Abstract:
An apparatus images a surface. An imager stage linearly translates the surface in a first direction. A light path has a first end defining an input aperture perpendicular to the first direction and parallel to the surface, and a second end defining an output aperture. A plurality of radiation beams linearly scan and interact in time-multiplexed alternating turns with the surface below the input aperture to produce a time-multiplexed light signal that is collected by the input aperture and transmitted by the light path to the output aperture. A photodetector arrangement detects the light signal at the output aperture. A processor processes the detected time-multiplexed light.

Description:
CROSS REFERENCE  
       [0001]     The following co-pending application U.S. Ser. No. 10/616,366, Filed Jul. 9, 2003, is hereby incorporated by reference in its entirety. 
     
    
     BACKGROUND  
       [0002]     The present exemplary embodiments relate to the imaging arts, and find particular application in conjunction with low and high-density cell detection, locating, and identifying in blood smears, biological assays, and the like across distinct imaging systems, and will be described with particular reference thereto. However, it is to be appreciated the exemplary embodiments will also find application in imaging, locating and identifying other types of low- or high-density features on various substantially planar surfaces and samples, such as imaging semiconductor wafers, imaging particulate contaminants in fluids or thin solid films, and so forth, with such imaging finding specific uses in the printing arts, electronic arts, medical arts, and other scientific and engineering areas.  
         [0003]     In rare cell studies, a particular problem arises due to the typically low concentration of the rare cells in the blood or other body fluid. In a typical rare cell study, blood is processed to remove cells that that are not needed. Then a fluorescent material is applied that attaches to antibodies, which in turn selectively attach to a cell surface or cellular protein of the rare cells. The cellular proteins may be membrane proteins or proteins within a cell, such as cytoplasm proteins. The antibodies may also attach to other types of molecules of the rare cell, as well as to DNA.  
         [0004]     The fluorescent material may be a fluorescent marker dye or any other suitable material which will identify the cells of interest. A smear treated in this manner, which may include the blood and/or components of the blood, is prepared and optically analyzed to identify rare cells of the targeted type. For statistical accuracy it is important to obtain as large a number of cells as required for a particular process, in some studies at least ten rare cells should be identified, requiring a sampling of at least ten million cells, for a one-in-one-million rare cell concentration. Such a blood smear typically occupies an area of about 100 cm 2 . It is to be understood, however, that this is simply one example and other numbers of cells may be required for statistical accuracy for a particular test or study. Other cell identifiers which are being used and investigated are quantum dots and nano-particle probes. Also, while a rare cell is mentioned as a one-in-one-million cell concentration, this is not intended to be limiting and is only given as an example of the rarity of the cells being sought. The concepts discussed herein are to be understood to be useful in higher or lower levels of cell concentration.  
         [0005]     In this regard, the ability to scan large numbers of cells at a high rate is considered a key aspect which increases the throughput of testing processes. Therefore, it is considered valuable to provide a system which improves the speed, reliability and processing costs which may be achieved by cell detection systems and/or processes.  
         [0006]     A number of cell detection techniques have been proposed including fluorescence in situ hybridization (FISH), flow cytometry, laser scanning cytometry (LSC), among others.  
         [0007]     While the above-noted systems are directed to creating faster scan rates, they nevertheless still have relatively small fields of view (FOV), such as microscopes. This will, therefore, still result in speeds which do not reach the desired scan rates.  
         [0008]     In view of this, the previously noted and incorporated U.S. application Ser. No. 10/271,347 discloses a fiber array scanning technology (FAST) that increases the speed at which scanning of a sample and the detection of potential or candidate rare cells may be accomplished, lending itself to the investigation of large samples. Still, while the aforementioned application provided an increased speed, a still further increase in speed can be accomplished by, e.g., providing a second laser that produces excitation light at a second wavelength or wavelength range and a second signal detector calibrated to sense a second fluorescence signal simultaneously with the first laser and signal detector. In this arrangement, each signal detector could be configured with a filter to sense only the desired respective fluorescence signal. This essentially doubles the amount of information that can be detected during a single scan.  
