Abstract:
A flow cytometer has a flow cell through which a sample flows and at least one laser emitting an excitation beam for illuminating a corresponding interrogation region in the flow cell. Scattered and fluorescence light from each interrogation region is collected by one or more input fibers for that region, and the input fiber(s) are fed to a dispersion module for that interrogation region that disperses the incoming light into different spectral regions. The dispersed light is conveyed, such as by a plurality of output fibers, to one or more photosensitive detectors. Thus, time multiplexed light signals may be delivered to a detector whereby several unique light signals can be measured by a single detector.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims benefit of U.S. Provisional Application Ser. No. 60/675,255 filed Apr. 27, 2005, which is hereby incorporated by reference herein. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH  
       [0002]     This work was supported by Grant No. DE-FG02-01ER83134 from the Department of Energy. The United States Government has certain rights in the invention. 
     
    
     FIELD OF THE INVENTION  
       [0003]     The invention relates to the field of flow cytometry.  
       BACKGROUND OF THE INVENTION  
       [0004]     Flow cytometry is a technique wherein light (usually focused laser beams) of specific wavelengths is used to illuminate cells, beads, macromolecules, etc. as they flow in a narrow stream. Scattered light is collected in the forward and side directions to provide information relating to the size, structure, etc. of the particles.  
         [0005]     A common practice in flow cytometry is to employ highly fluorescent dyes to label cells in specific ways. One common method is to attach these dyes to biologically active molecules such as antibodies which selectively attach themselves to specific sites on or inside of cells. In this way, cells having these binding sites will be labeled and demonstrate fluorescence of a particular emission color when illuminated by light of the appropriate wavelength.  
         [0006]     Regardless of how these dyes find themselves associated with cells, the strength of the measured signal is to a first approximation proportional to the amount of dye present with the cell. This method of labeling cells is now widespread in the study of biological specimens and is routinely done in samples prepared for fluorescent microscopes, flow cytometers, or other applications.  
         [0007]     Flow cytometers are unique among many of these instruments because they do not make aggregate measurements (signals coming from a number of cells). While fluorescent microscopy can also examine single cells, the process is usually characterized by small sample sizes, and poor quantitation.  
         [0008]     It is common to use more than one fluorescent dye in an effort to gain additional information about the cells as they pass through the illuminating light beam. To distinguish the signals from each other, the fluorescent labels are typically chosen so that they can be excited by the same light source, but fluoresce at different colors which can be separated using one or more techniques. Most dyes have a range of wavelengths at which they can be excited from their ground state to excited states. These molecules emit light over a range of wavelengths which is red shifted with respect to the illumination source when they return to their ground state.  
         [0009]     To more efficiently distinguish between different fluorescent light signals, the emission spectra of different dyes are chosen so there is little or no spectral overlap. A technique known as FRET (fluorescence resonance energy transfer) uses dye combinations to tune the emission spectra further away from the excitation light and thereby increase the range over which fluorescent signals can be distributed in the electromagnetic spectrum. Dyes that have narrower emissions are preferred to those with broad emissions since their use permits more dyes to be used at the same time. Armed with knowledge of the dyes&#39; emission spectra, it is possible and practical to account or compensate for the influence of an adjacent dye on another dye&#39;s signal provided the emission overlap is not too great.  
         [0010]     It is a common desire for flow cytometer practitioners to use several fluorescent labels at the same time. Rather than attempting to fit all these dyes along the electromagnetic spectrum with negligible emission overlap, a common technique is to use multiple light sources spatially separated along the stream of flowing cells. In this manner dyes that have the same emission spectra, but different excitation spectra can be used to label the cells and the signals can be more easily distinguished.  
         [0011]     A common flow cytometer design is to collect the signal light from all spatially separated interrogation regions and use wavelength dependent mirrors to separate the differently colored light signals. Apertures can be used to pass the light coming from a single interrogation region. In this manner, each detector would be used to detect a specific wavelength range coming from a single interrogation region. Using this type of arrangement common in flow cytometry, it takes n detectors to measure n different wavelength signals.  
         [0012]     The systems described above employ only free space optics. Recently, commercial flow cytometers have employed a linear array of optical fibers to separate light from different interrogation regions. The image of the flow cell is focused onto the input plane of a linear array of optical fibers. In this manner, light from each interrogation region enters a unique fiber.  
