Patent Publication Number: US-6211955-B1

Title: Imaging and analyzing parameters of small moving objects such as cells

Description:
RELATED APPLICATIONS 
     This application is a continuation-in-part application based on prior copending patent application Ser. No. 09/490,478, filed on Jan. 24, 2000, still pending the benefit of the filing date of which is hereby claimed under 35 U.S.C. §120. 
    
    
     FIELD OF THE INVENTION 
     This invention generally relates to imaging moving objects or particles for purposes of analysis and detection, and more specifically, to a system and method for determining and analyzing the morphology of moving objects, such as cells, and for detecting the presence and composition of Fluorescence In-Situ Hybridization (FISH) probes within cells. 
     BACKGROUND OF THE INVENTION 
     There are a number of biological and medical applications that are currently impractical due to limitations in cell and particle analysis technology. Examples of such biological applications include battle field monitoring of known airborne toxins, as well as the monitoring of cultured cells to detect the presence of both known and unknown toxins. Medical applications include non-invasive prenatal genetic testing and routine cancer screening via the detection and analysis of rare cells (i.e., low rate of occurrence) in peripheral blood. All of these applications require an analysis system with the following principal characteristics: 
     1. high speed measurement; 
     2. the ability to process very large or continuous samples; 
     3. high spectral resolution and bandwidth; 
     4. good spatial resolution; 
     5. high sensitivity; and 
     6. low measurement variation. 
     In prenatal testing, the target cells are fetal cells that cross the placental barrier into the mother&#39;s bloodstream. In cancer screening, the target cells are sloughed into the bloodstream from nascent cancerous tumors. In both of these applications of this technology, the target cells may be present in the blood at concentrations of one to five cells per billion. This concentration yields approximately 20-100 target cells in a typical 20 ml blood sample. The extreme rarity of the targeted cells demands that any detection and analysis system employed in these applications be capable of processing an enriched sample of approximately 100 million cells within a few hours, corresponding to a minimum throughput of 10,000 cells per second. Cell processing includes the determination of cellular morphology parameters such as overall size, nuclear size, nuclear shape, and optical density, the detection and characterization of numerous fluorescent markers and FISH probes, the quantification of the total amount of DNA in the nucleus, and the detection of other cellular components such as fetal hemoglobin. To accomplish these processing tasks, the system must be able to collect cell images with a spatial resolution of approximately 1 micron. Likewise, the system must have high spectral resolution and bandwidth to differentiate four or more fluorescent colors. Since some probes may label important cellular features with only a few thousand fluorescent molecules, the system must have high sensitivity and good measurement consistency to differentiate very weak signals. 
     The predominant research laboratory protocols for non-invasive prenatal diagnosis employ a complex series of process steps that include gradient centrifugation to remove unnucleated cells, high-speed cell sorting for fetal cell enrichment, and fluorescence microscopy for fetal cell identification and genetic analysis. These protocols often yield little or no fetal cells for analysis, because a fraction of the fetal cells are lost at each step of the protocol. Nevertheless, the protocols cannot be simplified because of limitations in existing analysis technology. Ideally, fetal cell identification and analysis would be performed in a few hours by a high-speed cell sorter having the necessary speed and sample handling capacity. This ideal is not possible with conventional systems, because conventional cell sorters lack the necessary imaging abilities, sensitivity, and repeatability to reliably identify fetal cells and enumerate the number and color of FISH probes used to make the diagnosis. Therefore, under current protocols, cells must be sorted onto slides and examined using fluorescence microscopy to establish their fetal origin and make a genetic diagnosis. The combination of low fetal cell yields and lengthy processing times precludes the clinical application of non-invasive fetal testing with existing technology. 
     No technology prior to the present invention incorporates all six of the principal characteristics of a viable fetal cell or cancer analysis system. In the prior art, there have been advances that might be applied to these applications, but significant limitations still remain. 
     A paper published by Ong et al. [Anal. Quant. Cytol. Histol., 9(5):375-82] describes the use of a time-delay and integration (TDI) detector in an imaging flow cytometer. A TDI detector is any pixilated device in which the signal produced in response to radiation directed at the device can be caused to move in a controlled fashion. Typically, the pixels of a TDI detector are arranged in rows and columns, and the signal is moved from row to row in synchrony with a moving image projected onto the device, allowing an extended integration time without blurring. The approach disclosed by Ong et al. advanced the art by addressing the need for spatial resolution and high sensitivity for cells in flow. However, this approach does not address the remaining principal characteristics. The authors of this paper cite an operating speed of 10 cells per second and a theoretical speed limitation of 500 cells per second, which is at least an order of magnitude slower than is required for non-invasive fetal testing. In addition, the system has no spectral resolution; laser scatter and fluorescent light are collected by the imaging system indiscriminately. 
     In more recent developments, U.S. Pat. No. 5,644,388 discloses an alternative approach to an imaging flow cytometer. The patent discloses the use of a frame-based image collection approach in which a video camera views cells in flow, in a freeze frame fashion. This method requires the image collection system to be synchronized with the presence of cells in the imaging area, unlike the case of TDI, wherein the detector readout rate is synchronized with the velocity of the cells. When a cell is imaged with the frame-based method, the integration period must be very short to prevent blurring. A short integration time is achieved either with a strobed light source, or a continuous light source combined with a shuttered detector. In either case, the short integration time reduces the signal-to-noise ratio and the ultimate sensitivity of the approach. Further, frame-based cameras require time to transfer data out of the camera, during which no images are acquired, and cells of interest can escape detection. Finally, like the work of Ong et al., this patent makes no provisions for acquiring data over a large spectral bandwidth and with sufficient spectral resolution to simultaneously resolve numerous and differently colored fluorescent probes and FISH spots. 
     Spectral discrimination is addressed in U.S. Pat. No. 5,422,712, in which the spectra of particles suspended in a fluid are collected as the particles flow through a detection region. However, there is no spatial representation of the object in the system disclosed in this patent, because the object is defocussed at the detector. In this system, light is collected from the object and an image is created at an intermediate aperture. The light continues through the aperture to a spectral dispersing element, which disperses the light spectrally along the axis of flow. The dispersed light is applied to an image intensifier in which it is amplified, and the light signal output from the image intensifier is finally directed to frame-based detector. At the intermediate aperture, prior to spectral dispersion, the image represents the spatial distribution of light in object space. The spatial distribution is blurred as the light propagates past the image plane, through the spectral dispersing element and onto the image intensifier. Because there is no provision for re-imaging the intermediate aperture at the intensifier, the resulting signal distribution at the intensifier represents only the spectral distribution of the light and does not preserve the spatial distribution of the light from the object. The loss of spatial information limits the utility of the invention for applications such as fetal cell analysis. If multiple identical FISH spots are present in a cell, their spectra can be ascertained using this approach, but the number of spots cannot be determined. In addition, this approach disperses the wavelength spectrum parallel to the axis of flow. If two particles are illuminated in the flow axis, their spectra can overlap on the detector. To prevent this problem, the patent discloses that a very short illumination height in the flow axis is used. The short illumination height decreases integration time, which necessitates the use of the image intensifier. Further, the short illumination height limits throughput by preventing the simultaneous imaging of multiple cells in the flow axis. 
     Accordingly, it will be apparent that an improved technique is desired that resolves the limitations of the conventional approaches discussed above. It is expected that the new approach developed to address these problems in the prior art will also have application to the analysis of other types of moving objects besides cells and may be implemented in different configurations to meet the specific requirements of disparate applications of the technology. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to an imaging system that is adapted to determine one or more characteristics of an object from an image of the object. There is relative movement between the object and the imaging system, and although it is contemplated that either (or both) may be in motion, the object will preferably move while the imaging system will be fixed. In addition, it should also be understood that while much of the following summary and the corresponding claims recite “an object,” it is clearly contemplated that the present invention is preferably intended to be used with a plurality of objects and is particularly useful in connection with imaging a stream of objects. 