         [0009]     One problem that arises with this arrangement, however, is that a portion of either stimulated fluorescence signal may significantly overlap the remaining fluorescence signal in terms of wavelength. Selective filtering can reduce this problem but at the cost of reducing the useful wavelength band that may be sensed. A second problem that arises is that this method would normally make use of a beam splitter or dichroic mirror in the light path of the fluorescent radiation in order to direct desired portions of the fluorescent signal to the respective signal detector, further reducing the intensity of the signal being sensed.  
       BRIEF DESCRIPTION  
       [0010]     In accordance with one aspect of the present exemplary embodiments, an imager for imaging a sample is disclosed. An imager stage supports the sample. A light path has a proximate end defining an input aperture viewing the sample on the microscope stage. The light path further has a distal defining an output aperture disposed away from the imager stage. A plurality of scanning radiation sources is arranged in fixed relative positions to the input aperture. The scanning radiation sources each scan a radiation beam on the sample in alternating turns within a viewing area of the input aperture. Each radiation beam interacts with the sample to produce a light signal that is received by the input aperture and transmitted via the light path to the output aperture. Each scanning radiation source rasters the radiation beam over a selected area of the sample. A photodetector arrangement detects the light signal at the distal light path end, and a processor processes the detected light signals. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0011]     The embodiments may take form in various components and arrangements of components, and in various steps and arrangements of steps. The drawings are only for purposes of illustrating preferred embodiments and are not to be construed as limiting the application.  
         [0012]      FIG. 1  shows a perspective view of an imaging apparatus suitable for incorporating features of a preferred embodiment;  
         [0013]      FIG. 2  shows an enlarged perspective view of the morphed fiber optic bundle of the imaging apparatus of  FIG. 1  in relation to the sample;  
         [0014]      FIG. 3  shows an enlarged end view of the first end that defines the input aperture of the morphed fiber optic bundle of the apparatus of  FIG. 1 ;  
         [0015]      FIG. 4  shows a side view of another embodiment, the view centered on the first end of the morphed fiber optic bundle;.  
         [0016]      FIG. 5  shows a plot of excitation radiation and resultant stimulated radiation;  
         [0017]      FIG. 6  shows a schematic view of features of a first embodiment;  
         [0018]      FIG. 7  shows a schematic view of features of an alternate embodiment; and  
         [0019]      FIG. 8  shows a schematic view of features of a further embodiment. 
     
    
     DETAILED DESCRIPTION  
       [0020]     With reference to  FIG. 1 , for purposes of understanding the embodiments described herein, an imaging apparatus is first described which utilizes a galvanometer-based laser scanning system with a single laser transmitter and a single detector. Various embodiments are then described which provide an increased scanning speed and which solve the aforementioned problems. In the figure, the imaging apparatus or imager  10  examines a sample  12  such as a biological smear  14  disposed on at least a portion of a surface of a slide  16 . Imaging apparatus or imager  10 , as expanded upon below, is designed for detection of minute or microscopic material.  
         [0021]     As is known in the art, for cell studies the sample  12  is suitably prepared by drawing a sample of a biological fluid such as, but not limited to, blood or parts of blood from a subject. In a preferred embodiment, the sample is a monolayer of cells. The fluid sample is treated with a fluorescent material, such as but not limited to a marker dye that selectively bonds to different kinds of biological molecules, which may be on the surface or inside the cell, such as proteins, nucleic acids or other molecules. Suitable markers are known in the art for marking a number of different cell types of clinical interest, including selected cancer cell types, fetal cells, or other appropriate cells to be considered. Work is also being undertaken to develop marking materials for numerous other cells such as brain cells, liver cells, as well as bacteria cells, among others. The material preferably emits a characteristic output, such as a fluorescence or a phosphorescence, responsive to a selected excitation irradiation, such as irradiation by a selected wavelength or spectrum of light, x-ray irradiation, electron-beam irradiation, or the like. The characteristic luminescence typically has a characteristic wavelength or spectral range of wavelengths. While dyes are the predominant tagging process, other techniques exist including the use of markers known as quantum dots and DNA nano-particle probes.  