         [0013]     Each of these fibers then delivers the light from a single interrogation region to an arrangement of wavelength dependent mirrors to direct light of different wavelength ranges each to a different detector. These arrangements of wavelength dependent mirrors are larger than desired (owing to the size of beam diameters, mirrors, and detector housings. Each of these arrangements require n detectors to measure n signals.  
         [0014]     The wavelength dependent mirrors are arranged such that incident light is not normal to them, but rather at some other angle. To the extent that incident light is not normal, the spectral properties of the mirror change and more importantly, the s and p polarizations tend to have different reflection/transmission properties. Since the signal light in a flow cytometer is upolarized, the transitions between reflected wavelengths and transmitted wavelengths become less sharp at off-normal incidence. To address this fact, manufacturers have increased the path length between wavelength selective mirrors to approach a closer-to-normal angle of incidence. While effective, the system becomes larger in size and more sensitive to misalignments and airborne dust.  
       SUMMARY OF THE INVENTION  
       [0015]     This application describes innovations to a flow cytometer&#39;s signal light collection/distribution and detection system which fundamentally change the way signals are detected. There are important new applications stemming from this technology and serious reductions in required space and component costs. The system is flexible in operation, modular and expandable.  
         [0016]     This patent application describes how the image of a flow cell can be focused onto a linear fiber array where each interrogation region is optically coupled with a different INPUT FIBER of the linear fiber array. The INPUT FIBERS are multimode fibers (larger core diameter and have numerical apertures which are compatible with the light collection and focusing lenses) to facilitate greater coupling efficiencies. The individual INPUT FIBERS are separated and fed into a DISPERSION MODULE which disperses multiwavelength light signals. These signals are coupled into an array of OUTPUT FIBERS, each containing a specific wavelength range, which are then directed to a set of detectors. The proposed arrangement involves fewer optical interfaces, which reduces optical losses, is more compact, more easily reconfigured, more robust, less prone to airborne dust, and can be made less expensively. This method permits time multiplexed signals to be delivered to detectors permitting several unique signals to be measured by a single detector. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0017]     The invention will be explained in more detail, by way of example, with reference to the figures in which:  
         [0018]      FIG. 1  is a block diagram illustrating an embodiment of the present invention; and  
         [0019]      FIG. 2  is a depiction of how signals from different interrogation regions in a flow cell can be combined and detected at single detectors.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0020]      FIG. 1  is a block diagram of a flow cytometer formed in accordance with an embodiment of the present invention. Lasers ( 1 ) are used as excitation sources. The use of more than one laser is common. Optics are typically used to combine the beams ( 2 ) and direct them ( 3 ) to the stream of flowing cells located in a flow cell ( 4 ). Laser light is scattered in all directions. Typically, light scattered in the forward direction (forward scatter) gives information different from that scattered at 90 degrees (side scatter). Both forward scatter and side scatter are commonly measured. Side scattered light and omnidirectional fluorescent light are collected and imaged using a set of optical elements (primarily lenses) ( 5 ) and efficiently coupled into an Input Fiber(s). One embodiment of the invention is to collect signal light from different sides of the flow cell in an effort to collect greater amounts of signal. For example, signal light is normally collected at 90 degrees to the direction of the lasers. An equivalent collection system can be placed at 270 degrees effectively doubling the light collection efficiency of the system. The two Input fibers contain the same information and can be combined effectively improving the sensitivity of the system. The multiwavelength light enters a Dispersion Module which houses an optical element capable of dispersing incoming light. Typically a diffraction grating or prism is used. Diffraction gratings typically have higher dispersion, have efficiencies that more wavelength dependent, and have more linear dispersive properties. Prisms are less wavelength dependent efficiencies, but require greater path lengths to disperse the light over a fixed distance and can have a nonlinear wavelength dependent dispersion.  
         [0021]     In either case, once the incoming light is dispersed, it is focused onto an array of output fibers wherein each output fiber contains a particular range of wavelengths. The Output Fibers can be placed in contact with one another in a linear array or each positioned uniquely. The Output fibers are typically multimode fibers to facilitate efficient coupling and overall alignment stability (less sensitive to temperature changes and other sources of misalignment). Arranging the Output Fibers in a close packed array minimizes losses at fiber to fiber junctions and permits all wavelengths over a fixed range to be efficiently collected into the Output Fibers.  
