Patent Publication Number: US-2007096039-A1

Title: Evaluation Of Multicomponent Mixtures Using Modulated Light Beams

Description:
CROSS-REFERENCES TO RELATED Applications  
      This application claims benefit as a continuation-in-part of copending U.S. patent application Ser. No. 10/429,426 filed May 5, 2003, which claims benefit of U.S. Provisional Application No. 60/377,935 filed May 3, 2002. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH  
      The U.S. government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Contract No. DE-FG02-01ER83134 awarded by the Department of Energy. 
    
    
     FIELD OF THE INVENTION  
      The invention relates to a method and apparatus for analyzing a sample material in which two or more fluorescent dyes are present.  
     BACKGROUND OF THE INVENTION  
      Fluorescence spectroscopy is now a fundamental analytical tool in the physical, chemical, and biological sciences. Analysis of a sample material commonly involves the use of more than one fluorophore. For example, in the biological sciences, it is common to label cells with more than one fluorochrome to facilitate the study of cellular properties. Modulated light sources are regularly employed with lock-in detection techniques to attain better signal to noise measurements. However, it is often difficult to find suitable fluorescent dyes that share common excitation spectra but have separate emission spectra, whereby a single excitation source can be used and the emission spectra can be detected by detecting different wavelength regions of the emitted fluorescence light.  
      Several approaches to overcoming this limitation have been described. In one flow cytometer approach described by Steinkamp et al., different wavelengths of excitation light are used to sequentially excite fluorescent dyes having separated excitation spectra, and the emitted fluorescence light is detected using a multichannel detector arrangement for sequentially detecting different wavelength regions. See J. Steinkamp et al.,  Improved Multilaser/Multiparameter Flow Cytometer for Analysis and Sorting of Cells and Particles , Rev. Sci. Instrum., Vol. 62 (11), pages 2751-2764 (November, 1991). In another approach, fluorescent dyes having overlapping emission spectra can be utilized by employing an intensity-modulated excitation beam in cooperation with phase-resolution techniques to discriminate between emissions having different fluorescence lifetimes. See the following references: D. Jameson et al.,  The Measurement and Analysis of Heterogeneous Emissions by Multifrequency Phase and Modulation Fluoremtry , Applied Spectroscopy Rev. Vol. 20 (1), pages 55-106 (1984); L. McGown et al., Phase-Resolved Fluorescence Spectroscopy, Analytical Chemistry, Vol. 56 No. 13 (November, 1984); J. Steinkamp, U.S. Pat. No. 5,270,548 issued Dec. 14, 1993 for Phase-Sensitive Flow Cytometer; and J. Keij et al.,  Simultaneous Analysis of Relative DNA and Glutathione Content in Viable Cells by Phase - Resolved Flow Cytometry , Cytometry, Vol. 35, pages 48-54 (1999).  
      While these approaches have broadened analytical possibilities, the first approach adds cost and complexity to instrumentation hardware, and the second approach requires that the chosen dyes have significantly different fluorescence lifetimes.  
     SUMMARY OF THE INVENTION  
      The present invention involves a method and apparatus for analyzing sample materials containing more than one fluorescing species. The invention is embodied in an apparatus generally comprising means for providing a plurality of intensity-modulated excitation light beams for interaction with the sample material, each beam being modulated at a respective unique frequency; a photosensitive detector receiving fluorescence light emitted by the sample material in response to interaction with the excitation light beams and providing signal information representative of the intensity of received light; and means connected to the detector for receiving and processing the signal information to extract a plurality of component signals respectively attributed to the plurality of excitation light beams. The distinctive modulation frequencies of the excitation beams are present in the fluorescence light emitted by the sample material and received by the detector. Consequently, the detector signal information can be processed to extract component signals corresponding to each excitation frequency, for example by Fourier transform analysis of the detector signal information. Because each excitation beam has a unique frequency, the fluorescence signal contribution attributable to each excitation beam can be determined. Assuming knowledge of the excitation spectra and fluorescence quantum yields at different wavelengths of each fluorescent dye present, information about the concentrations of each fluorescing species can be derived.  