         [0022]     The sample  12  is mounted on an imager translation stage, or slide holder,  20  (shown in part) which includes a linearly translatable track  22  that supports the sample  12 . A motor  24  connects with the track  22  via gearing  26  to translate the track  22  and the supported sample  12  along a y-direction (indicated by arrows  28 ) and a x-direction (indicated by arrows  29 ). Although translation stage  20  driven by a rotary motor  24  is shown in  FIG. 1 , it is also contemplated to employ other types of mechanical driving devices. Furthermore, other types of sample movement such as sample rotation are also contemplated.  
         [0023]     With continuing reference to  FIG. 1  and with further reference to  FIGS. 2 and 3 , a fiber optic bundle  40  includes a first end  42  that is proximate to the sample  12 , and a second end  44  that is distal from the sample  12 . The first end  42  includes a plurality of first fiber ends  46  arranged substantially parallel to one another in an arrangement that defines a generally linear or high-aspect-ratio rectangular input aperture  48  (best seen schematically in  FIG. 3 ) with a long dimension aligned with the x-direction. The input aperture  48  preferably includes a large number of first fiber ends  46 , i.e. thousands of fiber ends. In one suitable embodiment, 40,000 fibers each having an approximately 50 micron diameter are arranged into a 40 fiber-by-1000 fiber array to define the input aperture  48  with a long dimension of approximately 5 cm and a short dimension of about 0.2 cm corresponding to a 25:1 aspect ratio. The first fiber ends  46  can be arranged in a regular pattern, as shown in  FIG. 3 . Alternatively, the first fiber ends can be arranged in an irregular or non-periodic array and may have diameters which are greater or less than 50 microns. Although generally round fiber ends are shown, it is also contemplated to employ fibers with oval, square, hexagonal, or other cross-sectional shapes. The first fiber ends  46  are oriented substantially perpendicular to the plane of the biological smear  14  so as to view the smear  14 .  
         [0024]     The optical fiber bundle  40  “morphs” or changes cross-sectional dimensions and shape between the first end  42  to the second end  44  such that the second end  44  includes a plurality of second fiber ends  50  (best seen schematically in  FIG. 2 ) that define a compact, generally circular output aperture  52 . Preferably, there is a one-to-one correspondence between the first fiber ends  46  and the second fiber ends  50 , and each first fiber end connects with a second fiber end by an individual, distinct fiber having its own waveguiding cladding. Alternatively, each fiber can include only a light-transmissive fiber core, and an ambient/core interface functions to waveguide the light. Other optical fiber types can also be used, such fibers being well known in the art and typically formed of glass, plastic, or other light-transmissive materials by extrusion methods. In  FIG. 2 , the paths of two exemplary individual, distinct fibers  56 , 58  are indicated as dotted lines. The morphed shape of the fiber bundle  40  from an extended, generally linear first end  42  to a compact, generally circular second end  44  is preferably formed by varying a spatial arrangement of the fibers of the optical fiber bundle  40  in a continuous fashion. For the exemplary 40,000 fiber embodiment with each fiber having a 50 micron diameter, the generally circular output aperture  52  has a circular diameter of about 1.3 cm.  
         [0025]     It is particularly pointed out that the spatial relationship between the first fiber ends  46  and the second fiber ends  50  is generally arbitrary. For example, in  FIG. 2  the fibers  56 ,  58  run from approximately the same position in the input aperture  48 . However, the fiber  56  terminates near a top of the output aperture  52 , while the fiber  58  terminates near a middle of the output aperture  52 . Although for convenience in arranging the fibers it is contemplated to arrange the first and second fiber ends  46 ,  50  in the respective apertures  48 ,  52  with a selected correspondence relative to one another, the fiber ends  46 ,  50  can instead have a generally uncorrelated and arbitrary relationship therebetween. Morphed fiber optic bundles similar to the fiber optic bundle  40  are known and used in the optical arts for other applications such as transforming focused light into a linear illumination pattern, and for coupling a light beam into a linear slit of a monochromator or spectrometer.  