     The present invention provides a method and apparatus for the analysis of rare cells in the blood for the purposes of non-invasive fetal cell diagnosis and cancer screening, as well as other applications. To achieve such functions, the present invention is capable of rapidly collecting data from a large cell population with high sensitivity and low measurement variation. These data include simultaneous spatial and spectral images covering a large bandwidth at high resolution. Further, the present invention preserves the spatial origin of the spectral information gathered from the object. 
     Several different embodiments of the imaging system are provided. One preferred form of the invention includes a collection lens disposed so that light traveling from the object is collimated by passing through the collection lens and travels along a collection path. A spectral dispersing element is disposed in the collection path so as to spectrally disperse the collimated light that has passed through the collection lens in a plane substantially orthogonal to a direction of relative movement between the object and the imaging system, producing spectrally dispersed light. (As noted above, the object or the imaging system or both can be in motion relative to the other and for the sake of simplicity, this relative movement is hereinafter referred to simply as “the movement.”) An imaging lens is disposed to receive the spectrally dispersed light, producing an image from the spectrally dispersed light. Also included is a TDI detector disposed to receive the image produced by the imaging lens. As the movement occurs, the image of the object produced by the imaging lens moves from row to row across the TDI detector. The TDI detector produces an output signal that is indicative of at least one characteristic of the object, by integrating light from at least a portion of the object over time. 
     As a result of light collimation by the collection lens in this embodiment, all light emitted from a first point in the object travels in parallel rays. Light emitted from a second point in the object will also travel in parallel rays, but at a different angle relative to light from the first point. In this manner, spatial information in the object is transformed by the collection lens into angular information in the collection path. The spectral dispersing element acts on the collimated light such that different spectral components leave the spectral dispersing element at different angles, in a plane substantially orthogonal to the direction of the movement between the object and the imaging system. In this manner, both spatial and spectral information in the object are transformed into angular information. The imaging lens acts on the light from the dispersing element to transform different light angles into different positions on the detector. Spatial information is preserved by the system since light from the different positions in the object is projected to different positions on the detector, for both axes. In addition, light of different spectral composition that originates from the object is projected to different positions on the detector in an axis substantially orthogonal to the movement. In this manner, the spatial information from the object is preserved, while spectral information covering a large bandwidth is simultaneously collected at high resolution. 
     FIG. 16 further illustrates the simultaneous collection of spectral and spatial information by the present invention, when imaging male and female cells  200  and  208 , respectively. Light of shorter wavelength, such as green laser scatter  212 , will be focussed on the left side of the TDI detector. Light of slightly longer wavelength, such as yellow fluorescence  214  from a cell nucleus  202  or  210 , will be laterally offset to the right. Light of still longer wavelengths, such as orange fluorescence  216  from an X-chromosome FISH probe and red fluorescence  218  from a Y-chromosome FISH probe, will be focussed progressively farther to the right on the TDI detector. In this manner, different components of a cell that fluoresce at different wavelengths will be focussed at different locations on the TDI detector, while preserving the spatial information of those components. Each component image may be broadened laterally due to the width of its associated fluorescence emission spectrum. However, this broadening can be corrected based upon a priori knowledge of the emission spectra. Deconvolution of the emission spectrum from the broadened component image will yield an undistorted component image. Further, since the spectral dispersion characteristics of the spectral dispersing element are known, the lateral offsets of the different color component images can be corrected to reconstruct an accurate image of the cell. Using this embodiment of the invention, high spatial resolution information can be collected simultaneously with high spectral resolution over several hundred nanometers of spectral bandwidth. It should clear to those skilled in the art that the present invention can be employed to enumerate numerous and multicolored FISH probes to simultaneously determine many characteristics from cells. 
     In the following disclosure, for all forms of the present invention where a prism is used as a spectral dispersing element, it can be replaced with a spectral dispersing component having characteristics that ensure no distortion or convolution of the image occurs due to the emission bandwidth, and as a result, a deconvolution is not needed to correct the image. Preferably, the spectral dispersing component employed comprises a plurality of dichroic beam splitters, such as dichroic mirrors, which are arranged to reflect light within predefined bandwidths at predefined angles. Unlike a prism, where every wavelength leaves the prism at a different angle, all light within a predefined bandwidth incident on the dichroic beam splitter at a common angle leaves a given dichroic beam splitter at the same angle. Therefore, there is no convolution between the emission spectrum of the light leaving the object and the image of that object. When using such a spectral dispersing component, light of a first spectral bandwidth reflects off the first dichroic beam splitter at a predefined nominal angle. Light of a second spectral bandwidth is passed through the first dichroic beam splitter to the next dichroic beam splitter and is reflected therefrom at a different predefined nominal angle. Light of a third spectral bandwidth is passed through the first and second dichroic beam splitters to a third dichroic beam splitter and reflected therefrom at a third predefined nominal angle. The dichroic beam splitters are selected to cover the desired light spectrum with the appropriate spectral passbands. The angle of each dichroic beam splitter is set such that light reflected from it within the corresponding spectral bandwidth for the dichroic beam splitter is focussed onto a different region of the detector. Since the present invention uses a narrow field angle in object space along the axis perpendicular to the axis of motion, many different spectral bandwidths can be simultaneously imaged onto a single detector. In this manner, each region on the detector may cover a different spectral bandwidth while collecting light over the same field angle in object space. 
     Depending on the amount of out-of-band rejection required, a bandpass filter is optionally placed in front of the detector. In one embodiment, the bandpass filter comprises a plurality of narrow spectral filters placed side-by-side to cover regions of the detector in correspondence with the spectral information to be imaged in those regions. Since the position of each spectral bandwidth region is predefined, and since the present invention maintains the spatial integrity of the object, a full color, high spectral resolution representation of the object is generated from the spectral information imaged onto the detector. 
     The use of a TDI detector in the present invention results in an extended imaging region along the axis of motion and a correspondingly long integration time. Several light sources can be simultaneously projected into the imaging region, increasing the amount of light incident upon objects therein. In addition, the combination of an extended imaging region and the orthogonal orientation of the spectral dispersion axis relative to the axis of the motion allows multiple objects to be imaged simultaneously. The long integration time and parallel image acquisition of this embodiment allows sensitive and consistent imaging performance to be combined with high throughput. 
     There are several alternative ways to provide light from the object. In one case, the light from the object comprises an unstimulated emission from the object, i.e., the object emits light without requiring a light source to stimulate the emission. In another embodiment, a light source is disposed to provide an incident light that illuminates the object. In this case, the object may scatter the incident light so that the light scattered from the object at least in part passes through the collection lens, or the incident light illuminating the object may stimulate the object to emit the light that passes through the collection lens. Further, the incident light may at least be partially absorbed by the object, so that the light passing through the collection lens does not include a portion of the light absorbed by the object. Finally, the incident light from the light source may be reflected from the object toward the collection lens. The light source or sources that are used preferably comprise at least one of a coherent light source, a non-coherent light source, a pulsed light source, and a continuous light source. 
     Spectral dispersion may be accomplished by many means, including a prism or grating. Further, although one preferred form of the invention employs a spectral dispersing element, the present invention is not limited to imaging the spectral dispersion of light. Alternatively, a dispersing element can be used to disperse light as a function of position, angle, polarization, phase, and other properties. 
     The object may be entrained within a fluid stream that moves the object past the collection lens, or alternatively, can be carried on a support, or simply move without the benefit of a support or flowing medium. Moreover, the present invention is not limited to the imaging of microscopic or small objects. 