         [0022]     The diameter and number of fibers can vary to some degree. For a given range of emission wavelengths to be detected, for instance 400 nm, a grating is chosen that disperses this range of wavelengths efficiently and distributes them over a distance of (for instance) 1 cm at a distance 10 cm from the grating. A lens is placed after the grating to focus the signal light at the input faces of the Output Fibers. The result of the dispersion and focusing optics is a streak of light with the light distributed accorded to wavelength. Placing a close packed array of 1 mm diameter Output Fibers at this distance permits (in this example) approximately 40 nm bandwidth of light to be placed into each of 10 fibers. There is some small overlap depending upon the resolution attainable by the Dispersion Module which is largely dependent upon the Input Fiber&#39;s diameter (effectively the slit width of the Dispersion Module). In principle, a greater number of smaller diameter Output Fibers could be used to reduce the wavelength range in each output fiber. However, this strategy will ultimately increase the coupling losses at the junctions between adjacent fibers and ultimately above and below the Output Fibers when the Output Fiber diameters become smaller that the focused light (streak). Alternatively, a two dimensional bundle of smaller fibers can be used. Alternatively, for users who measure the same signals all the time, fibers of different sizes could be employed each placed in a fixed position along the streak in an effort to collect known signals.  
         [0023]     Alternatively, Output fibers of the same or different sizes could be freely positioned along the streak to optimize the collection of signals at known wavelengths.  
         [0024]     Alternatively, a one or two dimensional system of adjustable micromirrors could be used in place of the Output Fibers, reflecting the signal light to one or more detectors suitably arranged. This version of the device could either employ fixed position mirrors or computer controlled mirrors permitting the user greater flexibility. The mirrors could be free to move in one or two axes.  
         [0025]     Although it is common to have round fibers based upon the methods of fabrication and the need to minimize transmission losses, fibers of different cross-sectional shapes may be used to increase coupling efficiency by minimizing losses at fiber-to-fiber junctions.  
         [0026]     A given fluorescence light signal may be found in more than one fiber either because the spectral width of the light signal is larger than the input wavelength range of a single fiber or because no single fiber is positioned to precisely match the wavelength range of the fluorescence light. Therefore, to efficiently detect the weakest signals, it may be desirable to combine the light output from two or more adjacent fibers and direct their light to a common detector.  
         [0027]     Similarly, light signals from other interrogation regions and hence, from other Output Fiber sets, can be similarly combined at a common detector. If the detector&#39;s area is sufficiently large, it may be possible to physically bundle all the desired Output Fibers and place their terminal ends in such a manner to efficiently detect all the signals. If there are too many fibers to conveniently or efficiently detect all the signals adequately, it may be necessary to use lenses attached to the Output Fiber ends (or placed in close proximity) to collimate (or focus at some distance) the exiting light from each of the Output Fibers. It may be necessary to arrange the Output Fibers at different incidence angles and/or distances to the detector to accommodate all the fibers required. It may be desirable to use one or more wavelength selective filters between the Output Fibers and the detector to remove unwanted wavelengths to potentially improve signal-to-noise in the measurements.  
         [0028]     As discussed, it is possible to combine signals arising from different interrogation regions since they are generated at different times and hence distinguished from one another. The strength of scatter and fluorescence signals can vary over several orders of magnitude. While some effort should be made to combine signals of similar strength to the same detector, a logarithmic amplifier is capable of expanding the dynamic range of the detectors. It is also possible to optically attenuate bright signals through a variety of means including the use of filters, cross polarizers, the use of smaller Output Fibers, etc.  
         [0029]     It is important to appropriately position the focused laser beams defining the interrogation regions so that a cell or other object passes through the interrogation regions at known times. The time between interrogation regions is dependent upon the flow velocity and the distance between the interrogation regions. The Input Fibers are preferably arranged a fixed distance from each other and helps to define the spacing of the interrogation regions. In this example, the time elapsed when a cell travels from one interrogation region to the next is inversely proportional to the flow velocity. Preferable flow velocities are those that produce stable (laminar) flow conditions and permits sufficient numbers of cells to be measured per unit time. The elapsed time between interrogation regions must also permit the detector and amplifier circuits to sufficiently recover from the signal generated at the preceding interrogation region. The simplest embodiment is to hold the sheath liquid constant to establish a stable flow velocity and to alter the core pressure to adjust the core stream width and adjust the number of events recorded per unit time.  