         [0026]     To obtain good light transmission, the fiber optic bundle  40  preferably has a high fiber packing factor, for example, fiber optic bundle  40  has a packing factor of about 0.80 or higher. Other factors influencing the light transmission include the polishing or light transmission properties of the tips of the first and second fiber ends  46 ,  50 , the absorption per unit length of the fibers  56 ,  58 , and the overall length of the fibers  56 ,  58 . Fiber bending losses are preferably reduced by avoiding sharp bends of the fiber optic bundle  40 . For example, as seen in  FIGS. 1 and 2 , the difference in orientation of the input aperture  48  and the output aperture  52  is achieved by a gradual bend in the optical fiber bundle  40 .  
         [0027]     It is understood that while a fiber bundle has been described as the mode of transport of the acquired light, any other existing or subsequently developed light transmission component or light path or pipe which includes the appropriate characteristics may be employed as the light path or light pipe.  
         [0028]     With continuing reference to  FIGS. 1-3 , a scanning radiation (light) source  60  in a suitable embodiment includes a laser  62  that produces excitation light (radiation beam)  64  at a wavelength or wavelength range selected to excite the material used in marking the biological smear  14 . The excitation light  64  is angularly scanned by a galvanometer  66  that has a reflective surface that rotates (indicated by curved arrows  68 ) responsive to an electrical input. An optional focusing lens  70  focuses the angularly scanned excitation light  64  onto the sample  12 , and more particularly onto the biological smear  14 . The angular scanning produced by the galvanometer  66  translates into a linear sweeping or fast scanning (indicated by arrows  72 ) of the excitation light on the biological smear  14  along a linear trajectory  74  arranged below the input aperture  48  and parallel to the long dimension of the input aperture  48 . That is, using the coordinate system of  FIG. 1  the linear trajectory  74  is parallel to the x-direction. In a suitable embodiment, the trajectory  74  is disposed on the biological smear  14  about one millimeter below the input aperture  48 , although other distances will be appropriate dependant upon devices and the environment in which these concepts are implemented.  
         [0029]     For cell studies, the excitation radiation  64  preferably produces-a spot size on the biological smear  14  which substantially comports with a size of the cells, which may vary in size but are typically about one to thirty microns in size. To obtain such narrow beam focusing, the focusing lens  70  is typically included.  
         [0030]     With continuing reference to  FIGS. 1-3 , an electronic control unit  80  communicates with the galvanometer  66  and the translation stage  20  to coordinate the linear sweeping or scanning  72  of the radiation beam  64  along the trajectory  74  and the linear translation  28  of the sample  12  to effectuate a rastering of the radiation beam  64  across a selected area of the sample which is bounded in the x-direction by the smaller of a span of the trajectory  74  and the long dimension of the input aperture  42 . Preferably, the span of the trajectory  74  substantially comports with the long dimension of the input aperture  42 .  
         [0031]     Excitation radiation beam  64  is incident upon the biological smear  14  at an oblique angle which is larger than a collection angle θ of the input aperture  42 . The collection angle  0  depends upon a short dimension of the input aperture  42 , the distance between the input aperture  42  and the biological smear  14 , and the light collecting characteristics of the first fiber ends  46 . The latter is suitably characterized by a numerical aperture of the fiber ends. As is known in the art, an optical fiber end typically has a large numerical aperture corresponding to a large light collection angle which is particularly advantageous for collecting the typically weak characteristic luminescence of the cells. In a suitable embodiment, the radiation beam  64  impinges upon the sample  12  at 30°-90°. When beam  64  impinges upon sample  12  at approximately 90°, a bifurcated light path may be provided wherein light is collected on both sides of the scanning beam. One example of such-a bifurcated light path is shown in U.S. patent application Ser. No. (Attorney Docket D/A2247, XERZ 2-00868), entitled Improved Method and Apparatus for Scanning and Light Collection for a Rare Cell Detector, hereby fully incorporated by reference.  