     The TDI detector preferably responds to the image of the object by producing a signal that propagates across the TDI detector. Pixels of a typical TDI detector are arranged in rows and columns, and the signal propagates from row to row. However, the present invention is not limited to TDI detectors employing a rectilinear arrangement of pixels (e.g., a microchannel plate-based TDI detector). A propagation rate of the signal across the TDI detector can either be synchronized with a motion of the image of the object on the TDI detector as a result of the movement, or can be non-synchronized with the movement. 
     Other aspects of the present invention are directed to methods for imaging an object. These methods implement steps that are generally consistent with the imaging system discussed above. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING FIGURES 
     The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: 
     FIG. 1 is a plan view of a first embodiment of the present invention in which particles conveyed by a fluid stream depicted as flowing into the sheet; 
     FIG. 2 is a side elevational view of the first embodiment shown in FIG. 1; 
     FIG. 3 is an isometric view of the first embodiment of FIG. 1; 
     FIG. 4 is an isometric view of a confocal embodiment that includes a slit that is used for spatial filtering of extraneous light; 
     FIG. 5 is an isometric view showing different locations for a light source in connection with the first embodiment; 
     FIG. 6 is an alternative to the first embodiment in which a second set of imaging components and TDI detector is included for monitoring light from a particle, to avoid interference between FISH probes, and showing alternative locations for light sources; 
     FIG. 7 is an isometric view of an embodiment in which an object is supported by or comprises a slide that moves past a collection lens, showing different locations for a light source; 
     FIGS. 8A and 8B are respectively a plan view and a side elevational view of an alternative to the embodiment of FIG. 7 that is used to produce a scattered pattern on the TDI detector; 
     FIG. 9 is a plan view of yet a further embodiment in which light forming a scatter patterned image and spectrally dispersed light from the object are imaged on separate portions of a TDI detector; 
     FIG. 10 is a plan view of a still further embodiment in which light forming a scatter patterned image and spectrally dispersed light from the object are imaged by two different TDI detectors; 
     FIG. 11 is a schematic diagram illustrating the optical convolution of a narrow FISH emission spectrum by the present invention, to resolve two FISH probes in a cell; 
     FIG. 12 is a schematic diagram showing the optical convolution of two different colors of narrow FISH emission spectra, to resolve the image of the FISH probes on the TDI detector; 
     FIG. 13 is a schematic diagram illustrating how for a wider FISH emission spectrum, a deconvolution is provided by the present invention to resolve the image of two FISH probes of a single color; 
     FIG. 14 is a schematic diagram showing the deconvolution of two color FISH spectra that are relatively wide, to resolve the image of the FISH probes; 
     FIG. 15 is a schematic block diagram of the system used to process the signal produced by a TDI detector in the present invention; 
     FIG. 16 is a schematic diagram illustrating how the present invention is used to determine whether a cell is from a male or female; 
     FIG. 17 is a plan view of an alternate embodiment that employs a spectral dispersion component comprising a plurality of stacked dichroic filters employed to spectrally separate the light; 
     FIG. 18 is an X-Y plot of several typical passbands for the dichroic filters employed in the embodiment shown in FIG. 17; 
     FIG. 19 is a schematic illustration of a detection filter assembly that may optionally be placed in front of the TDI detector in the embodiment of FIG. 17 to further suppress out-of-band light; 
     FIGS. 20A-20E are X-Y plots of transmission vs. wavelength corresponding to corresponding passbands of the filter segments of the detection filter assembly that may optionally be placed in front of the TDI detector; 
     FIG. 21 is a plan view of another embodiment of the configuration of FIG. 17, wherein the spectral dispersion filter system comprises a plurality of dichroic cube filters orientated at various angles to create the spectral dispersing effect; and 
     FIG. 22 illustrates an exemplary set of images projected onto the TDI detector when using the spectral dispersing filter system of the FIG.  17 . 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The present invention offers considerable advantages over systems employed for cell and particle analysis in the prior art. These advantages arise from the use in the present invention of an optical dispersion system in combination with a TDI detector that produces an output signal in response to the images of cells and other objects that are directed on the TDI detector. Multiple objects can be imaged on the TDI detector at the same time. In addition, the image of each object can be spectrally decomposed to discriminate object features by absorption, scatter, reflection or probe emissions using a common TDI detector for analysis. 
     The present invention can be employed to determine morphological, photometric, and spectral characteristics of cells and other objects by measuring optical signals including light scatter, reflection, absorption, fluorescence, phosphorescence, luminescence, etc. Morphological parameters include nuclear area, perimeter, texture or spatial frequency content, centroid position, shape (i.e., round, elliptical, barbell-shaped, etc.), volume, and ratios of any of these parameters. Similar parameters can also be determined for the cytoplasm of cells with the present invention. Photometric measurements with the invention enable the determination of nuclear optical density, cytoplasm optical density, background optical density, and the ratios of any of these values. An object being imaged with the present invention can either be stimulated into fluorescence or phosphorescence to emit light, or may be luminescent, producing light without stimulation. In each case, the light from the object is imaged on the TDI detector of the present invention to determine the presence and amplitude of the emitted light, the number of discrete positions in a cell or other object from which the light signal(s) originate(s), the relative placement of the signal sources, and the color (wavelength or waveband) of the light emitted at each position in the object. 
     An initial application of the imaging system comprising the present invention will likely be employed as a cell analyzer to determine one or more of the parameters listed above, for cells entrained in a fluid flowing through the imaging system. However, it should also be understood that this invention can be used for imaging other moving objects. 
     First Preferred Embodiment 
     A first preferred embodiment of an imaging system  20  in accord with the present invention is schematically illustrated in FIGS. 1,  2 , and  3 , in connection with producing images of moving objects such as cells that are conveyed by a fluid flow  22  through the imaging system. In FIG. 1, fluid flow  22  entrains an object  24  (such as a cell, but alternatively, a small particle) and carries the object through the imaging system. The direction of the fluid flow in FIG. 1 is into (or out of) the sheet, while in FIGS. 2 and 3, the direction of flow is from top to bottom, as indicated by the arrow to the left of the Figures. Light  30  from object  24  passes through collection lenses  32   a  and  32   b  that collect the light, producing collected light  34 , which is approximately focussed at infinity, i.e. the rays of collected light from collection lens  32   b  are generally parallel. Collected light  34  enters a prism  36 , which disperses the light, producing dispersed light  38 . The dispersed light then enters imaging lenses  40   a  and  40   b , which focuses light  42  onto a TDI detector  44 . 
     As will be evident in FIG. 2, if the Figure depicts the imaging of object  24  over time, the object is shown at both a position  26  and a position  28  as it moves with fluid flow  22 . As a consequence, images of object  24  will be produced on the detector at two discrete spatial positions  26 ′ and  28 ′, as indicated on the right side of FIG.  2 . Alternatively, if FIG. 2 is depicting a single instant in time, positions  26  and  28  can represent the location of two separate objects, which are simultaneously imaged on the detector at positions  26 ′ and  28 ′. 
     In regard to imaging system  20  and all other imaging systems illustrated herein, it will be understood that the lenses and other optical elements illustrated are shown only in a relatively simple form. Thus, the collection lens is illustrated as a compound lens comprising only collection lenses  32   a  and  32   b . Lens elements of different designs, either simpler or more complex, could be used in constructing the imaging system to provide the desired optical performance, as will be understood by those of ordinary skill in the art. The actual lenses or optical elements used in the imaging system will depend upon the particular type of imaging application for which the imaging system will be employed. 
