavelength-selector;
• selecting a fraction of the fluorescence received at the fluorescence wavelength-selector that lies within a specified wavelength range using the fluorescence wavelength-selector and outputting fluorescence photons within the specified wavelength range from fluorescence wavelength-selector;
• receiving the fluorescence photons within the specified wavelength range from the fluorescence wavelength-selector at a photodetector;
• converting the fluorescence photons received by the photodetector into a time-dependent electrical signal using the photodetector and outputting the time-dependent electrical signal from the photodetector;
• receiving the time-dependent electrical signal from the photodetector at a digitizer;
• digitizing the time-dependent electrical signal;
• recording the digitized time-dependent electrical signal as a wavelength-time matrix that includes fluorescence decay curves for at least two excitation wavelengths;
• and
• analyzing the wavelength-time matrix to determine a numerical value, wherein the numerical value represents the contribution of at least one fluorescent component to the data contained within the wavelength-time matrix.