         [0032]     Because the incidence angle of the radiation beam  64  is larger than the collection angle θ of the input aperture  42 , specularly reflected radiation is not collected by the input aperture  42 . However, the characteristic luminescence produced by the treated cells generally emits uniformly in all spatial directions, i.e. each treated cell corresponds to a point light source. Hence, a substantial portion of the characteristic luminescence is collected by the input aperture  42  due to its close proximity to and alignment with the radiation beam trajectory  74  on the biological smear  14  as well as the large numerical aperture of the first fiber ends  46 . The collected light enters the first fiber ends  46 , transmits along the individual fibers, e.g. the fibers  56 ,  58  shown in  FIG. 2 , and exits the optical fiber bundle  40  at second fiber ends  50  that correspond to the collecting first fiber ends  46 .  
         [0033]     It will be appreciated that the characteristic luminescence produced by a particular cell will not generally be collected by all or even most of the first fiber ends  46 . Rather, only one or a few of the first fiber ends  46  which are closely proximate to the cell will collect the characteristic luminescence therefrom. In an exemplary embodiment, the radiation spot size is about 10-15 microns corresponding to a similarly sized cell, while each first fiber end  46  has a diameter of about 50 microns. Hence, only one or a few fibers may be needed to view and collect the characteristic luminescence for any given position of the sweeping radiation beam  64 .  
         [0034]     However, because at the second end  44  of the fiber bundle  40  the second fiber ends  50  are arranged to define the compact, output aperture  52 , the characteristic luminescence emanates from a small region of space corresponding to the output aperture  52  regardless of which of the first fiber ends  46  collected the characteristic luminescence. As the excitation beam  64  sweeps along its trajectory  74  parallel to and typically below the input aperture  48 , the proximate one or few of the first fiber ends  46  collect the characteristic luminescence, which is channeled by the fiber optic bundle  40  to the compact output aperture  52 .  
         [0035]     In one suitable embodiment, the blocking filter  94  is an interference filter with a reflectance peak coinciding with a center wavelength of the radiation beam  64  is employed. As is known in the art, optical interference filters have a rejection ratio that is strongly dependent upon the angle of incidence of the light. An exemplary interference filter used in one actually constructed embodiment exhibits a 106:1 or greater rejection ratio for light incident within ±14° of normal incidence. In this constructed embodiment, the first lens  92  includes a lens combination, designed using known optical design methods, that collimates light emanating from the output aperture  52  to within a ±10° angular divergence.  
         [0036]     With continuing reference to  FIG. 1 , a second lens  96  focuses the collimated collected light onto a photodetector arrangement  98 . By combining the compact output aperture  52  with focusing optics  92 ,  96 , photodetector  98 , which may be a single photodetector, provides signal detection for the spatially distributed linear input aperture  48 . Because of the typically low collected characteristic luminescence intensities produced by treated cells, the photodetector  98  is preferably a photomultiplier tube. As is known in the art, a photomultiplier tube provides substantial signal gain through cascade multiplication of electrons in a multi-stage high-voltage cathode. To further improve the signal-to-noise ratio, the optical path of the signal detector  90  is preferably enclosed to substantially reduce noise due to stray light.  
         [0037]     Those skilled in the art can suitably modify the signal detector  90  by addition, removal, or substitution of components to adapt it to specific imaging situations. For applications providing alternate signal-to-noise characteristics, a photodiode can be used for the photodetector  98 . Similarly, the single photodetector  98  and multiple focusing elements  92 ,  96  can be replaced by a photodetector array having an area that comports with an area of the output aperture  52 .  