     In each of the embodiments of the present invention, it will be understood that relative movement exists between the object being imaged and the imaging system. In most cases, it will be more convenient to move the object than to move the imaging system. However, it is also contemplated that in some cases, the object may remain stationary and the imaging system move relative to it. As a further alternative, both the imaging system and the object may be in motion but either in different directions or at different rates. 
     The TDI detector that is used in the various embodiments of the present invention preferably comprises a rectangular charge-coupled device (CCD) that employs a specialized pixel read out algorithm, as explained below. Non-TDI CCD arrays are commonly used for 2-dimensional imaging in cameras. In a standard CCD array, photons that are incident on a pixel produce charges that are trapped in the pixel. The photon charges from each pixel are read out of the detector array by shifting the charges from one pixel to the next, and then onto an output capacitor, producing a voltage proportional to the charge. Between pixel readings, the capacitor is discharged and the process is repeated for every pixel on the chip. During the readout, the array must be shielded from any light exposure to prevent charge generation in the pixels that have not yet been read. 
     In one type of TDI detector  44 , which comprises a CCD array, the CCD array remains exposed to the light as the pixels are read out. The readout occurs one row at a time from the top toward the bottom of the array. Once a first row is read out, the remaining rows are shifted by one pixel in the direction of the row that has just been read. If the object being imaged onto the array moves in synchrony with the motion of the pixels, light from the object is integrated for the duration of the TDI detector&#39;s total readout period without image blurring. The signal strength produced by a TDI detector will increase linearly with the integration period, which is proportional to the number of TDI rows, but the noise will increase only as the square root of the integration period, resulting in an overall increase in the signal-to-noise ratio by the square root of the number of rows. One TDI detector suitable for use in the present invention is a Dalsa Corp., Type IL-E2 image sensor, although other equivalent or better image sensors can alternatively be used. The Dalsa image sensor has 96 stages or rows, each comprising 512 pixels; other types of image sensors useable in the present invention may have different configurations of rows and columns or a non-rectilinear arrangement of pixels. The Dalsa sensor has approximately 96 times the sensitivity and nearly  10  times the signal-to-noise ratio of a standard CCD array. The extended integration time associated with TDI detection also serves to average out temporal and spatial illumination variations, increasing measurement consistency. 
     In imaging system  20  and in other embodiments of the present invention that employ a fluid flow to carry objects through the imaging system, a flow-through cuvette or a jet (not shown) contains the cells or other objects being analyzed. The velocity and cellular concentration of the fluid may be controlled using syringe pumps, gas pressure, or other pumping methods (not shown) to drive a sample solution through the system to match the pixel readout rate of the TDI detector. However, it should be understood that the readout rate of the TDI detector can be selectively controlled, as required, to match the motion of the sample solution. 
     Various optical magnifications can be used to achieve a desired resolution of the object that is being imaged on the light sensitive regions (pixels) of the TDI detector. It is contemplated that in most embodiments, the optical magnification will fall within a range of 1:1 to 50:1, providing a substantial range in the number of light sensitive regions on the TDI detector on which images of the object are formed, also depending of course, on the actual size of the object being imaged and its distance from the imaging system. It is envisioned that the present invention can have applications ranging from the analysis of cells and other microscopic objects to the imaging of stellar objects. 
     It should be emphasized that the present invention is not limited to CCD types of TDI detectors. Other types of TDI detectors, such as complementary metal oxide semiconductor (CMOS) and multi-channel plate imaging devices might also be used for the TDI detector in the present invention. It is important to understand that any pixellated device (i.e., having a multitude of light sensitive regions) in which a signal produced in response to radiation directed at the device can be caused to move through the device in a controlled fashion is suitable for use as the TDI detector in the present invention. Typically, the signal will move in synchrony with a moving image projected onto the device, thereby increasing the integration time for the image, without causing blurring. However, the motion of the signal can be selectively desynchronized from the motion of the radiation image, as required to achieve a desired affect. 
     Second Preferred Embodiment 
     FIG. 4 illustrates an imaging system  45 , which is a second preferred embodiment of the present invention and which is similar in many ways to imaging system  20 . However, imaging system  45  is a confocal embodiment that includes a slit  52  that substantially prevents extraneous light from reaching TDI detector  44 . In imaging system  45 , light  46  from object  24  is focussed by an objective lens  48  onto a slit  52 . Slit  52 , as shown in FIG. 4, is sufficiently narrow to block light which is not focussed onto the slit by objective lens  48  from passing through the slit. Light  30 ′ passes through the slit and is collected by collection lens  32  as discussed above, in regard to imaging system  20 . Collected light  34  is spectrally dispersed by prism  36 , and is imaged by imaging lens  40  onto TDI detector  44 , also as discussed above. By excluding light other than that from object  24  from reaching TDI detector  44 , the TDI detector produces an output signal that corresponds only to the actual images of the object, and the signal is not affected by the extraneous light, which has been excluded. If not excluded in this manner, the ambient light reaching TDI detector  44  might otherwise produce “noise” in the output signal from the TDI detector. 
     It should be noted that in the illustration of each of imaging systems  20  and  45 , a light source has not been shown. These first two embodiments have been illustrated in their most general form to make clear that a separate light source is not required to produce an image of the object, if the object is luminescent, i.e., if the object produces light. However, many of the applications of the present invention will require that one or more light sources be used to provide light that is incident on the object being imaged. The location of the light sources substantially affects the interaction of the incident light with the object and the kind of information that can be obtained from the images on the TDI detector. 
     In FIG. 5, several different locations of light sources usable to provide light incident on object  24  are illustrated. It should be understood, however, that light sources can be located at many other positions besides those shown in FIG.  5 . The location of each one or more light source employed will be dependent upon the kind of imaging of the object, and the kind of data for the object, to be derived from the signal produced by the TDI detector. For example, employing a light source  60   a  or a light source  60   b , as shown in the Figure, will provide light  58  that is incident on object  24  and which is scattered from the object into the optical axis of collection lens  32 . The optical axis of collection lens  32  is at about a 90° angle relative to the directions of the light incident upon object  24  from either light source  60   a  or  60   b.    
     In contrast, a light source  62  is disposed so that light  58  emitted from the source travels toward the object in a direction that is generally aligned with the optical axis of collection lens  32 , so that the image formed on TDI detector  44  will not include light absorbed by object  24 . Light absorption characteristics of the object can thus be determined by illuminating the object using a light source  62 . 
     A light source  64  is disposed to illuminate object  24  with light directed toward the object along a path that is approximately 30-45° off the optical axis of collection lens  32 . This light  58 , when incident on object  24  will be reflected (scattered) from object  24 , and the reflected or scattered light will be imaged on TDI detector  44 . A more directly reflected light is provided by an epi light source  66 , disposed so as to direct its light  58  toward a partially reflective surface  68  that is disposed so that a portion of the light is reflected through collection lens  32  and onto object  24 . The light reaching the object will be reflected from it back along the axis of collection lens  32  and will at least in part pass through partially reflective surface  68  to form an image of the object on TDI detector  44 . Alternatively, a dichroic mirror may be employed instead of, and in the position of, partially reflective surface  68  to direct light from epi light source  66  to excite fluorescence or other stimulated emission from object  24 . Emission from object  24  is then at least partially collected by collection lens  32  and passes through the dichroic mirror for spectral dispersion and detection by the TDI detector. 
     In addition to imaging an object with the light that is incident on it, a light source can also be used to stimulate emission of light from the object. For example, FISH probes that have been inserted into cells will fluoresce when excited by light, producing a corresponding characteristic emission spectra from any excited FISH probe that can be imaged on TDI detector  44 . In FIG. 5, light sources  60   a ,  60   b ,  64 , or  66  could alternatively be used for causing the excitation of FISH probes on object  24 , enabling TDI detector  44  to image FISH spots produced by the FISH probes on the TDI detector at different locations as a result of the spectral dispersion of the light from the object that is provided by prism  36 . The disposition of these FISH spots on the TDI detector surface will depend upon their emission spectra and their location in the object. Use of FISH probes in connection with producing images of FISH spots on the TDI detector with the present invention is discussed in greater detail below. 