      The present invention is embodied, for example, in a flow cytometer having a pair of laser light sources that are intensity-modulated at different frequencies, a flow cell through which a fluid sample material passes and interacts with the modulated excitation beams, a filter which receives both scattered excitation light and emitted fluorescence light from the sample material and blocks the excitation light wavelengths, a photomultiplier tube behind the filter for receiving the fluorescence light and generating intensity signal information, and a digital storage oscilloscope for processing the signal information to determine the signal contribution attributed to each modulated excitation beam. Other embodiments are disclosed, including systems for analyzing bulk sample material in a sample well and a fiber optic system.  
      The method of the present invention generally comprises the steps of providing a plurality of intensity-modulated excitation light beams each being modulated at a respective unique frequency; directing the intensity-modulated excitation light beams to interact with the sample material; detecting fluorescence emission light from the sample material to provide signal information representative of detected light intensity versus time; and extracting a plurality of component emission signals from the signal information, wherein each component emission signal corresponds to a respective one of the modulated excitation light beams. The strengths of the component emission signals can then be evaluated in view of known dye properties to determine the concentrations of the fluorescing species in the sample material.  
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      The nature and mode of operation of the present invention will now be more fully described in the following detailed description of the invention taken with the accompanying drawing figures, in which:  
       FIG. 1  is a plot showing excitation and emission spectra of a pair of fluorescing species in a mixture, wherein the wavelengths of first and second excitation light beams are also indicated;  
       FIG. 2  is a schematic diagram of a flow cytometer formed in accordance with a first embodiment of the present invention for analyzing a sample material having more than one fluorescing species;  
       FIG. 3  is a schematic diagram of a flow cytometer formed in accordance with a second embodiment of the present invention for analyzing a sample material having more than one fluorescing species;  
       FIG. 4  is a schematic diagram of a system formed in accordance with a third embodiment of the present invention for analyzing a bulk sample material having more than one fluorescing species;  
       FIG. 5  is a schematic diagram of a system formed in accordance with a fourth embodiment of the present invention for analyzing a bulk sample material having more than one fluorescing species;  
       FIG. 6  is a schematic view showing, in relevant part, a system for analyzing bulk samples contained in an well plate having an array of sample wells;  
       FIG. 7  is a schematic diagram of fiber optic system formed in accordance with a fifth embodiment of the present invention for analyzing a sample material having more than one fluorescing species;  
       FIG. 8  is a schematic diagram showing an alternative means for generating a plurality of modulated excitation light beams in accordance with the present invention; and  
       FIG. 9  is a schematic diagram showing another alternative means for generating a plurality of modulated excitation light beams in accordance with the present invention.  
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      Referring initially to  FIG. 1  of the drawings, a plot showing excitation and emission spectra of a pair of hypothetical fluorescent dyes A and B is presented in order to illustrate one situation in which the present invention proves useful. As can be seen, dyes A and B have overlapping excitation spectra and the same emission spectra. A pair of laser excitation light beams at different wavelengths are indicated by dotted lines L 1  and L 2 . From a broad standpoint, the present invention involves intensity modulation of L 1  and L 2  at different frequencies so that their respective contributions can be extracted from the detected fluorescence signal.  