         [0038]     Although the hereinbefore described embodiments show the stimulated emissions being collected by an aperture  48  arranged above the sample, it is to be appreciated that, as shown in  FIG. 4  an input aperture  48 ′ may be arranged to view the sample  12 ′ from below, i.e., from a side of the slide  16 ′ that is opposite the biological smear  14 ′. That is, the input aperture  48 ′ views the biological smear  14 ′ through the slide  16 ′, which is light transmissive for the characteristic luminescence of the cells. The slide  16 ′ may also include an optional laser blocking filter  110 , such as an absorption band pass filter, coating the surface below the biological smear  14 ′. The embodiment of  FIG. 4  may also include an optional cylindrical reflector  112  having a linear focal line generally coinciding with the radiation beam trajectory  74 ′ on the biological smear  14 ′. The cylindrical reflector  112  can improve the signal to noise ratio for certain imaging applications by increasing the amount of characteristic luminescence that is collected. It will be recognized that the cylindrical reflector  112  can also be used in conjunction with the configuration of  FIG. 1 .  
         [0039]     In the above-described embodiments, it would often be advantageous when scanning fluorescent probes decorating cells to use multiple probes, with each fluorescing at a different wavelength than the other. For example, multiple probes enable simultaneous measure of different cell properties. Multiple probes can also be used to identify and eliminate noise or artifacts. For efficient excitation, it is often desirable to excite each probe with a specific laser that is optimized for the probe&#39;s absorption range. It can also be desirable to measure emission from multiple probes excited by the same laser.  
         [0040]     An application of multiple excitation sources is used in conjunction with dual-labeling of cells to eliminate probe aggregate artifacts. Here two probes are chosen to have sufficiently different emission wavelengths that the collective emission can be effectively separated by conventional emission filters. The problem inherent in this approach is that the longer wavelength emission is inefficiently stimulated by a single excitation laser and would be more efficiently excited by a laser whose wavelength is closer to its emission. To obtain proper ratios of the emissions from the dual probes with one excitation source, the long wavelength emitter is desirably present at a higher concentration in the mixture. Such high concentrations can cause a spread of the ratio of the emissions and can also cause aggregate formation. Since lower concentrations can be used with more efficient excitation, it can be valuable to excite each probe with a different laser source.  
         [0041]     With reference to  FIG. 5 , the above-described problems associated with the use of dual probes, and dual laser sources, is shown. Transmission percentage  130  as a function of wavelength  132  is shown for exemplary wavelengths of interest. A first laser wavelength  134  is shown as a vertical line at 488 nm, which is a suitable laser wavelength for stimulating an FITC probe having a fluorescent emission  136  with peak emission intensity at approximately 520 nm. The difference in wavelength between the laser  134  and the peak emission wavelength of the FITC probe  136  is know in the art as the Stokes shift. Stokes shift is the difference in wavelength between absorbed and emitted quanta. The emitted wavelength is longer or equal to the incident wavelength due to energy conservation; the difference being absorbed as heat in the atomic lattice of the material. A first emission filter transmission curve  138  is shown which is suitable for filtering any undesirable reflections of the first laser, and other unwanted frequencies, while allowing substantial transmission of the desired probe fluorescence in the range of approximately 505 nm to 545 nm.  
         [0042]     If a second R-PE probe  140  having a peak emission intensity at 576 nm is added simultaneously with the first probe  136 , it may be observed that several problems arise. The emissions from the second probe overlap significantly with the emissions from the first probe (signal crosstalk) in the range of approximately 550 nm to 600 nm, making it difficult to differentiate between first and second probe emissions. In this exemplary case, a second emission filter  142  can be added with a transmission range from approximately 575 nm to 640 nm which partially alleviates the problem by blocking most of the first probe emissions. However, the same disadvantage still exists because of significant remaining crosstalk, even with the use of emission filters. For example, a significant portion of the first probe  136  emissions extends into the transmission band of the second emission filter  142  transmission curve, thereby reducing the sensitivity and signal-to-noise ration of the system.  