     Each of the light sources illustrated in FIG. 5 produces light  58 , which can either be coherent, non-coherent, broadband or narrowband light, depending upon the application of the imaging system desired. Thus, a tungsten filament light source can be used for applications in which a narrowband light source is not required. For applications such as stimulating the emission of fluorescence from FISH probes, narrowband laser light is preferred, since it also enables a spectrally-decomposed, non-distorted image of the object to be produced from light scattered by the object. This scattered light image will be separately resolved from the FISH spots produced on TDI detector  44 , so long as the emission spectra of any FISH spots are at different wavelengths than the wavelength of the laser light. The light source can be either of the continuous wave (CW) or pulsed type. If a pulsed type illumination source is employed, the extended integration period associated with TDI detection can allow the integration of signal from multiple pulses. Furthermore, it is not necessary for the light to be pulsed in synchronization with the TDI detector. 
     Pulsed lasers offer several advantages over CW lasers as a light source in the present invention, including smaller size, higher efficiency, higher reliability, and the ability to deliver numerous wavelengths simultaneously. Another advantage of pulsed lasers is their ability to achieve saturating levels of fluorescence excitation of fluorescent probes used in cells. Fluorescence saturation occurs when the number of photons encountering a fluorescent molecule exceeds its absorption capacity. Saturating excitation produced by a pulsed laser is inherently less noisy than unsaturating CW laser excitation because variations in pulse-to-pulse excitation intensity have little effect on the fluorescence emission intensity. 
     Prism  36  in the imaging systems discussed above can be replaced with a diffraction grating, since either is capable of spectrally dispersing the optical signals from the cells over the pixels of the TDI detector. In addition to providing useful data from a cell or other object, spectral dispersion can be used to reduce measurement noise. In cases where the light source wavelength differs from the emission spectra of the fluorescent probes, the light from the source that is scattered into the collection system is spatially isolated from the fluorescence signals. If the light source wavelength overlaps the emission spectra of the fluorescent probes, the pixels of the TDI detector in which light of the wavelength of the source falls can be isolated from those pixels on which the remaining fluorescence signals fall. Further, by dispersing the fluorescence signals over multiple pixels, the overall dynamic range of the imaging system is increased. 
     Third Preferred Embodiment 
     A third preferred embodiment is a stereoscopic arrangement  70  of the first preferred embodiment, as illustrated in FIG.  6 . This arrangement allows the imaging of the object from two different directions in order to distinguish features that would otherwise overlap when viewed from a single direction. While the third preferred embodiment can be employed for objects on moving substrates such as microscope slides, it is particularly useful for analyzing multi-component objects in solution, such as cells containing FISH probes. Such probes appear as point sources of light anywhere within the cell&#39;s three dimensional nucleus. In some cases, two or more FISH probes may appear in an overlapping relationship along the optical axis of the imaging system. In such cases, one of the FISH probes may obscure the others, making it difficult to determine the number of probes present in the cell. This is a key factor in the determination of genetic abnormalities such as trisomy 21, otherwise known as Down syndrome. Single-perspective systems may address this problem by “panning through” the object along the optical axis to acquire multiple image planes in the object. While this method may be effective, it requires a significant amount of time to collect multiple images and cannot be readily applied to a cell in flow. The stereoscopic imaging system  70  in FIG. 6 includes two TDI detectors  44   a  and  44   b , and their associated optical components, as discussed above in connection with imaging system  20 . 
     By positioning the optical axes of collection lenses  32  for the two TDI detectors so that they are spaced apart, for example, by 90°, it is possible to separately resolve the FISH spots imaged from two or more FISH probes on at least one of TDI detectors  44   a  or  44   b . If two or more FISH probes overlap in regard to the image produced on one of the detectors, they will be separately resolved in the spectrally dispersed images produced on the other TDI detector. Further, the use of two TDI detectors in imaging system  70  in what might be referred to as a “stereo or three-dimensional configuration” allows flexibility in the configuration of each leg of the system, including parameters such as the relative TDI readout rates, axial orientations, inclinations, focal plane positions and magnification. Multiple cells or other objects may be imaged onto each detector simultaneously in the vertical direction. Since the objects may move in synchronicity with the signal on the TDI, no gate or shutter is required to prevent blurring of the image. As previously noted, the present invention can use a pulsed or CW light source without need for a trigger mechanism to time a pulse coincident with particle arrival in the field of view. If a pulsed light source is used, the extended field of view in the axis of motion associated with TDI detection allows the cell or object in motion to be illuminated by multiple pulses during its traversal. In contrast to a frame-based imaging apparatus, a TDI system can produce a single unblurred image of the object that integrates the signal from multiple pulses. When a CW light source is used, the signal generated by the object will be collected throughout the entire traversal of the object through the field of view, as opposed to only a small segment in time when a shutter is open. Therefore, the amount of signal collected and imaged on the detector in the present invention is substantially greater than that of the prior art frame-based imaging systems. Consequently, the present invention can operate at very high throughput rates with excellent signal-to-noise ratio. 
     Also illustrated in FIG. 6 are several exemplary positions for light sources, which are useful for different purposes in connection with the imaging system illustrated therein. In connection with TDI detector  44   a,  light source  62  provides illumination of object  24  from a direction so that absorption characteristics of the object can be determined from the image produced on the TDI detector. At the same time, light provided by light source  62  that is scattered from object  24  can be used to produce a scatter image and spectrally dispersed images on TDI detector  44   b . Light source  74  can be employed to produce spectrally dispersed and scattered images on both TDI detectors  44   a  and  44   b . If light sources  62  and  72  are of different wavelengths and an appropriate filter is provided to block the wavelength from the light source aligned with the optical axis of the respective collections lenses  32 , these two light sources can be used for producing scattered light from the object. For example, suppose light source  72  produces light of a wavelength A that scatters from object  24  and is directed toward TDI detector  44   a . By including a filter (not shown) that blocks wavelength B produced by light source  62 , the light at wavelength B will not directly affect the images produced on TDI detector  44   a . Similarly, the light from light source  72  would be blocked with an appropriate filter (not shown) so that it does not interfere with the imaging of light produced by light source  62  that is scattered from object  24  onto TDI detector  44   b.    
     Epi light source  66  is also illustrated for use in producing images on TDI detector  44   a  in conjunction with partial reflector  68 . Light source  64  can be used to generate reflected light to produce images on TDI detector  44   a,  while scattered light from this source is directed toward TDI detector  44   b . These and other possible locations of light sources will be apparent to those of ordinary skill in the art, as appropriate for providing the incident light on the object needed to achieve imaging, depending upon the particular application and information about the object that is desired. 
     Imaging Slide or Object Carried by Slide 
     Turning now to FIG. 7, an imaging system  80  is illustrated that is similar to imaging system  20 , except that it is used for imaging object  24  on a slide  82 . Object  24  is supported by slide  82  and the slide moves relative to the imaging system as shown in FIG.  7 . Alternatively, slide  82  may be the object that is imaged. The object may be a semiconductor wafer, paper, or other object of interest since the object may be imaged using reflected incident light. 
     To provide light incident on either slide  82  or object  24  that is supported thereby, a light source placed at one of several different locations can be employed. Exemplary light sources  62 ,  64 , and  66  illustrate some of the locations at which light sources useful in this embodiment may be disposed. Light  58  emitted by any of the light sources can be either coherent or non-coherent light, pulsed or CW, and can be directed through slide  82  (if it is transparent) from light source  62  or can be reflected from the object or slide, if light sources  64  or  66  are employed. As noted previously, epi light source  66  illuminates the object in connection with a partially reflective surface  68 . 