      Attention is now directed to  FIG. 2 , which schematically depicts a flow cytometer  10  formed in accordance with a first embodiment of the present invention. Flow cytometer  10  generally comprises first and second light sources  12  and  14 , a flow cell  16  through which a sample material flows, a photosensitive detector  18  arranged to receive fluorescence light emitted from the sample material, and a digital storage oscilloscope  20  connected to detector  18  by communication line  19 .In the embodiment shown, light sources  12  and  14  are semiconductor lasers each emitting light at 635 nanometers, however the light sources  12  and  14  can also be chosen to emit light at different wavelengths as shown for example in  FIG. 1 . Light sources  12  and  14  are each energized by current from a respective drive circuit  22 . The drive current supplied to each light source  12 ,  14  is modulated in known fashion according to a periodic waveform (preferably sinusoidal), with each light source receiving current modulated at a different frequency and not harmonics of one another. Consequently, light emitted by light sources  12  and  14  is modulated at a respectively unique frequency corresponding to the associated drive current modulation frequency. As can be seen in  FIG. 2 , light sources  12  and  14  together with associated current drive circuits  22  serve as a means  24  for providing a pair of intensity-modulated excitation light beams L 1  and L 2  for interaction with the sample material flowing through an interrogation zone of flow cell  16 . As used herein, the term “light beam” is intended to have a broad meaning, and includes without limitation convergent, divergent, and collimated beams; electromagnetic flux by fiber optic transmission; and any flow of electromagnetic waves.  
      Excitation light from beam L 1  is reflected by a beam combining optical element  26  for travel along optical path  28 , whereas excitation light from beam L 2  is transmitted by optical element  26  for travel along the same optical path  28 . Beam combining optical element may be, for example, a polarizing cube beamsplitter where L 1  and L 2  have s and p polarizations. The differently modulated excitation light L 1  and L 2  is stretched along one axis by a cylindrical lens telescope system  30  and then focused on an interrogation zone in flow cell  16  by a focusing lens  32 .  
      A collector lens or lens system  34  is arranged adjacent to flow cell  16  along a path that is orthogonal to incident beam path  28  for collimating scattered excitation light and emitted fluorescence light coming from the sample material. A filter  36  is provided after collector lens  34  and in front of detector  18  to remove scattered excitation light at 635 nm.  
      In the present embodiment, detector  18  is a photomultiplier tube (PMT), for example a Perkin Elmer CPM C962-2. Detector  18  generates signal information representative of the intensity of received light, which signal information is transmitted over line  19  to digital storage oscilloscope  20 . The signal information from detector  18  is in the form of an aggregate emission signal wherein signal amplitude changes with time. The intensity modulation frequencies in L 1  and L 2  are chosen so that the modulation frequencies are reproduced in the fluorescence light emitted by the sample material. The signal information is processed by digital storage oscilloscope  20  to extract two component emission signals respectively corresponding to intensity modulated beams L 1  and L 2 . More specifically, digital storage oscilloscope  20  collects the signal data into a file which can be later analyzed using a Fast Fourier Transform (FFT) algorithm to find the contribution of each modulation frequency to the aggregate fluorescence emission signal generated by detector  18 .  
      The extracted component emission signals can be evaluated to derive the concentrations of the fluorescing species in the sample material. In the case of one dye, the strength of the collected signal for any one laser is a function of: 1) light intensity (laser power, focusing arrangement, optical losses); 2) probability of light absorption (absorption coefficient); 3) likelihood of a fluorescence emission (fluorescence quantum yield); 4) other factors affecting the measured intensity of fluorescence emission such as reabsorption of fluorescence light by the sample, quenching (especially as a function of increased concentration), and environmental factors such as complex formation; 5) probability that a fluorescence photon will reach the detector (efficiency of light collection system); 6) response of the detector at the fluorescence wavelength; and 7) signal electronics. It is a challenge to precisely relate signal intensity to concentration from consideration of the above parameters alone. However, one can calibrate the method and equipment using standards having the same dyes at known concentrations.  
      In the absence of any kind of self absorption the, fluorescence signal S f  is related to the excitation intensity I with the following relation. 
 