         [0030]     A logarithmic amplifier can require a longer time to recover particularly from a bright signal. We have found that slowing the flow velocity to create a 12 microsecond delay between signals from adjacent interrogation regions is adequate, but it could be as short as 3 and as long as 100 microseconds as long as this value is fixed and permits the amplifiers sufficient time to recover.  
         [0031]     Preferably, the scattered light signal from the first interrogation region is used as a trigger for the data acquisition system to initiate data collection. Preferably, an analog to digital converter operating at greater than 1 Megasamples per second begins to collect data. Data is collected for a time period equal to the number of the number of interrogation regions multiplied by the time interval between interrogation regions.  
         [0032]     The sample concentration can be adjusted so as to minimize the likelihood that two cells will be optically illuminated at the same time by any of the lasers. Being a statistical process, this may still occur at some frequency even at low sample concentrations. It is possible to detect most of these events and disregard the data by measuring the signal strength at the end of all time intervals and assuring oneself that the signal has dropped to a preset level consistent with anticipated detector/amplifier signal recovery.  
         [0033]     The collected data for a single event will then comprise of n peaks where n is the number of interrogation regions. The data is divided into the n intervals and the associated peaks analyzed for their heights and areas. In this manner n different signals are measured by each detector.  
         [0034]     There are important advantages of having multiple interrogation points and an equal number of spectrographs and Output Fiber arrays.  FIG. 2  illustrates the specific case where there are four interrogation points illuminated by five lasers (laser  4  (L 4 ) and laser  5  (L 5 ) illuminate the same point). The interrogation points are imaged onto four Input Fibers (IF 1 - 4 ) each delivering multispectral light to a different spectrograph. The dispersion gratings in each spectrograph can be chose to match the range of expected wavelengths delivered by the input fiber(s).  
         [0035]     In this example, there are only two detectors each of which has several output fibers leading to it. A given particle flowing through the flow cell will encounter each of the four interrogation zones in regular succession. The time interval between the interrogation points will depend directly upon the velocity of the stream and the distance between the interrogation points.  
         [0036]     Each of the detectors shown has at least one Output Fiber from each spectrograph. Detector  1  has Output Fibers S 1 - 1 , S 2 - 5 , S 3 - 8 , S 4 - 7  and S 4 - 4  and Detector  2  has Output fibers S 1 - 6 , S 2 - 6 , S 2 - 7 , S 3 - 9 , and S 4 - 9  where, for example, S 1 - 6  is the signal from the 6 th  Output Fiber in spectrograph  1 .  
         [0037]     Signal S 1 - 1  can be used to collect side scattered or forward scattered light. This relatively strong signal is easily detected and occurs first and can therefore be used to trigger data collection on all other detectors. The fact that the 6 th  and 7 th  fibers of spectrograph  2  are used in detector  1  reflects that the emission bandwith of that signal is wider than is distributed over a single Output Fiber. The fact that the 7 th  and 4 th  fibers (noncontiguous fibers) from spectrograph  4  deliver light to detector  1  is useful when: (1) one laser (L 4  or L 5 ) is generating two spectrally separated signals or (2) L 4  and L 5  produce two spectrally resolved signals. Since, in either of these two cases, the signals arrive at the same time, it is easier to interpret the data if the two possible signals are not present in the same event which is possible for certain sample types.  
         [0038]     For those events that occur at the same time (from the same event), it is better that they be divided among the different detectors so that they can be measured individually.  
         [0039]     It is recognized that the number of events per unit time may need to be adjusted (primarily through concentration and sample stream diameter) so that the chance of two events overlapping in time is low. If and when this does occur, these data points can be rejected as unreliable.  
         [0040]     A fast A-D system can be used to sample the output signal of the detector/amplifiers and be logically divided into different time domains (in this case  4  domains: i, ii, iii, iv). The peak height and area of each signal peak (and any other relevant parameters) can be calculated. These values can be stored in a much smaller computer file.