     Fourth Preferred Embodiment 
     FIGS. 8A and 8B show two different views of a fourth preferred embodiment, which is an imaging system  90  that produces a scattered pattern image of object  24  on TDI detector  44 . Light  30  from object  24  passes through collection lenses  32   a  and  32   b , and collected light  34  is directed onto a cylindrical lens  92 , as in the previous embodiments. Cylindrical lens  92  focuses light  94  on TDI detector  44 , generally along a line that is aligned with a central axis  96  of cylindrical lens  92 . Central axis  96  is shown in FIG. 8B, and it will be apparent that it is orthogonal to the direction in which object  24  moves through the imaging system. As object  24  moves downwardly, relative to its disposition as shown in FIG. 8A, the focus of cylindrical lens  92  on TDI detector  44  moves upwardly. Cylindrical lens  92  thus distributes an image of the object along a row or rows of the light sensitive regions or pixels of TDI detector  44 . 
     Fifth Preferred Embodiment 
     Referring now to FIG. 9, an illustration of a fifth preferred embodiment is provided of an imaging system  100  that produces both a scattered pattern image and a spectrally dispersed image of object  24  on TDI detector  44 . In imaging system  100 , light  30  from object  24  passes through collections lenses  32   a  and  32   b , which produce infinitely focussed light  34  directed toward a dichroic filter  102 . Dichroic filter  102  reflects light of a specific wavelength, e.g., the wavelength of a light source (not shown) that is incident upon object  24 . Light of any other wavelength is transmitted through dichroic filter  102  toward a diffraction grating  112 . Diffraction grating  112  spectrally disperses the light transmitted through dichroic filter  102 , which typically would be light produced by the fluorescence of FISH probes on object  24 , so that a plurality of FISH spots corresponding to the number of different FISH probes and objects being imaged are produced on TDI detector  44 . 
     Light  104 , which is reflected from dichroic filter  102  is transmitted into cylindrical lens  106  and is focussed along a line as a scattered pattern image in a region  110  on the TDI detector. The spectrally dispersed images of FISH spots or other aspects of object  24  having wavelengths different than that reflected by dichroic filter  102  are imaged as light  116  by imaging lenses  114   a  and  114   b  onto a region  118  of the TDI detector. Thus, signals corresponding to the scattered pattern image and the spectrally dispersed images are both produced by TDI detector  44 . 
     Sixth Preferred Embodiment 
     A sixth preferred embodiment, as illustrated in FIG. 10, is an imaging system  120  that is slightly different than the preceding fifth embodiment, since a dichroic filter  102 ′ is employed that is angled in a different direction, toward a second TDI detector  44   b . A dispersed pattern image represented by light  108 ′ is produced by a cylindrical lens  106 ′ in this embodiment. Just as in imaging system  100 , light transmitted through dichroic filter  102 ′ is focussed onto TDI detector  44   a . Aside from using two separate TDI detectors that are disposed at different sides of the imaging system, imaging system  120  is substantially identical in operation to imaging system  100 . However, just as in the third preferred embodiment, the use of two separate TDI detectors allows flexibility in the configuration of each leg of the system, including parameters such as the relative TDI readout rates, axial orientations, inclinations, focal plane positions, and magnification. It should also be noted that imaging system  100  could be constructed to include two separate TDI detectors instead of a single TDI detector, if desired. 
     Processing of Spectrally Dispersed Images on TDI Detector 
     When used for cell analysis, the present invention provides substantial utility in resolving FISH spots on the TDI detector, even when the FISH probes are disposed in spatially close relationship within the cell. When spectral imaging occurs in the present invention, the spatial distribution of light in the object is convolved with the spectral distribution of that light to produce the image of the object at the TDI detector. This convolution can result in blurring in the dispersion axis, depending on the spectral bandwidth of the light. Narrow spectral bandwidths will result in little or no blurring depending on the spectral resolution of the system. In the present invention, it is contemplated that the spectral resolution will be approximately 3 nm per pixel, with a spatial resolution in object space of approximately 1 micron. However, the spatial and spectral resolution can be adjusted to match the requirements of the particular application. 
     FIG. 11 illustrates the present invention with a spectral resolution of approximately 10 nm per pixel and a spatial resolution of approximately 0.5 microns. This Figure further illustrates how the present invention is used to image a cell  140  having a nucleus  142  in which are disposed two FISH probes  144   a  and  144   b  having the same emission spectrum. In FIG. 11, the emission spectrum  146  of the FISH probes  144   a  and  144   b  is approximately 10 nm in width, such as would be produced by “quantum dots” or a narrow-band fluorescent dye. The optical convolution of the narrow bandwidth spectrum results in minimal blurring of FISH spots  148   a  and  148   b , enabling them to be readily resolved on TDI detector  44 . 
     In FIG. 12, a cell  150  is illustrated having a nucleus  152  in which are disposed FISH probes  154  and  156  having different emission spectra. FISH probes are designed so that different emission spectra correspond to different DNA sequences. Each of the emission spectra of FISH probes  154  and  156  are relatively narrow, as indicated by wavebands  158  and  160 , and therefore, as in FIG. 11, minimal blurring occurs in FISH spots  162  and  164 . Furthermore, the spectral dispersion of the present invention, which maps wavelength into lateral position on TDI detector  44 , produces a relatively wide physical displacement of FISH spots  162  and  164 , despite the close proximity of FISH probes  154  and  156  in the cell. Taken together, FIGS. 11 and 12 illustrate how the present invention discriminates FISH probes of the same or different color, thereby enabling the simultaneous enumeration of numerous genetic traits. Those skilled in the art can appreciate that the present invention is well suited to the requirements of fetal cell analysis, where there may be ten or more probes of different colors present in the cell at one time. Further, those skilled in the art will appreciate that the present invention is not limited to the analysis of fetal cells using FISH probes. 
     FIGS. 13 and 14 illustrate that the present invention can also be used with light of wide spectral bandwidth. In this case an additional signal processing step is performed to correct for lateral blurring due to the wide emission spectra. In FIG. 13, a cell  140  having a nucleus  142  is shown, and FISH probes  170   a  and  170   b  having a common emission spectrum are disposed in the nucleus. FISH probes  170   a  and  170   b  are characterized by producing a relatively wide emission spectrum  172 . When optically convolved by the spectral dispersion provided by the present invention, FISH spots  174   a  and  174   b  are produced on TDI detector  44 , but their images are laterally blurred across TDI detector  44 , as a result of their relatively wide emission spectrum. To more clearly resolve the separation of FISH spots  174   a  and  174   b , a deconvolution is carried out on the signal produced by TDI detector  44 , with the known FISH emission spectrum, thereby producing accurate FISH spot representations  178   a  and  178   b  on a display  176 . The deconvolution step enhances the ability to enumerate the number of FISH spots. 
     FIG. 14 illustrates a corresponding relationship between FISH probes  180  and  182 , which are disposed within a nucleus  152  of a cell  150 . FISH probes  180  and  182  are characterized by each producing relatively wide band emission spectra  184  and  186 , as shown in the Figure. Optical convolution of the fluorescence emitted by the FISH probes, which are spectrally dispersed, produces FISH spots  188  and  190  on TDI detector  44 . Again, by deconvolving the known FISH emission spectra with the signal produced by TDI detector  44 , the corresponding images shown on display  176  of FISH spots  192  and  194  are recovered. Again, the spectral dispersion of the present invention, which maps wavelength into lateral position on TDI detector  44 , produces a relatively wide physical displacement of FISH spots  192  and  194 , despite the close proximity of FISH probes  180  and  182  in the cell. In this manner, it is possible to resolve these images of FISH spots produced by FISH probes having different and relatively wide emission spectra. 