 S   f   =KIη (1 −e   −94 Nl   (1)
 
 where K is a constant of the collection and detection system, η is the quantum efficiency, σ is the absorption cross-section, N is the concentration of the fluorescent dye and l is the length of the interaction volume. If the value of product σNl is very small, the above equation can be rewritten as 
 
S f ≈KINlησ  (2).
 
 If there are two types of fluorescent dye and two excitation lasers the signal due to both the lasers will be given as 
 
 S   1   =KlI   1 ( N   A η A σ A1   +N   B η B σ B1 )  (3)
 
 S   2   =Kll   2 ( N   A η A σ A2   +N   B η B σ B2 )  (4)
 
 where subscripts A and B refer to the two fluorescent dyes and the subscripts 1 and 2 refer to the two excitation beams respectively. Here it is assumed that the interaction length l for both the excitation beams is the same. It is clear from equation (3) and (4) that information about the concentration of two fluorescent dyes can easily be obtained from the two fluorescence signals (S 1  &amp; S 2 ) if the system parameter K, excitation intensities (I 1 , &amp; I 2 ) and the fluorophore parameters (η A , η B  &amp; σ A1 , σ A2 , σ B1 , σ B2 ) are known. 
 
      To further illustrate how this technique can be employed the following table based on the hypothetical system of  FIG. 1  is provided to illustrate how the extracted component emission signals can be evaluated to provide an indication of relative concentrations of fluorescing species A and B in a sample material.  
                                                                   1   2   3   4   5   6   7   8   9   10       Mixture   Conc. A   Conc. B   Signal L1-A   Signal L1-B   Signal L2-A   Signal L2-B   Total L1   Total L2   Ratio L1/L2                  #1   0   1   0*5   1*1   0*1   1*5   1   5   0.2       #2   0.1   1   0.1*5   1*1   0.1*1   1*5   1.5   5.1   0.3       #3   0.25   0.75   0.25*5   0.75*1   0.25*1   0.75*5   2   4   0.5       #4   0.5   0.5   0.5*5   0.5*1   0.5*1   0.5*5   3   3   1.0       #5   0.75   0.25   0.75*5   0.25*1   0.75*1   0.25*5   4   2   2.0       #6   1   0.1   1*5   0.1*1   1*1   0.1*5   5.1   1.5   3.4       #7   1   0   1*5   0*1   1*1   0*5   5   1   5.0       #8   1   1   1*5   1*1   1*1   1*5   6   6   1.0                  
 
 In this hypothetical system it is assumed that the absorption (molar extinction ratios), fluorescence quantum efficiencies, and fluorescence lifetimes of dyes A and B are equivalent. We have also assumed that fluorescence is not reabsorbed, and there are no quenching or other environmental factors altering the dyes&#39; properties. The concentration of dyes is low enough that the attenuation of excitation intensity is negligible. Also for purposes of simplicity, it is assumed that that the intensities of L 1  and L 2  are the same, their wavelengths are as shown, and their modulation frequencies are not. harmonics of one another. Consequently, excitation beam L 1  excites dye A five times more than it excites dye B, while excitation beam L 2  excites dye B five times more than it excites dye A. The detector receives a fraction of the fluorescence light that is generated from both sample dyes. The time varying signal from the detector has two components: a component at the frequency corresponding to modulated excitation beam L 1  and a component at the frequency corresponding to modulated excitation beam L 2 . 
 