         [0043]     The above-described signal loss is made more significant by the fact that only a single laser has been included in the system, and the second probe will not be stimulated as efficiently as the first probe because of the larger wavelength difference between the laser  134  and the second probe  140 . To more efficiently stimulate the second probe, a second laser  144  emitting with a wavelength of 532 nm may be added, emitting simultaneously with the first laser  134 . This, however, gives rise to another problem that is difficult to overcome. With a wavelength of 532 nm, the second laser falls within the transmission range of the first emission filter  138 . Because of this, reflections of the second laser  144  may be erroneously detected as stimulated emissions from the first probe  136 .  
         [0044]     With continuing reference to  FIG. 5 , and further reference to  FIG. 6 , shown schematically is an embodiment providing two or more signal detectors, and two or more lasers, which solves the aforementioned problems that arise when using dual probes (or multiple probes), by time-multiplexing the lasers. In this embodiment, the sample is stimulated sequentially, rather than simultaneously, with different laser wavelengths so there is no overlap of the probe&#39;s emission spectra by an excitation source. And also, signal crosstalk is reduced because the laser stimulating the first probe is only poorly stimulating the second probe which causes the crosstalk. To illustrate further, without time-multiplexing the lasers, a dichroic mirror having a transmission curve  146  would typically be used to separate the first and second probe emissions  136  and  140 . It may be observed in  FIG. 5  that a significant portion of the first probe emissions  136  fall undesirably within the transmission region of the dichroic mirror transmission curve  146 , i.e., for wavelengths greater than approximately 520 nm.  
         [0045]     The embodiment shown in  FIG. 6  is shown in simplified form in order to facilitate an understanding of the described embodiments. A first dichroic mirror  150  receives light  64 ′ from a first laser  62 ′ and light  64 ″ from a second laser  62 ″ operating at, e.g., 488 nm and 532 nm respectively. The received lights are selectively provided as beam  64 . The excitation signal from each laser is first passed through respective shutters  148 ′ and  148 ″. The shutters are coordinated so the single beam  64 , at any given time, only contains light from one of the laser sources. Beam  64  is reflected by a reflective device  152  to the galvanometer  66  which scans the reflected beam through the optional lens arrangement  70  to the sample  12 , generally operating as described previously with reference to  FIG. 1 .  
         [0046]     Stimulated fluorescence from first and second probes in the sample  12  is received and transmitted along a light path  154  to the first lens  92 . The focused light beam in light path  154  is then appropriately split by a second dichroic mirror  156 , and selective portions of the light beam of the light path  154  are received by the respective photodetectors  98 ′ and  98 ″. Each of the photodetectors may be preceded in the light path by respective blocking filters  94 ′ and  94 ″, and second lens arrangements  98 ′ and  98 ″. In this manner, each of the photodetectors  98 ′ and  98 ″ can detect the fluorescence from the respective probes, and communicate the detected intensity levels to the control unit  80 . Alternately, a single photodetector may be utilized by replacing the shown blocking filter arrangement with, e.g., a rotating disk having blocking filters  94 ′ and  94 ″ mounted in the disk, wherein the rotation of the disk is synchronized with the laser shutters  148 ′ and  148 ″.  
         [0047]      FIG. 7  shows an alternative embodiment which addresses the aforementioned issues, providing the benefits of a reduced total scanning time. In this embodiment, the sample is also stimulated sequentially, rather than simultaneously. This is accomplished by using a rotating polygon scanner arrangement  160 , shown in this exemplary system as a multi-sided mirror  160   a , rotated by a motor  160   b , and with a flywheel  160   c , where the motor and flywheel permit for smooth rotation which eliminates signal jitter. As shown, the scanner arrangement scans a laser beam across the sample surface. In this particular embodiment, one side of the polygon mirror  160  scans the beam from the first laser  62 ′ while another mirror of the polygon scanner scans the beam from laser  62 ″. This device enables the method of sensing emission by all probes from each laser with no laser interference. For example with dual probes as shown, it can be valuable to sense the emission from both probes excited by a first laser while also sensing the emission of a specific probe excited by a second laser. In the embodiment shown in  FIG. 7 , only one photodetector  98  has been shown, and time multiplexed filtering, if necessary, is accomplished by filter  162 . It is to be appreciated, however, that embodiments utilizing multiple photodetectors are intended to fall within the scope of this disclosure, and the use of the time-multiplexed filter  162  is optional.  