     A system  230  for analyzing the signal produced by TDI detector  44  and performing the deconvolution steps described above is illustrated in FIG.  15 . In this Figure, the signal from TDI detector  44  is applied to an amplifier  232 , which buffers the signal and amplifies it to achieve a level required by an analog to digital (A-D) converter  234 . This A-D converter converts the analog signal from amplifier  232  into a digital signal that is input into a TDI line buffer  236 . TDI line buffer  236  temporarily stores the digital signal until it can be processed by a CPU  238 . To carry out the deconvolution noted above, a spectral buffer  240  is loaded with the known emission spectrum for each of the FISH probes being used so that their emission spectra can be deconvolved with the signal stored in TDI line buffer  236 . CPU  238  is a high speed processor programmed to carry out the deconvolution and other analysis procedures, enabling the identification of desired characteristics or parameters of the object being imaged. The output from CPU  238  is temporarily stored in an image line buffer  242  that enables the image to be displayed or otherwise recorded for later analysis. 
     FIG. 16 illustrates a practical application of the present invention for identifying a male cell  200  and a female cell  208  and for producing their corresponding scatter images  212  and  220 . Male cell  200  includes a nucleus  202  that has been stained with a yellow fluorescent dye. In addition, a FISH probe  204  produces a fluorescent orange emission, indicating the presence of an X-chromosome in the nucleus, while a FISH probe  206  produces red fluorescence emission, indicating the presence of a Y-chromosome. Spectral decomposition of the fluorescence emissions from male cell  200 , when the cell is illuminated with light from a green laser, results in a series of images on TDI detector  44 , separated as a function of the wavelength of the light that is imaged. Laser light that is incident on the cells has an extremely narrow waveband, and image  212  of male cell  200  produced by laser scatter is only slightly convoluted by the spectral decomposition process. Green laser scatter image  212  of cell  200  and its nucleus  202  appear on the left side of the TDI detector, while a fluorescent spot  214  corresponding to the yellow fluorescence emitted by nucleus  202  appears in the next few columns on the TDI detector. Furthermore, as a function of the different wavelengths of the fluorescence emitted by FISH probes  204  and  206 , FISH spots  216  and  218  appear at locations spaced apart on the detector, but slightly blurred across the columns of TDI detector  44  due to the widths of their respective emission spectra. By analyzing the signals produced by the TDI detector, the FISH probes responsive to X and Y chromosomes are detected, enabling the user to determine that cell  200  is a male cell, since it includes both the X and Y chromosome. Similarly, female cell  208 , when spectrally decomposed, also includes the characteristic yellow fluorescence of nucleus  210 , but unlike the male cell, includes two FISH spots  216  corresponding to FISH probes  204 , which indicates the presence of two X-chromosomes. Because TDI detector  44  also distinguishes the spatial position of male cell  200  and female cell  208 , the corresponding spectral decompositions for these cells are readily separately resolved as both cells pass through the imaging system in the direction indicated by the arrow to the lower left of FIG.  16 . Again, it should be noted that a deconvolution can be applied to the signal produced by TDI detector  44  to provide better resolution of the corresponding FISH spots that are illustrated. 
     Non-Distorting Spectral Dispersion Systems 
     The present invention can be provided with a spectral dispersion filter assembly that does not convolve the image with the emission spectra of the light forming the image, thereby eliminating the need for deconvolution of the emission spectra from the image. FIG. 17 illustrates a seventh preferred embodiment of the invention corresponding to such a non-distorting spectral dispersion system  250  that employs a five color stacked wedge spectral dispersing filter assembly  252 . This seventh embodiment is substantially similar to the embodiment shown in FIGS. 1,  2 , and  3 , except that spectral dispersing prism element  36  (of FIGS. 1,  2  and  3 ) is replaced by spectral dispersing filter assembly  252 . The spectral dispersing filter assembly splits the light into a plurality of light beams having different bandwidths. Each light beam thus produced is directed at a different nominal angle so as to fall upon a different region of TDI detector  44 . The nominal angular separation between each bandwidth produced by the spectral dispersing filter assembly  252  exceeds the field angle of the imaging system in object space thereby preventing overlap of the field images of various bandwidths on the detector. 
     Spectral dispersing filter assembly  252  comprises a plurality of stacked dichroic wedge filters, including a red dichroic filter R, an orange dichroic filter O, a yellow dichroic filter Y, a green dichroic filter G, and a blue dichroic filter B. Red dichroic filter R is placed in the path of collected light  34 , oriented at an angle of approximately 44.0° relative to an optic axis  253  of collection lenses  32   a  and  32   b . Light of red wavelengths and above, i.e., &gt;640 nm, is reflected from the surface of red dichroic filter R at a nominal angle of 1°, measured counter-clockwise from a vertical optic axis  257 . Example spectral reflectance characteristics R′ of red dichroic filter R are plotted in FIG. 18, along with example spectral reflectance characteristics corresponding to the other dichroic filters used in spectral dispersing filter assembly  252 . In FIG. 18, O′ indicates the spectral reflectance characteristics of orange dichroic filter O, Y′ indicates the spectral reflectance characteristics of yellow dichroic filter Y, etc. The light reflected by red dichroic filter R leaves spectral dispersing filter assembly  252  and passes through imaging lenses  40   a  and  40   b , which cause the light to be imaged onto a red light receiving region of TDI detector  44 , which is disposed toward the right end of the TDI detector, as shown in FIG.  17 . 
     Orange dichroic filter O is disposed a short distance behind red dichroic filter R and is oriented at an angle of 44.5 degrees with respect to optic axis  253 . Light of orange wavelengths and greater, i.e., &gt;610 nm, is reflected by orange dichroic filter O at a nominal angle of 0.5° with respect to vertical optic axis  257 . Because the portion of collected light  34  comprising wavelengths longer than 640 nm was already reflected by red dichroic filter R, the light reflected from the surface of orange dichroic filter O is effectively bandpassed in the orange colored region between 610 nm and 640 nm. This light travels at a nominal angle of 0.5° from vertical optic axis  257 , and is imaged by imaging lenses  40   a  and  40   b  so as to fall onto an orange light receiving region disposed toward the right hand side of TDI detector  44  between a center region of the TDI detector and the red light receiving region, again as shown in FIG.  17 . 
     Yellow dichroic filter Y is disposed a short distance behind orange dichroic filter O and is oriented at an angle of 45° with respect to optic axis  253 . Light of yellow wavelengths, i.e., 560 nm and longer, is reflected from yellow dichroic filter Y at a nominal angle of 0.0° with respect to vertical optic axis  257 . Wavelengths of light reflected by yellow dichroic filter Y are effectively bandpassed in the yellow region between 560 nm and 610 nm and are imaged by imaging lenses  40   a  and  40   b  near vertical optic axis  257  so as to fall on a yellow light receiving region toward the center of TDI detector  44 . 
     In a manner similar to dichroic filters R, O, and Y, dichroic filters G and B are configured and oriented so as to image green and blue light wavebands onto respective green and blue light receiving regions of TDI detector  44 , which are disposed toward the left-hand side of the TDI detector. By stacking the dichroic filters at different predefined angles, spectral dispersing filter assembly  252  collectively works to focus light within predefined wavebands of the light spectrum onto predefined regions of TDI detector  44 . Those of ordinary skill in the art will appreciate that the filters used in the spectral dispersing filter assembly  252  may have spectral characteristics that differ from those described above and in FIG.  18 . Further, the spectral characteristics may be arbitrary and not limited to dichroic in order to achieve the desired dispersion characteristics. 