      In the above table, column 1 assigns a case number to specific concentrations of dye A (column 2) and dye B (column 3). Columns 4-7 calculate the contribution to the total signal for each dye-laser combination. These values are the product of the concentration and a measure of the amount of absorbed light at the laser wavelength. Column 8 is the sum of columns 4 and 5 and column 9 is the sum of columns 6 and 7. The values of columns 8 and 9 are equivalent to the two frequency components of the aggregate or total detector signal and can be experimentally determined. As mentioned above, these component signal values can be derived by Fourier transform analysis of the signal information from detector  18 . The ratio of column 8 to column 9 is entered in column 10  
      The table illustrates several important points. First, samples containing either A alone or B alone have widely different characteristic ratios (column 10), namely 5.0 for A alone (Mixture #7) in contrast to 0.2 for B alone (Mixture #1). Thus, if a cell or bead marked by species A alone or species B alone passes through the interrogation zone of flow cytometer  10 , the particle&#39;s identity can be readily ascertained by the ratio. This development is already an important accomplishment of the present invention and permits particles marked by dyes having overlapping spectral properties to be mixed together and be readily distinguished from one another. Second, it will be observed that the mixtures of species A and B also give rather distinct ratio values. When the absolute amounts of A and B are different, but the ratio of their concentrations is the same (Mixtures #4 and #8), the characteristic ratio L 1 /L 2  is the same, but not the values in columns 8 and 9 which are directly proportional to dye concentrations.  
      In the embodiment described above, the detector  18  is free to detect the entire bandwidth of the fluorescence emission light from the sample material, except for the spectral region of scattered light blocked by filter  36 . The capabilities of the basic system can be improved dramatically by adding further light sources providing excitation light at different wavelengths along with further detectors to detect different wavelength regions. In this manner, a large number of light sources and detectors can be used to obtain even greater amounts of information. The option of spatially separating different laser beams is envisioned in order to improve performance.  
       FIG. 3  shows a flow cytometer  40  formed in accordance with a second embodiment of the present invention. Flow cytometer  40  generally comprises first and second light sources  12  and  14 , a flow cell  16  through which a sample material flows, a photosensitive detector  18  arranged to receive fluorescence light emitted from the sample material, signal electronics  42  connected to detector  18  by communication line  19 , and a computer  44  having an internal analog-to-digital conversion card  46 . In the embodiment shown, light sources  12  and  14  are continuous wave lasers each emitting excitation light at different frequencies from one another. The excitation light beams from light sources  12  and  14  are each modulated by a respective beam modulator  21  acting on the associated excitation light beam. Each modulator  21  functions to modulate the intensity of the excitation light beam coming from an associated light source  12  or  14  according to a periodic waveform, such that the excitation light beams are modulated at respectively unique frequencies corresponding to the setting of the associated modulator  21 . Thus, light sources  12  and  14  together with associated beam modulators  21  function as a means  24  for providing a pair of intensity-modulated excitation light beams L 1  and L 2  for interaction with the sample material flowing through an interrogation zone of flow cell  16 .  
      Modulated excitation light beam L 1  passes through a telescope system  23  and is reflected by a mirror  29  in the direction of beam combining optical element  26 . Likewise, modulated excitation light beam L 2  passes through a telescope system  23  and is reflected by a pair of mirrors  29  such that beam combing optical element  26  will transmit light from beam L 2  and reflect light from beam L 1  along a common optical path  28 . The differently modulated excitation light L 1  and L 2  is stretched along a single axis by cylindrical lens telescope system  30  and then focused on an interrogation zone in flow cell  16  by focusing lens  32 .  
      As in the first embodiment described above, a collector lens  34  is arranged adjacent flow cell  16  along a path that is orthogonal to incident beam path  28  for collimating scattered excitation light and emitted fluorescence light coming from the sample material. A filter  36  is provided after collector lens  34  and in front of detector  18  to remove scattered excitation light. In the present embodiment, the signal information generated by detector  18  is transmitted over line  19  to signal electronics  42 , which extracts and amplifies those portions of the detector&#39;s signal attributed to L 1  and L 2 . These two analog component signals are then digitized by an analog-to-digital conversion card  46  installed in computer  44 . As discussed above, this information is useful for determining the presence and concentrations of fluorescing species excited by beams L 1  and L 2 .  
      Flow cytometry is an established method wherein cells, macromolecules, polymer beads, or other definite objects are made to flow in a narrow stream and are optically illuminated so as to produce light signals indicative of size, molecular composition, and other structural or functional properties. A very common signal measured in a flow cytometer is the fluorescence coming from a dye introduced to measure some property of the definite object. It is important to recognize that these events occur randomly.  
      The need to produce a sufficiently strong fluorescence signal with a cost effective light source and produce a signal that does not significantly vary depending upon the definite object&#39;s position in the stream, necessitates that the stream be narrow.  
      Preferred stream widths are typically between 5 and 100 microns in diameter. The need to maintain stable flow conditions and provide sufficiently fast acquisition (events/sec) suggests preferred flow rates of 0.5 to 20 m/s. The time that an object actually spends being illuminated (event duration) varies and depends upon several parameters, but is typically and preferably only a few (for example, 2) microseconds.  