         [0048]      FIG. 8  shows a further embodiment which also addresses the aforementioned issues. In this embodiment, the sample is also stimulated sequentially, rather than simultaneously. This embodiment also uses a polygon scanner arrangement which scans the laser beam across the sample surface, however, in this embodiment, an additional scan is,automatically inserted for each laser between scans provided by the embodiment of  FIG. 7 , thereby doubling the scan rate without increasing the speed of the polygon mirror  160 . By proper placement of the angles of incidence from the lasers, the polygon automatically accomplishes the sequential scanning, including the additional inserted scans, without the use of shutters at each of the lasers. In the example shown, because of the placement of the lasers  62 ′ and  62 ″ each side of the polygon scanner arrangement  160  first scans the beam from the first laser  62 ′ and then sequentially scans the beam from the second laser  62 ″ for the direction of rotation shown. The additional scan for each side of the mirror effectively doubles the number of scans. It is to be noted that the concept may be extended to any number of appropriately positioned lasers. The polygon scanner may also have any desired number of sides.  
         [0049]     The embodiment shown in  FIG. 8  works particularly well if the scanning efficiency is less than 50%, thereby allowing room for the automatically inserted scan. An advantage of this system and method is that an extra scan is inserted without increasing the polygon rotation rate or lowering the overall process speed. An additional advantages lies in the fact that the rotating scanner arrangement  160  produces less jitter that the oscillating galvanometer used in a previously described embodiment. Still further, the embodiment shown in  FIG. 8  can potentially double the amount of information detected in a single scan, thereby reducing total scanning time significantly. It is to be appreciated that the arrangement and order of components provided in the figure represent an exemplary embodiment only, and the present disclosure is not limited by the arrangement of components.  
         [0050]     With any of the embodiments shown in  FIGS. 6-8 , the degradation of fluorescence signal due to an overlapping laser frequency is essentially eliminated. Further, signal crosstalk is reduced because the desired probe can be stimulated with a laser frequency that maximizes its emission, while adjacent crosstalk-producing probes are stimulated at inefficient emission frequencies. Each of these solutions significantly lowers the cost of the filtering system while upgrading its performance in a multi-probe, multi-laser fluorescence system. The embodiment shown in  FIG. 8  can double the number of scans provided by the embodiment shown in  FIG. 7 , without increasing the speed of rotation of the polygon mirror or the scan rate.  
         [0051]     Another aspect of the time-multiplexed scanning as described in the embodiments lies in the fact that autofluorescence noise is higher near the excitation wavelength and diminishes for stimulated emission wavelengths farther from the excitation wavelength. As was shown with reference to  FIG. 5 , the first emission filter transmission curve  138  was necessarily configured to block a significant portion of the longer wavelengths of the FITC fluorescent emission  136  in order to filter out the crosstalk with the simultaneous R-PE fluorescent emission  140 . Because the crosstalk is essentially eliminated by time-multiplexed scanning, it is no longer necessary to block the longer wavelength emissions of a particular probe, thereby improving the signal-to-noise ratio which is valuable in certain image filtering operations.  
         [0052]     Although the embodiments have been described with particular reference to cell identification, the described concepts are not limited in application thereto. The imager apparatus configurations shown in  FIGS. 6-8  are suitable for many imaging applications in which features are to be identified or located. In one such application lying in the biomedical arts, typically ten to ten thousand DNA elements are arranged in an array known in the art as a DNA chip. The DNA elements are processed so that selected elements include a fluorescent tag. The embodiments shown are suitable for identifying the tagged DNA elements in a DNA chip that includes a large number of DNA elements. Implementing the concepts described in the foregoing permits for an imaging apparatus that can access the sample several times faster than existing technology.  
         [0053]     The application has been described with reference to the preferred embodiments. Obviously, modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the disclosure be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.