     The wedge shape of the dichroic filters in the preceding discussion allows the filters to be placed in near contact, in contact or possibly cemented together to form the spectral dispersing filter assembly  252 . The angle of the wedge shape fabricated into the substrate for the dichroic filter allows easy assembly of the spectral dispersing filter assembly  252 , forming a monolithic structure in which the wedge-shaped substrate is sandwiched between adjacent dichroic filters. If the filters are in contact with each other or cemented together, the composition of the materials that determine the spectral performance of the filter may be different from those which are not in contact. Those of ordinary skill in the art will appreciate that flat, non wedge-shaped substrates could be used to fabricate the spectral dispersing filter assembly  252 . In this case another means such as mechanically mounting the filters could be used to maintain the angular relationships between the filters. 
     In addition to the foregoing configuration, non-distorting spectral dispersion system  250  may optionally include a detector filter assembly  254  to further attenuate undesired signals in each of the light beams, depending upon the amount of rejection required for out-of-band signals. FIG. 19 illustrates the construction of an exemplary detector filter  254  corresponding to the five color bands discussed above and includes a blue spectral region  256 , a green spectral region  258 , a yellow spectral region  260 , an orange spectral region  262 , and a red spectral region  264 , all of which are disposed side-by-side, as shown in the Figure. The corresponding spectral characteristics of the blue, green, yellow, orange, and red spectral regions or wavebands are respectively shown in FIGS. 20A-20E. The detection filter assembly shown in FIG. 19 may be constructed by cementing separate filters in side-by-side arrangement on a common substrate or by other means well known to those or ordinary skill in the art. Additionally, the ordinary practitioner in the art will understand that the filter may alternatively be placed at an intermediate image plane, instead of directly in front of TDI detector  44 . 
     In the embodiment shown in FIG. 17, light may pass through each dichroic filter in the spectral dispersing filter assembly  252  twice before exiting the spectral dispersing filter assembly  252 . This condition will further attenuate out-of-band signals, but will also attenuate in-band signals. FIG. 21 illustrates an eighth embodiment of the present invention in which the light does not pass through another dichroic filter after reflection. In this embodiment, a plurality of cube dichroic filters, including a red cube filter  266 , a yellow cube filter  268 , a green cube filter  270 , and a blue cube filter  272  are spaced apart sufficiently to ensure that light does not pass through any of the cube filters more than once. As with the embodiment of FIG. 17, the cube dichroic filters are oriented at appropriate angles to image light within a predefined bandwidth to distinct regions on a TDI detector  274 . As the light is reflected from each of cube dichroic filters  266 ,  268 ,  270  and  272 , it is directed toward imaging lenses  40   a  and  40   b , and different bandpass portions of the light are focussed upon corresponding red, yellow, green, and blue light receiving segments or regions defined on a light receiving surface of TDI detector  274 . If desired, an optional detector filter assembly  276  of similar construction to detector filter assembly  254  (but without the orange spectral region) may be used to increase the rejection of out-of-band signals. It should be apparent to those skilled in the art that separate spaced apart plate, or pellical beam splitters could also be used in this application instead of the cube filters. In the eight embodiment illustrated in FIG. 21, the image lenses  40   a  and  40   b  must be placed a sufficient distance away from the plurality of cube filters to minimize the clear aperture requirement for lenses  40   a  and  40   b . Those skilled in the art will appreciate the clear aperture in the plane orthogonal to the page can increase as the distance between the lenses and plurality cube filters increases. Therefore, the placement of lenses  40   a  and  40   b  must be chosen to appropriately accommodate the clear aperture in both planes. 
     The foregoing descriptions of the seventh and eighth preferred embodiments illustrate the use of four and five color systems. Those skilled in the art will appreciate that a spectral dispersing component with more or fewer filters may be used in these configurations in order to construct a system covering a wider or a narrower spectral region, or different passbands within a given spectral region. Likewise, those skilled in the art will appreciate that the spectral resolution of the present invention may be increased or decreased by appropriately choosing the number and spectral characteristics of the dichroic and or bandpass filters that are used. Furthermore, those skilled in the art will appreciate that the angles or orientation of the filters may be adjusted to direct light of a given bandwidth onto any desired point on the TDI detector. In addition, there is no need to focus the light in increasing or decreasing order by wavelength. For example, in fluorescence imaging applications, one may wish to create more spatial separation on the TDI detector between the excitation and emission wavelengths by changing the angles at which the filters corresponding to those wavelengths are oriented with respect to the optic axes of the system. Finally, it will be clear to those skilled in the art that dispersion of the collected light may be performed on the basis of non-spectral characteristics, including angle, position, polarization, phase, or other optical properties. 
     As with the earlier embodiments discussed above, many applications of the seventh and eighth preferred embodiments will require that one or more light sources be used to provide light that is incident on the object being imaged. Accordingly, various light sources disposed at different positions, such as those shown in FIGS. 5-7 and discussed above, may be used to enhance the image quality produced by each of these embodiments. For clarity and to simplify the explanation of these embodiments, the light sources have been omitted in FIGS. 17 and 21; however, it will be recognized by those skilled in the art how such light sources may be employed in these embodiments, based on the previous discussion of the use of the light sources with respect to the earlier embodiments. 
     FIG. 22 illustrates the distribution of images on TDI detector  44  corresponding to imaging a plurality of cells  200  when using non-distorting spectral dispersion system  250 . As will be evident by comparing FIG. 22 to FIG. 16, the resultant images on the TDI detector are similar in many ways. However, when using the non-distorting spectral dispersion system, there is no image broadening as is seen in FIG. 22, which would otherwise result due to the convolution of the emission spectrum and the object. Instead, all wavelengths within the predefined bandwidth of each dichroic filter are reflected from the filter at the same nominal angle, so image components that fall within that passband suffer no positional distortion on the detector. The field angle orthogonal to flow in object space is also indicated on FIG.  22 . In this particular configuration, the field angle in object space is less than +/−0.25°. Those skilled in the art will appreciate that the field angle can be made larger or smaller. To the extent that the field angle is made larger, for example, to image cells over a wider region on a slide or in a broad flat flow, the field angle at the detector will increase in proportion to the number of colors used. FIG. 22 illustrates the image projected onto the detector when three cells  280 ,  282  and  284  are flowing through the field of view. Light scatter images of cells  280 ,  282 , and  284  are seen on the left hand side of the detector denoted as the BLUE area. Images of cell nuclei  202  stained with a green fluorescent dye are seen in the GREEN area of the detector. Three differently-colored genetic probes  204 ,  205 , and  206  are also employed for the analysis of the sex chromosomes within the cells. Probe  204  stains the X chromosome with an orange fluorescing dye, probe  205  stains the Y chromosome with yellow fluorescing dye, and probe  206  stains the inactive X chromosome in female cells with a red fluorescing dye. Cell  282  is imaged onto the detector as shown in FIG.  22 . An image  286  of probe  204  from cell  282  is seen in the ORANGE area of the detector. Likewise an image  288  of probe  205  is seen in the YELLOW area of the detector. The signal on the detector is processed to determine the existence and position of these images on the detector to determine that cell  282  is a male cell. In a similar manner, cells  280  and  284  contain probes  204  and  206 , which create images  290  and  292  in the ORANGE area of the detector, and images  294  and  296  in the RED area of the detector, indicating that these cells are female, respectively. 
     Although the present invention has been described in connection with the preferred form of practicing it, those of ordinary skill in the art will understand that many modifications can be made thereto within the scope of the claims that follow. Accordingly, it is not intended that the scope of the invention in any way be limited by the above description, but instead be determined entirely by reference to the claims that follow.