      The application of the modulation technique described in this patent application to these transient events is not trivial and requires careful attention to operational parameters. The modulation frequency must be sufficiently high so as to preferably have a minimum of two modulations of light intensity during the event duration. For example if a event duration is 1 us (baseline to baseline), a modulation frequency of 2 MHz will provide only 2 periods of intensity modulation. In practice, additional periods of modulation are preferred to more accurately determine the frequency components of the recorded signal. While it is possible to modulate illuminated light beams at higher frequencies than 2 MHz, it becomes more of a challenge.  
      The more serious challenges, however rest with other processes and components of the flow cytometer. Every fluorescent dye has a characteristic fluorescence lifetime which is a measure of the time necessary for a electronically excited dye to return to its ground state. Most organic dyes have fluorescence lifetimes that are between 0.5-100 ns. If the modulation frequency is too high, the fluorescence signal&#39;s modulation depth will decrease and the signal will begin to become other than sinusoidal and make detection and analyses more difficult and, in the extremely high frequency case, impossible. In addition, the frequency response of the detector and amplification circuits must be sufficiently high to faithfully record the modulation signal. Furthermore, high frequency noise is common in electronic circuitry, has a variety of sources, and can be confused with signals at these higher frequencies. An additional point is that many amplifier circuits trade gain for increased frequency response which will tend to prevent weaker signals from being detected as efficiently.  
      When all these considerations are taken into account, the preferred maximum modulation frequency is 100 MHz. Thus, there is a narrow range (2-100 MHz) of preferred modulation frequencies and the need to use two or more distinguishable frequencies to practice the method. Given the limited sampling available, modulation frequencies (and their harmonics) should be preferably be far enough apart. In practice, one should preferably have the modulation frequencies separated by 1 MHz or more to minimize the crosstalk between signals arising from different modulation frequencies.  
      Given the preferred separation between frequencies and the need to use two or more different frequencies it is fortunate for this technique that there is sufficient preferred range of frequencies available (2-100 MHz). Attempting to use a modulation frequency below 2 MHZ would require impractically slow stream velocities which would produce lower signal to noise measurements. Attempting to modulate above 100 MHz would be expensive, confuse signal with high frequency noise, tend to limit amplifier gain and start to distort the sinusoidal nature of the recorded signal (different for each dye having a different characteristic fluorescent lifetime which can also vary upon the dye&#39;s local conditions).  
       FIGS. 4 and 5  illustrate further embodiments of the present invention for analyzing a bulk quantity of sample material. In the system of  FIG. 4 , two light sources  12  and  14  are arranged on opposite sides of a sample well  52  containing sample material in bulk. The light sources are internally modulated lasers each modulated at a fixed frequency that is different from the frequency of the other laser and not harmonics of one another. System  60  of  FIG. 5  is generally similar to system  50 , however light source  12  is a blue LED and light source  14  is a green LED. A function generator  33  modulates current supplied to each LED, such that the light emitted by each source is intensity modulated at a frequency that differs from the modulation frequency of the other source and are not harmonics of one another. The detection arrangements are the same for each system, and include a filter  36  for blocking excitation wavelengths, a detector  18  after the filter, and a digital storage oscilloscope  20  receiving fluorescence signal information over line  19 .  
       FIG. 6  shows an application wherein numerous sample materials are analyzed automatically. A well plate  51  is shown as including an array of sample wells  52  for holding sample materials. Excitation light traveling along path  28  strikes dichroic mirror  31  and is reflected to the sample material in an aligned sample well  52 . Fluorescent emission light is transmitted through dichroic mirror  31  and filter  36  to detector  18 . Well plate  51  can be moved automatically in a horizontal X-Y plane by an automatic drive  55  to align a different sample well  52  for analysis. Of course, dichroic mirror  31 , filter  36 , and detector  18  could also be moved while well plate  51  remains fixed to align another sample well  52 .  
      Turning now to  FIG. 7 , a system  70  of the present invention incorporating fiber optic transmission is shown. This embodiment is primarily intended to increase sensitivity. System  70  comprises a modulated light sources  12  and  14 , a bifurcated optical fiber  72 , and optical means  37  associated with each light source for coupling light into optical fiber  72 . Bifurcated optical fibers are commercially available, one example being the SPliT200-UV-VIS bifurcated fiber available from Ocean Optics Inc. A distal end of the optical fiber is coated with sample material  74 , such as an antibody that specifically binds an antigen. If the antigens are already fluorescently labeled, then the fluorescent labels will be excited by modulated light near the surface of fiber  72  (fluorescent dyes any distance from the surface of fiber  72  are not excited). Some of the fluorescent light is trapped in the fiber and is transmitted back through decoupling optics  39  to detector  18  connected to signal processing electronics  76 . In a situation wherein the sample material is a dilute solution of fluorescently labeled antigen in water, the distal end of optical fiber  72  could be immersed in the solution to provide sufficient illumination and improve collection efficiency with respect to fluorescent light.  
      While the embodiments described above show the use of multiple light sources to generate excitation beams at different wavelengths, the present invention can also be practiced using a single light source as shown in  FIGS. 8 and 9 . In  FIG. 8 , single light source  12  is a multiline argon ion laser emitting light grouped in bands about several different strong wavelength lines. The laser light is passed through a dispersive optical element  25 , such as a prism or grating, to spectrally separate the laser light into a plurality of excitation beams each at a different central wavelength. Each of the excitation beams is reflected by a mirror  29  and passed through a beam modulator  21  and an attenuator  27 . The respective modulators  21  are set at different modulation frequencies. The modulated excitation beams are reflected by further mirrors  29  to pass in reverse fashion through another dispersive element  25 , whereby the modulated excitation beams are recombined and directed along the same optical path. A similar system is shown in  FIG. 9 , however a series of dichroic mirrors  31  is used to separate out different wavelength bands from argon ion laser  12 , and another series of dichroic mirrors  31  recombines and directs the modulated beams along the same optical path. Dichroic mirrors  31  could also be interference filters, fiber optic Bragg filters, or any optical element that reflects a portion of incident light and transmits a portion of incident light depending on wavelength. As will be appreciated, the single light source systems of  FIG. 8  and  FIG. 9  can be substituted for a multiple light source system as a means for providing a plurality of intensity-modulated excitation light beams.  
      It will be understood that various types of light sources, modulation techniques, detectors, and processing means can be employed to practice the present invention. By way of non-limiting example, possible light sources include internally modulated lasers such as Power Technology&#39;s IQ series lasers and LaserMax, Inc.&#39;s LSX series lasers; single wavelength continuous wave (cw) or quasi-cw lasers; multiline argon ion lasers such as the Spectra Physics Stabilite 2017; light emitting diodes (alone or equipped with filters to narrow the emission wavelength range); and white light sources combined with selectively transmitting wavelength filters.  
      Modulation techniques include all possible techniques for creating an intensity modulated beam, including the use of internal modulation at the sources or external modulation downstream from the source. As used herein, “internal” modulation refers to any modulation technique that causes light to leave its source in modulated form. Examples of internal modulation techniques include direct modulation using current drive electronics such as a Wavetek Model 178-50 MHz Programmable Waveform Synthesizer, and the use of internally modulated lasers. As used herein, “external” modulation refers to any modulation technique that modulates light after it has left its source. Examples of external modulation devices include mechanical choppers, variable attenuators such as a liquid crystal devices, electro-optic modulators, acousto-optic modulators such as IntraAction&#39;s ATM200C1 modulator and Model ME driver, Mach-Zehnder interferometers, and rotating polarizers.  
      Various types of photosensitive detectors can be used depending upon the application. These include, without limitation, photomultiplier tubes such as the Model HC120-15 by Hamamatsu or Perkin Elmer PMTs; photodiodes and avalanche photodiodes; photoresistive detectors, charge coupled devices and CCD arrays.  
      Signal processing to derive the respective modulation frequencies in the fluorescence light can be accomplished using hardware and software techniques. A digital storage oscilloscope having FFT capability is readily useful for this purpose. Other possibilities include the use of lock-in amplifiers such as the EG&amp;G Princeton Applied Research Model 5302, frequency selective analog circuits akin to those used in radios to select stations at known frequencies, and analog-to-digital conversion by a PC Card in combination with execution of Fourier transform software.  
      In accordance with the apparatus embodiments described above, the present invention further encompasses a method for analyzing sample material having more than one fluorescing species. Stated broadly, the method comprises the steps of providing a plurality of intensity-modulated excitation light beams each being modulated at a respective unique frequency; directing the intensity-modulated excitation light beams to interact with the sample material; detecting fluorescence emission light from the sample material to provide signal information representative of detected light intensity versus time; and extracting a plurality of component emission signals from the signal information, wherein each component emission signal corresponds to a respective one of the modulated excitation light beams. The central wavelength of each excitation light beam can be chosen depending upon the spectral properties of the fluorescent species involved for optimal signal-to-noise characteristics in the detection signal. The intensity-modulated excitation light beams can be directed along a common optical path to interact with said sample material, or they can be directed along separate optical paths to interact with said sample material. As described above in connection with the hypothetical analysis table, the method may further comprise the step of evaluating the plurality of component emission signals to determine concentration information regarding the fluorescing species in said sample material.  
      As will be appreciated from the foregoing description, the present invention is widely applicable in fluorescence spectroscopy. One application that is contemplated for biotechnology research is the identification of several naturally florescent proteins. A common procedure in biotechnology is to introduce foreign genetic matter into cells that codes for the production of a naturally fluorescent protein. Many such proteins exist, but generally only a few are employed at one time. Mutants of the most commonly used proteins are known to exist and often have different excitation and emission spectra. It is desirable to employ more of these proteins at one time, however a central problem to this approach is the spectral overlap of the dyes. By employing a number of excitation beams at different wavelengths and modulation frequencies, a flow cytometer operator can discriminate between dyes with similar spectroscopic properties. Information about which proteins are present in what quantities and at what time is useful for measuring one or more properties of a cell&#39;s genome. Specific applications include drug discovery, disease studies, and genetic modification studies. The present invention can also be used for chromatography and time dependent analysis of molecular processes.  
      From a general standpoint, the invention is suitable for analyzing gas and liquid mixtures in a flow stream. It is also suitable for measuring solid, liquid or gas mixtures in bulk. Of particular interest is the case of flowing liquids which may have dissolved or suspended fluorescing species.